Alpha-amylase Production by Bacillus licheniformis 1331 using Jackfruit Seeds as Substrateby Rica Mae A. Palongpalong An Undergraduate Thesis Submitted to the School of Chemical Engineering and Chemistry in Partial Fulfillment of the Requirements for the Degree Bachelor of Science in Biotechnology Mapúa Institute of Technology December 2008 ii APPROVAL SHEET This is to certify that we have supervised the preparation of and read the thesis / practicum or research report prepared by Rica Mae A. Palongpalong entitled Alpha-amylase Production by Bacillus licheniformis 1331 using Jackfruit Seeds as Substrate and that the said thesis / practicum or research report has been submitted for final examination by the Oral Examination Committee. Percival G. Garcia Adviser Flordeliza C. De Vera Course Adviser As members of the Oral Examination Committee, we certify that we have examined this report and hereby recommend that it be accepted as a fulfillment of the practicum for the Degree Bachelor of Science in Biotechnology. Kevin B. Dagbay Panel Member 1 Arturo L. Tapas Jr. Panel Member 2 Russell S. Julian Panel Member 3 This practicum paper is hereby approved and accepted by the School of Chemical Engineering and Chemistry as fulfillment of the practicum requirement for the Degree Bachelor of Science in Biotechnology. Isidro C. Medina, Jr. Head (OIC), Biotechnology Program Luz L. Lozano Dean iii ACKNOWLEDGEMENT I would like to express my sincere gratitude to the following people who have contributed in the completion of my thesis: First, to God Almighty, for all the countless blessings and graces through the following people who have been instrumental in the completion of my thesis. To my mother, Dr. Beauty A. Palongpalong, for all the support that she has given me. Thank you for the encouragement, the sleepless nights of coaching and correcting my sentence construction, for pushing me hard to sustain my spirit to reach the finish line. Most of all the friendship, unconditional love, care, and for nurturing me to be the person that I am today. To my late father, Dr. Artemio D. Palongpalong, PhD. whose memory has served as my inspiration to finish my thesis, he being a professor and Dean of the prime university of the country. To my uncle and aunt, Engr. Ruel Janolino and Grace Janolino, for being my second father and mother respectively. Thank you so much for the financial support, paying my tuition fees, giving my daily allowance and other personal needs, for lending me the coffee grinder for grinding the jackfruit seeds and the money for the chemicals used in the study. Most of all the strong moral support and encouragement and cannot be overemphasize is the pampering just like a spoiled brat kid. The gesture of treating me like your own baby girl will stay in my heart forever. To my brother, Heinrich Victor Valery A. Palongpalong for being understanding that he gives way every time I need the computer for my thesis. To Prof. Percival G. Garcia, my very supportive thesis adviser, for initially approving my thesis topic, purchasing the bacteria from UPLB, patiently checking the English grammar, giving me protocols for the methodology, help me analyze the data and for all the advises, guidance, and continuous support will not be forgotten. To Prof. Kevin B. Dagbay, Prof Russell S. Julian, and Dr. Arturo L. Tapas Jr. my respective panellists, for sharing their ideas, screening my thesis paper, their objective criticisms and suggestions on the improvement of the study. Thank you so much. To Prof. Herbert J. Santos and Prof. Flordeliza C. De Vera, my thesis 1 and thesis 3 course coordinator respectively, for sharing suggestions and ideas regarding the study. To Engr. Ariziel Ruth Marquez, Former Head of Advance Chemistry Laboratory, for giving me permission to use the UV-Vis spectrophotometer and analytical balance. To Ate Lolit, for helping me operate the UV-Vis spectrophotometer in Physical Chemisty Laboratory. iv To Ate Thelma, for helping me make the KH2PO4-NaOH buffer for my alpha-amylase assay and for the friendship we had since I was a freshman in Mapua. To the Mapua guards, for ensuring our safety 24 hours a day when we did our laboratory work overtime. To Jerico Jayson D.C. Uy, for helping me in doing the culture of my bacteria, doing the preparation of standard curve for Bradford assay, and assisting me in my experiment. To Jansen Jan T. Chua, for helping me also in doing the culture of my bacteria To Edison I. Pineda, for writing the letter allowing us to use the N313 or Microbiology Laboratory overnight. To Kathrina R. Siongco, for helping me make the combined graph of Alpha-amylase assay, Bradford assay, and Biomass versus time in hours. To Biologic and Chemsoc family, for being part of your family and letting me serve my two organizations as an Executive Secretary for Chemsoc or Chemistry Society of Mapua and Assistant Secretary and Secretary for Biologic, Association of Biotechnology students of Mapua. You have inspired me to go on up to the final stage of my stay in Mapua. To UPIS Batch 2004, for the friendship, bonding, and unity that we had since our Kindergarten days, partly you have encouraged me during my lowest points in Mapua. To Elaine L. Cunanan, for being my best friend since freshmen through thick and thin, through highs and lows, for always being there for me, and for your loyalty. To Aldrich Thomas R. Agtarap and Alberto L. Abadiano Jr., for being such a good and loyal friend, and for being always ready to lend your helping hand in times of my needs. To those whom I forgot to mention, thank you for everything. For this achievement, I give back all the glory and praises to the omnipotent Father Almighty. Rica Mae A. Palongpalong v TABLE OF CONTENTS TITLE PAGE APPROVAL PAGE ACKNOWLEDGEMENT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABSTRACT Chapter 1: INTRODUCTION Chapter 2: REVIEW OF LITERATURE Jackfruit seed Amylase Alpha (α )-amylase Alpha (α )-amylase Production Chapter 3: ALPHA-AMYLASE PRODUCTION BY BACILLUS LICHENIFORMIS 1331 USING JACKFRUIT SEEDS AS SUBSTRATE Abstract Introduction Methodology Preparation of jackfruit seed Bacterial Strain Medium Preparation Preparation of inoculum i ii iii v vii viii ix 1 3 3 6 7 8 21 21 21 23 23 23 24 24 vi Optimization of cultural conditions Preparation of crude extract enzyme Alpha (α )-amylase assay Iodine reagent Buffer solution Substrate: Starch solution Analytical procedure Bradford assay Preparation of Standard Curve Determination of Unknown Protein Content Biomass determination Results and Discussion Conclusion Recommendation References Chapter 4: CONCLUSION Chapter 5: RECOMMENDATION REFERENCES APPENDICES A: Composition of HIMEDIA® M001 B: Assay on Alpha-amylase Activity C: Assay on Protein Concentration 25 26 26 26 26 26 26 27 27 28 28 29 36 36 36 42 43 44 49 50 51 52 D: Alpha-amylase activity, Protein Concentration, and Biomass Concentration of Samples Taken at Regular Intervals 54 vii LIST OF TABLES Table 1: Factorial design at pH 7 and 30°C Table 2: Factorial design at pH 7 and 37°C Table 3: Alpha-amylase activity at pH 7 and 30°C Table 4: Alpha-amylase activity at pH 7 and 37°C Table 5: Protein concentration at pH 7 and 30°C Table 6: Protein concentration at pH 7 and 37°C Table 7: Alpha-amylase activity, Protein Concentration, and Biomass Concentration of samples taken at regular intervals Table 8: Yields and Productivity Table A.1: Composition of HIMEDIA® M001 Table B.1: Alpha-amylase activity at pH 7 and 30°C Table B.2: Alpha-amylase activity at pH 7 and 37°C Table C.1: Tabulation of absorbance versus BSA concentration Table C.2: Protein concentration at pH 7 and 30°C Table C.3: Protein concentration at pH 7 and 37°C Table D.1: Alpha-amylase activity of samples taken at regular intervals Table D.2: Protein concentration of samples taken at regular intervals Table D.3: Biomass concentration of samples taken at regular intervals Table D.4: Specific enzyme activity (Yamylase/protein), Yp/x, and Productivity of samples taken at regular intervals 25 25 29 30 31 32 33 35 45 46 46 47 48 48 49 50 52 viii LIST OF FIGURES Figure 1: Alpha-amylase activity at pH 7 and 30°C Figure 2: Alpha-amylase activity at pH 7 and 37°C Figure 3: Protein concentration at pH 7 and 30°C Figure 4: Protein concentration at pH 7 and 37°C Figure 5: Plot of Alpha-amylase activity, Protein Concentration, and Biomass Concentration during course of fermentation Figure C.1: Plot of BSA standard curve Figure D.1: Plot of Alpha-amylase activity of samples taken at regular intervals Figure D.2: Plot of Protein concentration of samples taken at regular intervals Figure D.3: Plot of Biomass concentration of samples taken at regular intervals 29 30 31 32 33 47 49 50 51 ix ABSTRACT Amylases have great significance in biotechnology and are commercially important enzymes in various starch processing industries. The microbial amylases meet industrial demands and have almost completely replaced chemical hydrolysis of starch in starch processing industry. This study explored the use of jackfruit seed as carbon source in the production of alpha-amylase. A production medium composed of ground jackfruit seed and yeast extract powder was mixed with 10% inoculum of Bacillus licheniformis 1331 cultured in nutrient broth at 30 for 18 hours. The effects of temperature and carbon to nitrogen C ratio were determined. Triplicate analysis was done at 10%, 15%, 20% carbon source with 20% nitrogen source set at pH 7 and temperature of 30C and 37C. It was found out that alpha-amylase production was optimal at 10% carbon, 20% nitrogen, pH 7, and 37C. Amylase production was conducted in 1000 ml medium and incubated with shaking. Samples were taken at regular intervals at 0, 1, 2, 4, 6, 8, 18, 24, 27, and 30 hours of fermentation. Enzyme activity and protein concentration were determined using Alphaamylase assay and Bradford assay, respectively. Biomass was determined by dry cell weight (DCW) analysis. Alpha-amylase production was highest during exponential growth at 4-8 hours of fermentation. Keywords: alpha-amylase, Bacillus licheniformis, jackfruit seed, yeast extract x Chapter 1 INTRODUCTION In the environment abound possible sources of essential materials or substances necessary for the development or production of certain products like enzymes, which will be useful to humans, plants, and other animals. These substances can be produced in significantly large amounts with the advent of biotechnology, but primarily the materials and the factors that influence its production source must be identified. Papain was prepared from high quality latex of Carica papaya (Baines, B. S. and Brocklehurst, K., 1978). Some wastes from fruits and vegetables can also be a good substrate for enzyme production. One study was done on banana fruit stalk, which was used as a substrate for alpha-amylase production by Bacillus subtilis under solid-state fermentation (Krishna and Chandrasekaran, M., 1996). A similar study was also done using potato peelings (Shukla, J. and Kar, R., 2000). Trypsin/ chymotrypsin inhibitor was isolated from jackfruit by ammonium sulfate fractionation and chromatography (Sai Annapurna, S. and Siva Prasad, D., 1990). These studies showed that some enzymes could be produced using components of fruits and vegetables as substrate. This particular study aims to produce an enzyme by a Bacillus sp. using the seeds of a locally grown fruit as substrate. This particular fruit is jackfruit scientifically known as Artocarpus heterophyllus or locally known as “nangka” or “langka”, which is a favorite dessert of Filipinos and is a widely grown fruit crop in the Philippines. It contains carbohydrates, protein, calcium, iron, sodium, potassium, B-complex, ascorbic acid, and small amounts of fats, ash, and iron. Analysis of jackfruit food composition per 100 gram edible portion showed that the carbohydrate content of the seed is 34.90 g (Agriculture and Fisheries Information Science, Department of Agriculture). 1 Based on the above composition, the jackfruit seed having a high content of carbohydrate can be used as a substrate for the production of the enzyme amylase. The production of amylase is of great significance in biotechnology. It is commercially important in some processing industries such as beverages, food, textile, detergents, and paper industries (Pandey et al., 2000). This enzyme can be produced using a specific microorganism or fungi under controlled conditions. The amylases can be derived from several sources ranging from bacteria to plants to humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extracellular digestion. When they have broken down the insoluble starch, the soluble end products such as glucose or maltose are absorbed into their cells. This particular study will use Bacillus licheniformis for amylase production. This is an exploratory study to determine alpha-amylase production by Bacillus licheniformis using jackfruit seed as substrate. This study is limited to optimization of cultural and environmental conditions such as temperature and carbon/nitrogen ratio. No enzyme purification and characterization will be done. The significance of this study is that another substrate will be added to the existing list of carbon sources for amylase production by microorganisms. Many industrial processes use waste materials from plants or animals. Part of solving the environmental problem of waste management can be addressed by the utilization of waste products. Jackfruit seeds as waste from fruit can be a potential material for enzyme production. Being a substrate it will become a source of income for fruit vendors and farmers. This study becomes important to the biotechnology industry and likewise contributes to environmental management and economic stability. 2 Chapter 2 REVIEW OF LITERATURE Almost all of the chemical changes brought about by the cells of all living organisms such as animals, plants, and microorganisms are mediated by appropriate catalyst. Enzymes are the protein biocatalysts produced by living cells. Considerable quantities of enzymes are currently produced commercially from animal and plant sources. To produce such enzymes there must be a substrate as a carbon source and a nitrogen source processed under controlled conditions. This study identified jackfruit seed as a substrate to make seeds useful since these are thrown away after eating the pulp. Jackfruit seed Several studies about jackfruit were done in Asia particularly in South and Southwest Asia and South America. S. Sai Annapurma and D. Siva Prasad (1991) made a study on the purification of trypsin/chymotrypsin inhibitor, which was isolated from jackfruit seeds, by ammonium sulfate fractination and chromatography on DEAE cellulose and SephadexG-100. During all stages of purification the ratio of trypsin and chymotrypsin inhibitory activities remained constant. Vanna Tulyathan et al. (2002) of Chulalongkorn University in Thailand did some investigation studies on physicochemical properties of jackfruit seed flour and starch. Flour from jackfruit has high protein and carbohydrate content. It also has good water and oil absorption abilities. From the result of the study, amylose content of jackfruit seed starch was 32% and is higher than tapioca starch (17%) and corn starch (26%). These starchy substances can be used as substrate for carbon, a necessary element for the production of amylase by Bacillus. Jackfruit can be a potential source of 3 carbon in amylase production since it has a high protein and carbohydrate content (Tulyathan et al., 2001). A study on the factorial design evaluation of some experimental factors for phenol oxidation using crude extracts from jackfruit was published in the Journal of the Brazilian Chemical Society (2002). Another study involving jackfruit seed was conducted by Bhat AV and Pattabiraman (1989) where the protease inhibitory activity of jackfruit seed (Artocarpus integrifolia) was investigated. The inhibitory with a molecular weight of 26 kilodalton was found to be a glycoprotein. Galactose, glucose, mannose, fructose, xylose, glucosamine, and uronic acid were identified as constituent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. It was published in Bangladesh Journal of Agricultural Research (1991) a biochemical study on jackfruit seed meal that reveals its proximate composition, soluble protein distribution pattern, peptisation, starch pattern and mineral composition. The meal was found to contain 7.44% moisture, 1.86% ash, 2.88% crude fiber, 1.48% lipids, 75.03% carbohydrate, and 11.31% crude protein. Protein distribution pattern according to Osborn scheme revealed that the major protein fractions were albumin and glutelin, which together constituted 54.73% of the extracted protein. Globulin and prolamin were the minor fractions, which together comprised 6.63% of the extracted protein. A considerable percentage of protein (38.64%) could not be extracted by the solvents used. Peptisation of nitrogen as a function of pH in water revealed that the maximum extraction of 73.47% was obtained at pH 13, while the minimum extraction of 11.55% at pH 3. The meal contained 60.7% starch 4 consisting of 56.29% amylose and 43.71% amylopectin, their ratio being 100:78. Starch granules were oval in shape. Mineral composition of the meal were P(0.13%), Ca (0.59%), Mg (0.41%), K(0.45%), S(0.15%), B(0.08%), Fe (120 ppm), Cu(50 ppm), Mn (1.47 ppm) and Zn (56 ppm). Evidently, jackfruit seed meal can be used as ingredient for the products that require easy dispersion. There were several studies on the properties of jackfruit seed flour. One of which was done by S.A. Odoemelam (2005) where functional properties of raw and heat processed jackfruit flour were determined. The functional properties, water and oil absorption, gelation, bulk density, foaming, emulsification, and nitrogen solubility were studied. The effects of pH and NaCl concentration on some of these functional properties were also investigated. Emulsification, foam capacity, and nitrogen solubility were pH dependent with minimum values at pH 4. Addition of NaCl up to 0.4 M improved emulsification capacity of both raw and heat processed flours whereas a decrease was observed in foam capacity at 0.2 M. The foam of the raw flour was more stable than that of the heat processed flour. Heat processing of jackfruit flour increased the water and oil absorption capacity but lowered nitrogen solubility, foam capacity, and emulsification capacity. Water and oil absorption capacities of raw jackfruit flour were 2.3 ml/g and 2.8 ml/g, respectively, while heat processed flour sample gave 3.5 ml/g and 3.1 ml/g. The water and oil absorption capacities of the heat processed jackfruit flour were significantly higher (p<0.05) than those of the raw flour. Least gelation concentration of raw jackfruit was found to be 16% and heat processed flour, 18% while bulk densities of 0.61 g/ml were calculated for raw and heat processed flours. A comparative study of the chemical composition and mineral element content of jackfruit and breadfruit seeds and seed oils was conducted by Ibironke Adetolu Ajayi (2006). 5 The two seeds from Moraceae family, jackfruit and breadfruit are quite rich in oil, protein, carbohydrate, and some mineral elements. The oil content of the seeds classifies them as average oil yielding. Potassium is high in jackfruit seed as compared to breadfruit followed by sodium, magnesium, calcium, iron, zinc, and copper in descending order. The previous studies showed that there were existing researches done involving jackfruit. This particular study on alpha-amylase production using jackfruit as a substrate is new and relatively unexplored study in the Philippines. Amylase Enzymes are named according to the substrate on which they act, therefore, the term amylase indicates action on starch, which contains two types of polysaccharides: 15- 20% of amylose and 80-85% of amylopectin (Harger, 1982). Gupta et al. (2003) and Pandey et al. (2005) described that amylase catalyzes hydrolysis of starch molecules liberating diverse products, including dextrins and progressively, small glucose polymers. Amylases are classified according to how it break down starch molecules. Alphaamylase reduces the viscosity of starch by breaking down the bonds at random producing varied sized chains of glucose. Beta-amylase breaks the glucose-glucose bonds down by removing two glucose units at a time, thereby producing maltose. The third type is the amyloglucosidase, which breaks successive bonds from the non-reducing end of the straight chain, producing glucose. Bacteria and fungi secrete amylases to the outside of their cells to carry out extracellular digestion. When the insoluble starches are broken down, the soluble end products such as glucose or maltose are absorbed into the cells of the organisms. These enzymes present great importance in biotechnology with applications in the processing of fermented beverages, food, and additives to detergents for removing stains, saccharification 6 of starch for alcohol production, brewing, textile industries, and paper industries. Despite being able to be extracted from diverse sources, including plants, animals, and microorganisms, microbial enzymes generally find great industrial demand. A large number of microbial amylases are available commercially and have almost completely replaced chemical hydrolysis of starch in starch processing industry (Pandey et al., 2000). Amylases are among the most important enzymes used in biotechnology, particularly in process involving starch hydrolysis. Though amylases originate from different sources like plants, animals, and microorganisms, the microbial amylases are the most produced and used in industry, due to their productivity and thermostability (Burhan et al., 2003). Alpha (α )-amylase Pandey et al., (2005) define α -amylase (1,4-α -glucan-4-glucanohydrolase, EC 3.2.1.1.) as an enzyme that breaks the α (1,4) bonds of polysaccharides that have ten or more units of D-glucose united by α -1,4 bonds. The attack occurs in a non-selective form (as endoenzyme) on different points of the chain simultaneously, so that the first hydrolysis products are oligosaccharides of 5 to 7 units of glucose. This represents a preferential attack on each step of the helix, of the amylose or amylopectin spiral chain. The α -amylase cleaving point, which after its attack on the represented links, originate fragments of 5 to 7 units of glucose. After hydrolysis, units of glucose, oligosaccharides of different molecular weights and dextrins are released. It acts, isolatedly or simultaneously, with other amylolytic enzymes, presenting important applications in the food, drinks, textile, and pharmaceutical industries. Endogenous α -amylase of cereal seeds are used in baking and beer industry, while of microbial enzymes are used in processes that require saccharification and liquefaction of starch. 7 Alpha (α )-amylase Production Bacterial amylase could be produced by Bacillus species, Pseudomonas, Saccharophila, and Clostridium species. But on industrial scale the strains of Bacillus species seem to be preferred. Fukumoto (1951) carried out extensive work on the production of alpha-amylase by Bacillus species. Later on, many other scientists attempted towards the production of alpha-amylase by Bacillus species. Pretorius et al. (1986) have isolated 134 alpha-amylase producing strains of Bacillus. The strains were divided into 12 groups and their biochemical and morphological characterizations were carried out. The isolates were related to Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens. Using enrichment techniques a large number of microorganisms were isolated and their alpha-amylase activity was detected by flooding the plates with a weak iodine solution. Several alpha-amylase producing strains of yeast, fungi, and actinomycetes were isolated. Among them a mesophilic strain of Bacillus licheniformis B-29 produced the maximum enzyme activity extracellularly in submerged conditions. The productivity of enzyme was optimum at pH 7.0 after 72 h of cultivation with 2% rice slurry or sorghum in the medium (Rehana et al., 1989). Borchet et al. (1995) stated that the addition of amylase to detergent would be possible means for the removal of starch strains. The Bacillus licheniformis was chosen as the most suitable source of alpha-amylase for this purpose. Ednord and Dietrich (1996) observed that Bacillus licheniformis showed high capacity for the production of alphaamylase. The resulting enzyme preparation was stabilized by CaCl2 at concentration as low as 10-30 ppm and was active at temperature up to 98°C. 8 Niziolek (1998) have studied the production of extracellular amylolytic enzymes in 41 strains of the genus Bacillus representing 13 species using different liquid media and cultivation temperature of 30°C and 38°C. It was found that 8 strains were amylase-negative, 19 strains were low-productive and 12 were medium-productive strains (10-25 U/ml). Bacillus subtilis AS-1-108, Bacillus subtilis NCIB 8159, and Bacillus licheniformis NCIB 7198 strains were included among the higher-productive as they produced about 370, 170, and 40 U/ml of alpha-amylase, respectively. The enzymes from Bacillus subtilis AS-1-108 and NCIB 8159 strains were more thermosensitive than those of the medium-productive strains of Bacillus subtilis. An acid stable alpha-amylase hyperproducing strain, designated as MIR-61, was isolated in a screening procedure from South American soil samples. MIR-61, a 60°C thermo-resistant strain, was identified using 98 biochemical and morphological tests and characterized as Bacillus licheniformis by numerical taxonomy. Batch cultures of Bacillus licheniformis MIR-61 showed extracellular alpha-amylase and alpha-glucosidase activities during exponential growth phase. The production of alpha-amylase was studied at constant pH values at 37°C and 45°C. Maximum alpha-amylase activity (4,767 kU/dm3 in a liquid medium) was detected at 45°C at a constant pH 7.0 in the late exponential phase. The alphaamylase production of Bacillus licheniformis MIR-61 was 10 to 300 times higher than the enzyme production reported in strains of the same species. Optimum alpha-amylase activity was found at 50 to 67°C in an acid pH range from 5.5 to 6.0 (Castro et al., 1999). Khajeh et al. (2001a) made the comparative study on limited and extensive proteolysis of mesophilic alpha-amylase from Bacillus amyloliquefaciens (BAA) and thermophilic from Bacillus licheniformis (BLA) using trypsin. As expected, the thermophilic 9 enzyme showed greater resistance to digestion by the protease. While the catalytic potential of BLA was enhanced by proteolysis, that of BAA was diminished owing to this process. Combined with greater catalytic activity, a lower thermal stability was observed for BLA on proteolytic treatment. For both enzymes, the extent of proteolytic cleavage was reduced in the presence of various stablizing agents. The productivity of alpha-amylase is affected by various carbon and nitrogen sources. Fukumoto et al. (1957) found that lactose and galactose were most effective in stimulating amylase production. Glucose and fructose were most effective in promoting respiration but were almost ineffective with regards to enzyme formation. However, glucose, which was rather inhibitory at high concentration, was found to become available at low concentration. Nomura et al. (1956) reported on the effect of several substrates on amylase formation. Glucose and casein hydrolysate stimulate the alpha-amylase formation. The addition of amino acid causes only a slight inhibition of enzyme formation. The effects of different oilseed cakes on alpha-amylase production by Bacillus licheniformis CUMC305 were investigated by T. Krishnan and A.K. Chandra (1982). The oilseed cakes came from different sources namely groundnut, coconut copra, sesame, madhuca, cotton mustard and linseed. All enhanced production of thermostable extracellular alpha-amylase by Bacillus licheniformis CUMC305 but on varying degrees. The effects of the different concentrations of each oilseed cake was compared to the results of a control experiment in which Bacillus licheniformis was grown in conventional medium without any oilseed cake. The saccharolytic alpha-amylase activity in the control experiment was 6 U/ml, which was considered as 100%, and the relative enhancement of enzyme production was calculated accordingly. The best effect was obtained with mustard seed cake, 10 which caused an increase in enzyme production of almost two-fold at concentration above 2%. Cottonseed cakes showed the next best effect as determined by direct correlation of the concentration gradient with the enzyme at 1.75 fold increase. It was evident from the study that oilseed cakes may serve as ideal fermentation bases for obtaining high yields of alpha-amylase from Bacillus licheniformis CUMC305. The existence of any relationship between extracellular alpha-amylase production and sporulation in Bacillus licheniformis CUMC305 when grown in the presence of different carbohydrates was investigated. It was noted that alpha-amylase production in the organism was almost complete during the period of maximum sporulation, irrespective of the carbon source used (Krishnan and Chandra, 1983). Votruba et al., (1984) have studied the kinetics of alpha-amylase production in continuous cultivation of Bacillus licheniformis on a semi-synthetic medium (glucose or maltose as C source). The specific rate of alpha-amylase production was proportional to growth rate but it was repressed by higher substrate concentrations. Besides glucose or maltose, peptone was also used as an alternative carbon source during cultivation. The specific rate of production of the enzyme on maltose was half that found with glucose. Pratima and Umender (1989) studied the production of amylase in a low cost medium by Bacillus licheniformis TCRD-B-13 that was isolated from soil. The alpha-amylase of this strain showed excellent stability at high temperature and over a wide pH range. Elimination of yeast extract and peptone from the basal cornstarch medium and substitution by gluten resulted in higher enzyme concentration in the fermented broth. The addition of corn flour to the medium further improved alpha-amylase production. 11 Ramesh and Lonsane (1990) have studied the critical importance of moisture content of the medium in alpha-amylase production by Bacillus licheniformis M27 in a solid-state fermentation system. A large reduction (about 30-78%) was observed in the production of alpha-amylase by Bacillus licheniformis M27 in standardized wheat bran medium under solid-state fermentation when the moisture content of the medium was higher than the standardized value (65%). However, a surge in enzyme production in the first 24 h of fermentation was observed in media with 75% and 85% moisture. The role of decreased oxygen transfer in reducing enzyme titers by about 78% in the medium containing 95% moisture was evident, since the enzyme titer can be effectively increased by agitating the medium during fermentation. Ramesh and Lonsane (1991) found that amylase production by Bacillus licheniformis M27 in submerged fermentation was reduced from 480 to 30 U/ml when soluble starch concentration in medium was increased from 0.2 to 1.0%. In contrast the enzyme production increased with 29-fold increase in the concentration of soluble starch and other starch substrates in solid-state fermentation systems. From the investigation of some properties of the alpha-amylase and proteinase in the culture filtrate from Bacillus licheniformis MB 80 strain it has been established that the alpha-amylase activity is the highest at pH 6.0 to 6.5 and at 90°C (Emanuilova et al., 1984). Leaching of alpha-amylase from bacterial bran, produced by Bacillus licheniformis M27 in solid-state fermentation was about 2.2 times higher at 50°C as compared to that at 30°C. Further increase by about 19% in leaching efficiency was observed when contact time was extended from 60 to 120 min. The overall increase of 2.54 times under these strategies is 12 of economic importance and no information was available earlier on enhanced leaching of enzyme from fermented bran at elevated temperatures (Padmanabhan et al., 1992). Ivanova et al. (1993) purified the extracellular alpha-amylase by the Bacillus licheniformis strain. The enzyme was stable at pH 6.5-8.0, while its optimum temperature was 90°C. The thermostability was Ca2+ dependent. The half-life of the purified enzyme was 10 min at 85°C in buffer without Ca2+. The half-life at pH 6.5 with 1.0 mM CaCl 2 added was 30 min., and over 120 min. with 5.0 mM CaCl 2. the purified enzyme was strongly inhibited by N-bromosuccinimide (NBS) and by EDTA. Anyangwa et al. (1993) studied the effect of removal of starch in the sugar refining industry by alpha-amylase from the bacterium Bacillus licheniformis. The optimum condition for use of the enzyme was at temperature 80-95°C, pH 5.0-7.0 and the reaction time 30 minutes. The activity was increased with concentration of the enzyme. The influence of polyhydric alcohols and carbohydrates on the thermostability and the heat inactivation kinetics of Bacillus licheniformis alpha-amylase was studied in the temperature range 96°C to 130°C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration (Dc-Cordt et al., 1994). Hendriekx et al. (1994) studied the influence of polyhydric alcohols and carbohydrates on the thermostability of alpha-amylase. The heat inactivation kinetics of Bacillus licheniformis alpha-amylase was studied in the temperature range 96°C to 130°C. 13 High concentrations of glycerol, mannitol, sucrose or starch can markedly decrease the inactivation rate. Weemaes et al., (1996) have compared three different alpha-amylases from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis with respect to thermal stability, pressure stability, and combined pressure-temperature stability. Measurements of residual enzyme activity and residual denaturation enthalpy showed that the alpha-amylase from Bacillus licheniformis has by far the highest thermostability and that two other alphaamylases have thermostabilities of the same order magnitude. FTIR spectroscopy showed that changes in the conformation of the alpha-amylases from Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis due to pressure occurred at about 6.5, 7.5, and 11 kilobar, respectively. It seemed that for the enzymes studied, thermal stability was correlated with pressure stability. As to the resistance under combined heat and high pressure, the alpha-amylase from Bacillus licheniformis was much more stable than the alpha-amylases from Bacillus amyloliquefaciens and Bacillus subtilis, the latter two being about equally stable. It appears that under high pressure or temperature, Bacillus licheniformis alphaamylase was most resistant among the three enzymes studied. A study on the selection of a suitable low cost fermentation medium for the production of alpha-amylase by Bacillus licheniformis GCBU-8 was conducted by Ikram-ulHaq et al. (2002). It utilized different agricultural by products such as wheat bran, sunflower meal, cotton seed meal, soybean meal, and rice husk or rice bran. Wheat bran was found to be the best basal and standardized medium for optimal production of alpha-amylase. The production was increased 2-fold when soluble starch was replaced with pearl millet starch at 1% level and nutrient broth concentration was reduced from 1% level to 0.5%. The newly 14 selected fermentation medium contained (%w/v) wheat bran 1.25, nutrienth broth 0.5, pearl millet starch 1.0, lactose 0.5, NaCl 0.5, CaCl2 0.2 in 100 ml of phosphate buffer. The production of the enzyme was greater in the newly selected medium compared to the conventional medium. A follow-up study was conducted by Ikram-ul-Haq et al. (2005) on starchy substrates for the production of alpha-amylase by parental and mutant derivatives of Bacillus licheniformis. The starches were added to the fermentation medium at 1% level. The production of enzyme by the parental strain was higher in the presence of soluble starch. However, its mutant derivatives gave optimum production of alpha-amylase in the presence of pearl millet starch. This was attributed to the adequate nutrient content of pearl millet (carbohydrates 67.1%, protein 11.6%, minerals 2.7%) for the growth of microorganism as well as for the production of alpha-amylase. The parental and its mutant derivatives were compared for the production of alpha-amylase in the presence of pearl millet. The pearl millet was added to the fermentation medium at 0.5-3.0% levels. Maximum enzyme activity by mutant strain GCBU-8 was achieved when 1.0% starch was added to the medium, while in the other mutant derivatives optimum alpha-amylase production was when 1.5% pearl millet was supplemented in the medium. As the amount of the starch was further increased, the growth of the organism and the enzyme production were significantly inhibited. The effect of different concentrations of nutrient broth was also investigated. The nutrient broth was added to the medium at 0.25-1.25% levels. The parental strain gave maximum enzyme production in the presence of 1.0% nutrient broth and 0.5% in the mutant derivatives, but for Bacillus licheniformis GCUCM-30 it was 0.25% of nutrient broth. As the amount of the nutrient broth was increased, the productivity was significantly decreased, possibly due to the 15 fact that the higher concentration of nitrogen source has an adverse effect on the growth of microorganisms as well as on the production of alpha-amylase (Hewitt and Solomos, 1996; Pedreson and Nielson, 2000). P.V. Dharani Aiyer (2004) studied the effect of C:N ratio on alpha-amylase production by Bacillus licheniformis SPT 27, an isolate obtained from the soil of Cambay, in the western region of Gujarat, India. It produces extra cellular alpha-amylase exhibiting activity at a wide pH range and was relatively stable. The Bacillus licheniformis isolate, however, produces low yields of the amylase. Different types of starchy grains and tubers were tested on its effect on the amylase activity. Amaranthus paniculatus has the highest activity followed by Zea mays, potato, and Metroxylan remphii. Of the nitrogen sources tested, peptone and ammonium hydrogen phosphate were best organic and inorganic sources, respectively. The C: N ratio found to be optimum was 1:1. A study conducted by David M. Rothstein et al. (1986) showed that alpha-amylase production varied more than 100-fold depending on the presence or absence of a cataboliterepressing carbon source in the growth medium with Bacillus licheniformis as the microorganism. Alpha-amylase production by Bacillus licheniformis is related to the nature of carbon source used for growth. If glucose is present in ample amounts, alpha-amylase is repressed, even if starch is present. When glucose is the carbon source, but present in growthrate-limiting concentrations, then alpha-amylase production increases. The alpha-amylase in Bacillus licheniformis 5A1 was produced predominantly during the growth phase and not at the onset of the stationary phase. It was also shown in the study that transcription was enhanced at least 50-fold when glutamate was used as carbon source, consistent with the observed increase in secreted alpha-amylase. Cells utilizing glutamate as a carbon source 16 initiate a considerable number of alpha-amylase transcripts consistent with strong expression of the alpha-amylase gene. Regulation of alpha-amylase clearly occurs at the level of transcription. In Central Amazonia, Brazil a study was conducted using six isolates of indigenous rhizobia, which were screened for the production of amylases in liquid media using various starchy substances as carbon sources (Oliveira A. N. et al., 2006). The effects of the wheat bran, cassava, oat, peach palm, potato, tapioca flour, corn starch, and maltose on growth and amylase production were studied after adding 1.0% (w/v) substrates to the modified YM medium. All rhizobia strains could produce more extracellular protein, biomass and amylases with the different kinds of carbon substrates. Among the carbon sources tested, maltose was the best substrate for protein and amylase production. In general, peach, palm flour and cornstarch were also considered to be good carbon sources for rhizobia amylases. The biomass production by the rhizobia isolates was higher in the presence of oat flour. The results obtained in the study revealed several Central Amazonian rhizobia strains as promising sources of amylase for biotechnological applications especially in starch industry. Saito and Yamamoto (1975) investigated the effects of various carbohydrates on alpha-amylase synthesis. Polysaccharides such as glycogen, starch, and dextrin induced alpha-amylase formation when such polysaccharides were added to a culture pre-grown on sorbital medium. Induction of alpha-amylase also resulted from addition of maltose oligosaccharides, maltotriose, maltotetraose, maltopentose, maltohexaose, and maltoheptaose. All have been found to be superior to maltose as inducers of alpha-amylase synthesis in Bacillus licheniformis 584. Amylase synthesis in the parent strain resulted by the 17 addition of compounds having linkages of α -1,4-, β -1,4-, and β -1,6- glucosyl glucose, or α -1,6- galactosyl glucose. Studies conducted by D. Dhanasekaran et al. (2006) compared the production of alpha-amylase using Bacillus species on free and immobilized state. It has utilized Bacillus although the particular specie was not specified. Cells of Bacillus species were immobilized by entrapment in sodium alginate for the production of alpha-amylase. Cell growth rate was reduced when the cells were immobilized as compared to free cells. Reduced growth rate of immobilized cells may be attributed to the mass transfer limitation of oxygen. Maximum concentration of alpha-amylase was obtained at 48 hours by the isolates. Immobilized cells did not show any appreciable alpha-amylase production. The alpha-amylase of Bacillus sp. had the optimum pH at 6.5-7.5 and temperature optima at 45°C, with the maximum activity in substrate concentration of 0.1% to 1.0%. The characterization of the type of amylase was done using thin layer chromatography. Bacteria can only grow and multiply in the presence of a source of energy, which can be derived from the controlled breakdown of various organic substrates present in the external environment such as polysaccharides, lipids, and proteins. It is a known fact that some members of genus Bacillus are able to breakdown starch and utilize it as source of energy. Organisms unable to produce amylase cannot utilize starch as energy. Students of Emphoria State University, Emporia, Kansas (USA) examined the ability of various Bacillus species to regulate expression of the enzyme amylase (D. M. Nickless et al., 2001). This was done by growing the organisms in the presence of different sugars and amylase activity was assayed on a starch plate. 18 Alpha-amylase levels in Bacillus licheniformis 5A1 were compared in culture grown in medium containing either glucose or starch as a sole carbon source. Cells were harvested during exponential growth and in stationary phase. Approximately five times as much alphaamylase was present per cell mass when cells were grown in medium containing starch instead of glucose. For a given carbon source alpha-amylase synthesis remained constant during exponential growth. In the study of D. Rothstein et al. (1986) it was shown that alphaamylase production in Bacillus licheniformis is related to the nature of carbon source used for growth. If glucose is present in ample amounts alpha-amylase is repressed, even if starch or other carbon source are also present. The extent to which catabolite repression is relieved correlates with the degree of growth limitation of the carbon source. Growth medium containing starch is not a particularly depressing condition for alpha-amylase. Bacillus licheniformis 5A1 is regulated differently from most Bacilli. Strain 5A1 and other Bacillus licheniformis strain grow well in defined medium unlike other Bacilli that require undefined supplements that could influence alpha-amylase production. It was also showed that growth conditions resulting in the appearance of alpha-amylase in the culture medium result in a corresponding increase in transcription from the alpha-amylase promoter. Regulation of alpha-amylase occurs at the level of transcription. The data confirmed the presence of a transcription termination site upstream of the alpha-amylase promoter at –6S at a location following inverted repeats and a poly (T) tract, typical of prokaryotic termination sites. The appearance of alpha-amylase in the culture medium of Bacillus licheniformis 584 has the characteristics of de novo protein synthesis during the period of secretion and controlled by induction, catabolite repression, apparent temperature-sensitive repression, and culture age (Saito and Yamamoto, 1974). Amylase formation in the parent strain of Bacillus 19 licheniformis was immediately suppressed by the addition of 0.5% glucose, glycerol, acetate or succinate, and no measurable enzyme synthesis occurred when glucose or starch were added simultaneously to non-induced cultures of the parent strain. These observations suggest that the secretion of alpha-amylase in the parent strain is sensitive to catabolite repression. The addition of cyclic AMP to a growing culture of the parent strain at various points stimulated the enzyme production by about 40-70% but could neither shorter the lag period after which the enzyme synthesis starts nor result in an alleviation of the repressive effect by glucose. This observation did not occur in other mutant strains in which alphaamylase formation was not repressed by 0.5% glucose but was sensitive to 2% glucose. It was observed that mutants F-12 and F14 could synthesize alpha-amylase when grown on a medium containing glucose as the sole carbon source. The age of the culture fluid is important factor in promoting alpha-amylase synthesis by Bacillus licheniformis (Saito and Yamamoto, 1974). In the study, it showed that the best combination for alpha-amylase formation was two-day-old cells and one day-old culture fluid. In this case, however, alpha-amylase formation was increased by the addition of either 0.5% starch or glucose and no catabolite repression was observed at this concentration. 20 Chapter 3 ALPHA-AMYLASE PRODUCTION BY BACILLUS LICHENIFORMIS 1331 USING JACKFRUIT SEEDS AS SUBSTRATE Abstract Amylases have great significance in biotechnology and are commercially important enzymes in various starch processing industries. The microbial amylases meet industrial demands and have almost completely replaced chemical hydrolysis of starch in starch processing industry. This study explored the use of jackfruit seed as carbon source in the production of alpha-amylase. A production medium composed of ground jackfruit seed and yeast extract powder was mixed with 10% inoculum of Bacillus licheniformis 1331 cultured in nutrient broth at 30C for 18 hours. The effects of temperature and carbon to nitrogen ratio were determined. Triplicate analysis was done at 10%, 15%, 20% carbon source with 20% nitrogen source set at pH 7 and temperature of 30C and 37C. It was found out that alpha-amylase production was optimal at 10% carbon, 20% nitrogen, pH 7, and 37C. Amylase production was conducted in 1000 ml medium and incubated with shaking. Samples were taken at regular intervals at 0, 1, 2, 4, 6, 8, 18, 24, 27, and 30 hours of fermentation. Enzyme activity and protein concentration were determined using Alphaamylase assay and Bradford assay, respectively. Biomass was determined by dry cell weight (DCW) analysis. Alpha-amylase production was highest during exponential growth at 4-8 hours of fermentation. Keywords: alpha amylase, Bacillus licheniformis, jackfruit seed, yeast extract Introduction In the environment abound possible sources of essential materials or substances necessary for the development or production of certain products like enzymes, which will be useful to humans, plants, and other animals. These substances can be produced in significantly large amounts with the advent of biotechnology, but primarily the materials and the factors that influence its production source must be identified. Papain was prepared from high quality latex of Carica papaya (Baines, B. S. and Brocklehurst, K., 1978). Some wastes from fruits and vegetables can also be a good substrate for enzyme production. One study was done on banana fruit stalk, which was used as a substrate for alpha-amylase production 21 by Bacillus subtilis under solid-state fermentation (Krishna and Chandrasekaran, M., 1996). A similar study was also done using potato peelings (Shukla, J. and Kar, R., 2000). Trypsin/ chymotrypsin inhibitor was isolated from jackfruit by ammonium sulfate fractionation and chromatography (Sai Annapurna, S. and Siva Prasad, D., 1990). These studies showed that some enzymes can be produced using components of fruits and vegetables as substrate. This particular study aims to produce an enzyme by a Bacillus sp. using the seeds of a locally grown fruit as substrate. This particular fruit is jackfruit scientifically known as Artocarpus heterophyllus or locally known as “nangka” or “langka”, which is a favorite dessert of Filipinos and is widely grown fruit crop in the Philippines. It contains carbohydrates, protein, calcium, iron, sodium, potassium, B-complex, ascorbic acid, and small amounts of fats, ash, and iron. Analysis of jackfruit food composition per 100 gram edible portion showed that the carbohydrate content of the seed is 34.90 g (Agriculture and Fisheries Information Science, Department of Agriculture). Based on the above composition, the jackfruit seed having a high content of carbohydrate can be used as a substrate for the production of the enzyme amylase. The production of amylase is of great significance in biotechnology. It is commercially important in some processing industries such as beverages, food, textile, detergents, and paper industries (Pandey et al., 2000). This enzyme can be produced using a specific microorganism or fungi under controlled conditions. The amylases can be derived from several sources ranging from bacteria to plants to humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extracellular digestion. When they have broken down the insoluble starch, the 22 soluble end products such as glucose or maltose are absorbed into their cells. This particular study will use Bacillus licheniformis for amylase production. This is an exploratory study to determine alpha-amylase production by Bacillus licheniformis using jackfruit seed as substrate. This study is limited to optimization of cultural and environmental conditions such as temperature and carbon/nitrogen ratio. No enzyme purification and characterization will be done. The significance of this study is that another substrate will be added to the existing list of carbon sources for amylase production by microorganisms. Many industrial processes use waste materials from plants or animals. Part of solving the environmental problem of waste management can be addressed by the utilization of waste products. Jackfruit seeds as waste from fruit can be a potential material for enzyme production. Being a substrate it will become a source of income for fruit vendors and farmers. This study becomes important to the biotechnology industry and likewise contributes to environmental management and economic stability. Methodology Preparation of jackfruit seed One hundred (100) g of jackfruit seeds was autoclaved in 1L beaker at 121°C (15 lbs psi) for 15 minutes. The outer seed coating was removed. Seeds were grinded by using a coffee grinder. Grinded seeds were placed in zip locked plastic bag and stored in the freezer. Bacterial strain 23 The bacteria, Bacillus licheniformis 1331, was obtained from the Philippine National Collection of Microorganisms (PNCM), BIOTECH, University of the Philippines, Los Baños, Laguna. Medium Preparation HIMEDIA® M001 was used for maintenance of organism. The composition of nutrient agar is shown in Appendix A. Twenty-eight (28) g of nutrient agar was mixed with 1L-distilled water in 1L cotton plugged Erlenmeyer flask. The medium was sterilized by autoclaving at 121°C (15 lbs psi) for 15 minutes. From the pure culture, cells were subcultured by streaking them on nutrient agar plate and incubated at 30°C for 30 h. Several growing colonies were transferred to nutrient agar slants and incubated at 30°C for 18 h. Nutrient agar slants were stored in refrigerator and subcultured every three (3) weeks. Preparation of inoculum Nutrient broth was used for preparation of inoculum. The medium contains (in g/L): HIMEDIA® RM 027 yeast extract, 3; peptone, 5; and NaCl, 8. The medium was sterilized at 121°C (15 lbs psi) for 15 minutes. Three (3) loopfuls of B. licheniformis 1331 from nutrient agar slant was inoculated in 100 ml nutrient broth in 250 ml cotton plugged Erlenmeyer flask. It was incubated at 30°C for 18 h. Production medium 24 The production medium consists of grinded jackfruit seeds as carbon source and HIMEDIA® RM 027 yeast extract powder as nitrogen source. One hundred (100) ml of production medium in 250 ml Erlenmeyer flask contains jackfruit seed as carbon source at 10%, 15%, and 20% and HIMEDIA® RM 027 yeast extract powder as nitrogen source at 20%. The pH of the production medium was 7. The inoculum size was 10% (v/v) of the production medium. The culture was incubated at 30°C for 24 h. Optimization of cultural conditions Factors such as temperature and source of carbon and nitrogen affecting production of amylase were optimized by varying parameter one at a time. The experiment was conducted in 250 ml cotton plugged Erlenmeyer flask containing 100 ml of production medium. The pH of 7, temperature at 30°C and 37°C, carbon source at 10%, 15%, and 20%, and nitrogen source at 20% were used. The optimum C/N ratio was determined by varying carbon and nitrogen source of production medium. A factorial design shown below was conducted. Table 1. Factorial design at pH 7 and 30°C Carbon source (w/v) Nitrogen source (w/v) 20% 10% 15% 20% (10%, 20%) (15%, 20%) (20%, 20%) Table 2. Factorial design at pH 7 and 37°C Carbon source (w/v) Nitrogen source (w/v) 20% 10% 15% 20% (10%, 20%) (15%, 20%) (20%, 20%) 25 The experiment was done in triplicate. After determining the optimum temperature and C/N ratio, 500 ml of production medium in 1L cotton plugged Erlenmeyer flask was made. Samples were taken at regular intervals (0, 1, 2, 4, 6, 8, 18, 24, 27 and 30h) and analyzed for biomass, protein concentration, and alpha-amylase activity. Preparation of crude enzyme extract Five (5) ml culture broth in micro test tube was centrifuged for 20 minutes at 5000 rpm. The supernatant was used as crude enzyme solution to determine protein concentration (mg/ml) and alpha-amylase concentration (units/ml). Alpha (α )-amylase assay Iodine reagent The stock solution was a mixture of two solutions in separate 100 ml volumetric flask. The two solutions were 0.5 g iodine in 100 ml water and 5 g Potassium iodide in 100 ml water. The freshly prepared working solution was a mixture of 1 ml stock solution, 5 ml HCl (5N), and 500 ml deionized water in 500 ml volumetric flask. Buffer solution The KH2PO4 solution was a mixture of 0.6803 g of KH 2PO4 (0.05M) and distilled water in 100 ml volumetric flask. The NaOH solution was a mixture of 0.1999 g NaOH and distilled water in 100 ml volumetric flask. One-hundred (100) ml KH2PO4 solution was placed in 250 ml beaker and 10-11 ml of NaOH solution was added to have 0.05M KH2PO4-NaOH buffer at pH 6.0. Substrate: Starch solution 26 0.2 g soluble starch was dissolved in boiling 0.05M KH 2PO4-NaOH buffer at pH 6.0 and was cooled to 40°C. Analytical procedure For the assay, 1.0 ml of the diluted enzyme solution (0.5 ml crude enzyme and 0.5 ml buffer) was placed in a test tube and warmed to 40°C in a water bath for 10 min. Two (2.0 ml) of starch solution was added and incubated for 10 minutes. Then 0.2 ml was removed from the test tubes and placed in another tube containing 5.0 ml of iodine reagent. The absorbance at 620 nm was measured against a blank (0.2 ml buffer + 5 ml of iodine reagent). The substrate control contains 1.0 ml buffer + 2 ml substrate in place of diluted enzyme solution. α -Amylase activity was calculated from the absorbances by using the equation: α -Amylase activity (U/ml)= Ac − At × 40 D , where: Ac is the absorbance of Ac substrate control, At is the absorbance of test sample, 40 represents 4.0 mg starch present in the reaction tube times 10, and D is the enzyme dilution factor. One unit of alpha-amylase is defined as the amount of enzyme that will hydrolyze 0.1 mg of starch in 10 min at 40°C when 4.0 mg of starch is present. Activities which resulted in absorbances of less than 0.125 after 10 min required dilution to give linear reactions over the 10-min period (Smith and Roe, 1949). Protein determination by Bradford assay Preparation of Standard Curve Twenty (20) µ l of each standard solution of bovine serum albumin (BSA) was prepared at 0 (blank), 125, 250, 500, 1000 and 1500 µ g/ml. Two (2) ml of Bradford dye reagent was added to each solution. The solution was equilibrated for two minutes to one 27 hour, after which the absorbance of each solution was measured at 595 nm, using distilled water as a blank. Determination of unknown protein content Two (2) ml of Bradford dye reagent was added to 400 µ l of the crude enzyme solution. The solution was equilibrated for two minutes to one hour, after which the absorbance of each solution was measured at 595 nm, using distilled water as a blank. If the enzyme solution has absorbance reading outside the range established by the standard curve, the sample enzyme solution was diluted with water and absorbance measurement at 595 nm was redone. Dilution factors were taken into account when calculating the actual protein concentration of the enzyme solution. Biomass determination Using a pipette, 5 ml of culture broth was placed in pre-weighed micro test tubes that have been dried overnight at 105°C and cooled in a desiccator. The sample was centrifuged for 5 minutes at 5000 rpm. The supernatant was discarded. The pellet was resuspended in 5 ml distilled water using a vortex mixer. It was again centrifuged for 5 minutes and the supernatant was discarded. The micro test tubes with pellets were dried at 105°C overnight and cooled in a desiccator. The sample was weighed and the dry cell weight (DCW) of biomass was calculated. 28 Results and Discussion The starch content of jackfruit seeds was verified at the Bureau of Plant Industry (BPI) before the actual experiment. The starch content was 37.68% by using Luffshoorl method. The average of the results of the alpha-amylase activity for three trials at pH 7 and 30°C is shown in Table 5. It can be seen that the highest alpha-amylase activity was at 10% carbon and 20% nitrogen. Table 3. Alpha-amylase activity at pH 7 and 30˚C Alpha-amylase activity (U/ml) 10% Carbon, 20% Nitrogen 0.1592 15% Carbon, 20% Nitrogen 0.059 20% Carbon, 20% Nitrogen 0.1061 29 Alpha-amylase activty (U/ml) 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 1 2 % Carbon, % Nitrogen 3 Figure 1. Alpha-amylase activity at pH 7 and 30°C The average of the results of the alpha-amylase activity for three trials at pH 7 and 37°C is shown in Table 4. The highest alpha-amylase activity was also at 10% carbon and 20% nitrogen. Table 4. Alpha-amylase assay at pH 7 and 37˚C 10% Carbon, 20% Nitrogen 15% Carbon, 20% Nitrogen 20% Carbon, 20% Nitrogen Alpha-amylase activity (U/ml) 0.6374 0.609 0.1658 30 Alpha-amylase activity (U/ml) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 % Carbon, % Nitrogen 3 Figure 2. Alpha-amylase activity at pH 7 and 37°C Figure 1 and Figure 2 shows that the higher concentration of carbon source has an adverse effect on the production of alpha-amylase particularly at 37°C. It may be due to the thickness of the fermentation medium that resulted in the decreased agitation and poor mixing of air, which were essential for the growth of organism and production of enzyme (Haq et. al., 1998). Also, alpha-amylase production was possibly affected by the presence of a catabolite repressing carbon source in the growth medium. Table 5. Protein concentration at pH 7 and 30°C 10% Carbon, 20% Nitrogen 15% Carbon, 20% Nitrogen 20% Carbon, 20% Nitrogen Protein concentration (mg/ml) 3.1338 3.3168 3.1464 31 Protein Concentration (mg/ml) 3.35 3.3 3.25 3.2 3.15 3.1 3.05 3 1 2 % Carbon, % Nitrogen 3 Figure 3. Protein concentration at pH 7 and 30°C Table 6. Protein concentration at pH 7 and 37°C Protein concentration (mg/ml) 10% Carbon, 20% Nitrogen 2.8833 15% Carbon, 20% Nitrogen 2.7915 20% Carbon, 20% Nitrogen 3.0993 32 3.15 3.1 3.05 3 2.95 2.9 2.85 2.8 2.75 2.7 2.65 2.6 1 2 % Carbon, % Nitrogen 3 Figure 4. Protein concentration at pH 7 and 37°C The total protein concentration in the sample was determined by the Bradford assay. As shown in Table 5 and Table 6, the highest protein concentration was obtained at 30°C, 15% carbon and 20% nitrogen. Table 7. Alpha-amylase activity, Protein concentration, and Biomass concentration of samples taken at regular intervals Time (h) Alpha-amylase activity (U/ml) Protein concentration (mg/ml) Biomass (mg/ml) 0 0.2879 2.6988 0.2 1 0.3165 2.9745 0.26 2 0.3275 3.0455 0.2 4 0.4045 3.2888 0.36 6 0.5868 3.3288 0.38 Protein Concentration (mg/ml) 33 8 18 24 27 30 0.6484 0.6593 0.6593 0.6659 0.6725 3.224 3.2825 3.2975 3.2318 3.2688 0.3 0.22 0.32 0.12 0.38 3.5 Concentrations (mg/ml) 3 2.5 2 1.5 1 0.5 0 0 10 20 time (h) 30 40 Alpha-amylase activity (U/ml) Protein concentration (mg/ml) Biomass concentration (mg/ml) Figure 5. Plot of Alpha-amylase activity, Protein Concentration, and Biomass Concentration during course of fermentation The production of alpha-amylase was conducted in 500 ml of medium at pH 7 and optimum temperature of 37°C and C/N ratio of 10% carbon and 20% nitrogen. Samples were taken at regular intervals (0, 1, 2, 4, 6, 8, 18, 24, 27, and 30h) for analysis of alpha-amylase activity, protein concentration and biomass (DCW), as shown in Table 7. Figure 5 shows the alpha-amylase activity in the course of fermentation as shown in Alpha-amylase activity was highest during exponential growth at 4-8 hours and protein concentration was highest at 6 hours of fermentation. The values of protein concentration in mg/ml were higher than the values of alphaamylase in U/ml. It may be due to the fact that protein concentration in jackfruit seeds was high. 34 Ajayi (2008) reported that the moisture, ash, protein, crude oil, crude fiber, and carbohydrate contents of Artocarpus heterophyllus seeds to be 2.78%, 6.72%, 20.19%, 11.39%, 7.10%, and 51.82%, respectively. Bobbio et al., (1978) reported protein, crude lipids, and carbohydrate contents of jackfruit seeds as 31.9%, 1.3%, and 66.2%, respectively. Kumar et al. (1988) also reported composition of seeds from two varieties of jackfruit. Protein, crude lipids, and carbohydrate content were 17.8-18.3%, 2.1-2.5%, and 76.1%, respectively. The protein content of Artocarpus heterophyllus reported by Bobbio et al. (1978) was very high; however the seeds were reported to have been collected from fruits of various varieties of jackfruit. Table 8. Yields and Productivity Time (h) 0 1 2 4 6 8 18 24 27 Yamylase/protein 0.1067 0.1064 0.1075 0.123 0.1763 0.2011 0.2009 0.2 0.2061 Yp/x Productivity 1.4395 N/A 1.2173 0.3165 1.6375 0.1637 1.1236 0.1011 1.5442 0.0978 2.1613 0.081 2.9968 0.0366 2.0603 0.0274 5.5492 0.0247 35 30 0.2057 1.7697 0.0224 Table 8 shows the yields and productivity in the course of fermentation. It can be seen that alpha-amylase production was highest during exponential growth and was almost complete during the stationary phase. 36 Conclusion Alpha-amylase was successfully produced using jackfruit seed as solid substrate having a starch content of 37.68% in 100 grams seeds. The optimum conditions for alphaamylase production were pH of 7, temperature of 37°C and C/N ratio of 1:2. Alpha-amylase activity was highest during exponential growth at 4-8 hours of fermentation. Protein concentration was highest at 6 hours of fermentation. Recommendation In this study, the production medium contained the basic ingredients that would make the bacteria produce alpha-amylase enzyme. Determining the best carbon source and nitrogen source could improve alpha-amylase production by Bacillus licheniformis 1331. In the study, 10% carbon and 20% nitrogen (1:2) was found as the best C/N ratio. It is recommended that other less expensive nitrogen sources such as corn steep liquor, peptone, and ammonium hydrogen phosphate be used. It is also recommended that other investigators do following study on thermal stability, characterization of alpha-amylase by using SDS-PAGE and zymogram, as were as the production and purification of enzyme in large scale. References Agriculture and Fisheries Information Science, Department of Agriculture, Philippines. Ajayi, I. A. (2008). Comparative study of the chemical composition and mineral element content of Artocarpus heterophyllus and Treculia africana seeds and seed oils. Bioresource Technology, Volume 99, 5125-5129. Anyangwa, E. M., Mapsev, C., Musanage, P. and M. Elemva (1993). The effect and removal of starch in the sugar refining industry. J. Int. Sugar., Volume 95, 210-213. Arunava, B., Pal, S.C. and S.K. Sen (1993). Alpha-amylase production in lactose medium by Bacillus circulanse. J. Microbiologia., Volume 9 (2),142-148. 37 Awal, H. M. A. and S. Gheyasuddin (1991). Biochemical parameters of jackfruit seed-meal. Bangladesh Journal of Agricultural Research, Volume 16 (1), 17-22. Baines, B. S. and K. Brocklehurst (1979). A Necessary Modification to the Preparation of Papain from Any High-Quality Latex of Carica papaya and Evidence for the Structural Integrity of the Enzyme Produced by Traditional Methods. Biochem J., Volume 177 (2), 541548. Bhat, A. V. and T. N. Pattabiraman (1989). Protease inhibitor from jackfruit seed (Artocarpus integrifolia). Journal of Biosciences, Volume 14 (4), 351-365. Bernfeld, P. (1955). Amylases, α , β , Methods in Enzymology. Volume 1, 149-155. Bobbio, F. O., El-Dash, A. A., Bobbio, P. A., and L. R. Rodrigues (1978). Isolation and characterization of the physico-chemical properties of the starch of jackfruit seed (Artocarpus heterophyllus). Cereal Chem, Volume 55, 501-511. Borchet T.V.F., Lassen, S. E., Svendsen, A. and H.B. Frantzen (1995). Oxidation stable amylase for detergent. J. Biotechnol., Volume 10,175-179. Burhan A., Nisa U., Gokhan C., Omer C., Ashabil A., Osman G. (2003). Enzymatic properties of a novel thermostable, thermophilic, alkaline, and chelator resistant amylase from an alkaliphilic Bacillus sp. Isolate ANT-6. Process Biochem, Volume 38 (10), 13971403. Castro, G. R., Biagori, M. D. and F. Sineriz (1999). Studies on alpha-amylase production by Bacillus licheniformis MIR-61. Acta. Biotechnol., Volume 19 (3), 263-272. De-Cordt, S., Vanhoof, K., Hu, J., Maesmans, G., and P. Tobback (1994). The influence of polyalcohols and carbohydrate on the thermostability of alpha-amylase. J. Bioeng., Volume 43 (2), 107-114. de Oliveira A. N., de Oliveira L. A., Andrade J. S., and A. F. C. Junior (2007). Rhizobia amylase production using various starchy substances as carbon substrates. Brazilian Journal of Microbiology, Volume 38, 208-216. Dharani Aiyer, P. V. (2004). Effect of C:N ratio on alpha-amylase production by Bacillus licheniformis SPT 27. African Journal of Biotechnology, Volume 3 (10), 519-522. Ednord, R. and K. Dietrich (1996). Kinetics of starch hydrolysis with Bacillus amyloliquefaciens alpha-amylase under high hydrostatic pressure. J. Biotechnol., Volume 48 (11-12), 409-414. Emanuilova, E. I. and K. Toda (1984). Alpha-amylase production in batch and continuous culture by Bacillus caldolyticus. Appl. Microbiol. Biotechnol., Volume 19, 301-305. 38 Ekunsaumi, T. Laboratory Production and Assay of Amylase by Fungi and Bacteria. UWWashington country. Fukumoto, J., Yamamoto, T., and K. Ichikawa (1951). Crystallization of bacterial saccharogenic amylase and the properties of the cyrstalline amylase. Proc. Jap. Acad., Volume 27, 352-358. Fuwa, H. (1954). A new method for microdetermination of amylase activity by the use of amylose as the substrate. J. Biochem., Volume 41, 583-603. Harger, C., Sprada, D., and E. Hiratsuka (1982). Amilase Fungica. In: Bioquimica das Fermentacoes. 56. Haq, I., Ashraf, H., Zahara, R., and M. A. Qadeer (1998). Biosynthesis of alpha-amylase by Bacillus subtilis GCB-12 using agricultural by products as substrates. Biologia, Volume 44 (1&2), 154-163. Haq, I., Ashraf, H., Zahara, R., and M. A. Qadeer (2003). Production of alpha-amylase by Bacillus licheniformis using an economical medium. Bioresource Technology , Volume 87, 57-61. Haq, I., Ashraf, H., Iqbal, J.., and M. A. Qadeer (2005). Pearl millet, a source of alphaamylase production by Bacillus licheniformis. Bioresource Technology , Volume 96, 12011204. Hendrickx, M., Tobback, P., Avila, I., and S. Cordt (1994). DSC and protein-based timetemperature integrators: Case study of alpha-amylase stabilized by polyols and/or sugar. Biotechnol. Bioeng., Volume 4 (7), 859-865. Ivanova, V., Yankov, D., Kabaivanova, L. and D. Pashkkoulov (2001). Simultaneous biosynthesis and purification of two extra cellular Bacillus hydrolases in aqueous two alphaamylase. J. Biochem. Eng., Volume 8 (1), 61-81. Kathiresan K. and S. Manivannan (2006). Alpha-amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. African Journal of Biotechnology Volume 5 (10), 829-832. Khajeh, K., Naderi, H., Ranjbar, B., Moosavi, A., and M. Nemat (2001). Chemical modification of lysine residues in Bacillus alpha-amylases: Effect on activity and stability. Enzyme. Microbiol. Technol., Volume 28 (6), 543-549. Krishnan, T. and A. K. Chandra (1982). Effect of oilseed cakes on alpha-amylase production by Bacillus licheniformis CUMC 305. Appl. Environ. Microbiol., Volume 44 (2), 270-274. Krishnan, T. and A. K. Chandra (1983). Correlation between alpha-amylase production and sporulation in Bacillus licheniformis CUMC 305 with respect to the effect of some 39 carbohydrates and phenylmethylsulfonyl flouride treatment. Zentralbl Mikrobiol., Volume 138 (6), 475-485. Kumar, S., Singh, A. B., Abidi, A. B., Upadhyah, R. G., and A. Singh (1988). Proximate composition of jackfruit seeds. J. Food Sci. Tech, Volume 25, 308-309. Madigan, Michael T., Martinko, John M., and Jack Parker (2003). Brock Biology of Microorganisms. 10th Edition. Pearson Education, Inc. 980-981. Nickless, D. M., Sobieski, R. J., and S. S. Crupper (2001). Genetic Regulation of Amylase Expression in Bacillus. Bioscene, Volume 27 (4), 27-29. Niziolek S. (1998). Production of extracellular amylolytic enzymes by some species of the genus Bacillus. Acta. Microbiologica. Polonica., Volume 47 (1), 19-29. Nomura, M., Moruo, B., and S. Akabor (1956). Studies on amylase fermentation by Bacillus subtilis. Effect of high concentration of polyetylene glycol on amylase formation by Bacillus subtilis. J. Biochem., Volume 43, 143. Odoemalam, S. A. (2005). Functional Properties of Raw and Heat Processed Jackfruit (Artocarpus heterophyllus). Pakistan Journal of Nutrition, Volume 4 (6), 366-370. Onyeike, E. N., Olungwe, T., and A. A. Uwakwe (1995). Effect of heat treatment and defatting on the proximate composition of some Nigerian local soup thickeners. Food Chem, 53, 173-175. Padmanabhan, S., Ramakrishna, M., Lonsane, B.K., and M. M. Krishnaiah (1992). Enhanced leaching of product at elevated temperatures: Alpha-amylase produced by Bacillus licheniformis M27 in solid state fermentation system. Lett. Appl. Microbiol., Volume 15 (6), 235-238. Pandey, A. (1990). Aspects of Fermenter Design for Solid State Fermentation, Process Biochemistry. Volume 26, 335-361. Pandey, A., Nigam P., Soccol C. R., Soccol, V. T., Singh D., and R. Mohan (2000). Advances in microbial amylases. Biotechnol. Appl. Biochem, Volume 31, 135-152. Pandey, A., Webb, C., Soccol, C. R., and C. Larroche (2005). Enzyme Technology. New Delhi: Asiatech Publishers, Inc. 197. Pretorius, S., De-Kock, M. J. Britz, T. J., Potgieter, H. J. and P. M. Lategan (1986). Numerical taxonomy of alpha-amylase producing strain of Bacillus species. J. Appl. Bacteriol., Volume 60 (4), 351-360. Pratima, B. and Umender, S. (1989). Production of alpha-amylase in a low cost medium by Bacillus licheniformis TCRC-B13. J. Ferment. Bio. Eng., Volume 67 (6), 422-423. 40 Ramesh, M. V. and B. K. Lonsane (1990). Critical importance of moisture content of the medium in alpha-amylase production by Bacillus licheniformis M27 in a solid-state fermentation system. Appl. Microbiol, Biotechnol., Volume 33 (5), 501-505. Ramesh, M.V. and B. K. Lonsane (1991). Ability of solid state fermentation technique to significantly minimize catabolic repression of alpha-amylase production by Bacillus licheniformis M26. Appl. Microbiol. Biotechnol., Volume 35 (5), 591-593. Rehana, F., Venkatasubbaiah, P. and K. Nand (1989). Preliminary studies on the production of thermostable alpha-amylase by a mesophilic strain of Bacillus licheniformis. Chem. Mikrobiol. Technol. Lebensem., Volume 12 (1), 8-13. Rothstein, D. M., Devlin, P. E., and R. L. Cate (1986). Expression of alpha-amylase in Bacillus licheniformis. American Society for Microbiology, Volume 168 (2), 839-842. Sai Annapurna S. and D. Siva Prasad (1991). Purification of trypsin/ chymotrypsin inhibitor from jackfruit seeds. Journal of the Science of Food and Agriculture, Volume 54 (3), 399411. Saito N. and K. Yamamoto (1975). Regulatory factors affecting alpha-amylase production in Bacillus licheniformis. American Society for Microbiology, Volume 121 (3), 848-856. Shukla J. and R. Kar (2006). Potato peel as a solid substrate for thermostable alpha-amylase production by thermophilic Bacillus isolates. World Journal of Microbiology and Biotechnology, 417-422. Smith, B. W. and J. H. Roe (1949). A photometric method for the determination of alphaamylase in blood and urine with the use of the starch-iodine color. J. Biol. Chem., Volume 179, 53-56. Spier, M. R., Vandenberghe L. P. D. S., Woiciehowski A. L., C. R. Soccol (2006). Production and Characterization of Amylases by Aspergillus niger under Solid State Fermentation Using Agro Industrial Products. International Journal of Food Engineering, Volume 2 (3), 6. Tulyathan V., Tananuwong K., Songjinda P., and N. Jaiboon (2002). Some Physicochemical Properties of Jackfruit (Artocarpus heterophyllus Lam) Seed Flour and Starch. Science Asia, Volume 28, 37-41. Vortruba, J., Emanuilova, E., Kaymakchiev, A. and J. Pazlarova (1984). Kinetics of alphaamylase production in a continuous culture of Bacillus licheniformis. Folia. Microbiol., Volume 29 (1), 19-22. 41 Weemaes, C., De-Cordt, S., Goossens, K., Ludikhuyze, L., Hedrickx, M., Heremans, K., and P. Tobback (1996). High pressure, thermal, and combined pressure temperature stabilities of alpha-amylases from Bacillus species. Biotechnol. Bioeng. Volume 50 (1), 49-56. Wilson, J. J. and W. M. Ingledew (1982). Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes. Appl. Env. Microbiol., Volume 44, 301-307. 42 Chapter 4 CONCLUSION Alpha amylase was successfully produced using jackfruit seed as substrate having a starch content of 37.68% in 100 grams seeds. The optimum conditions for alpha-amylase production were pH of 7, temperature of 37°C and C/N ratio of 1:2 (10% carbon and 20% nitrogen). Alpha-amylase activity was highest during exponential growth at 4-8 hours of fermentation. Protein concentration was highest at 6 hours of fermentation. 43 Chapter 5 RECOMMENDATION In this study, the production medium contained the basic ingredients that would make the bacteria produce alpha-amylase enzyme. Determining the best carbon source and nitrogen source could improve alpha-amylase production by Bacillus licheniformis 1331. In the study, 10% carbon and 20% nitrogen (1:2) was found as the best C/N ratio. It is recommended that the other less expensive nitrogen sources such as corn steep liquor, peptone, and ammonium hydrogen phosphate be used. It is also recommended that other investigators do follow-up study on thermal stability, characterization of alpha-amylase by using SDS-PAGE and zymogram, as well as production and purification of enzyme in large scale. 44 REFERENCES Agriculture and Fisheries Information Science, Department of Agriculture, Philippines. Ajayi, I. A. (2008). Comparative study of the chemical composition and mineral element content of Artocarpus heterophyllus and Treculia africana seeds and seed oils. Bioresource Technology, Volume 99, 5125-5129. Anyangwa, E. M., Mapsev, C., Musanage, P. and M. Elemva (1993). The effect and removal of starch in the sugar refining industry. J. Int. Sugar., Volume 95, 210-213. Arunava, B., Pal, S.C. and S.K. Sen (1993). Alpha-amylase production in lactose medium by Bacillus circulanse. J. Microbiologia., Volume 9 (2),142-148. Awal, H. M. A. and S. Gheyasuddin (1991). Biochemical parameters of jackfruit seed-meal. Bangladesh Journal of Agricultural Research, Volume 16 (1), 17-22. Baines, B. S. and K. Brocklehurst (1979). A Necessary Modification to the Preparation of Papain from Any High-Quality Latex of Carica papaya and Evidence for the Structural Integrity of the Enzyme Produced by Traditional Methods. Biochem J., Volume 177 (2), 541548. Bhat, A. V. and T. N. Pattabiraman (1989). Protease inhibitor from jackfruit seed (Artocarpus integrifolia). Journal of Biosciences, Volume 14 (4), 351-365. Bernfeld, P. (1955). Amylases, α , β , Methods in Enzymology. Volume 1, 149-155. Bobbio, F. O., El-Dash, A. A., Bobbio, P. A., and L. R. Rodrigues (1978). Isolation and characterization of the physico-chemical properties of the starch of jackfruit seed (Artocarpus heterophyllus). Cereal Chem, Volume 55, 501-511. Borchet T.V.F., Lassen, S. E., Svendsen, A. and H.B. Frantzen (1995). Oxidation stable amylase for detergent. J. Biotechnol., Volume 10,175-179. Burhan A., Nisa U., Gokhan C., Omer C., Ashabil A., Osman G. (2003). Enzymatic properties of a novel thermostable, thermophilic, alkaline, and chelator resistant amylase from an alkaliphilic Bacillus sp. Isolate ANT-6. Process Biochem, Volume 38 (10), 13971403. Castro, G. R., Biagori, M. D. and F. Sineriz (1999). Studies on alpha-amylase production by Bacillus licheniformis MIR-61. Acta. Biotechnol., Volume 19 (3), 263-272. 45 De-Cordt, S., Vanhoof, K., Hu, J., Maesmans, G., and P. Tobback (1994). The influence of polyalcohols and carbohydrate on the thermostability of alpha-amylase. J. Bioeng., Volume 43 (2), 107-114. de Oliveira A. N., de Oliveira L. A., Andrade J. S., and A. F. C. Junior (2007). Rhizobia amylase production using various starchy substances as carbon substrates. Brazilian Journal of Microbiology, Volume 38, 208-216. Dharani Aiyer, P. V. (2004). Effect of C:N ratio on alpha-amylase production by Bacillus licheniformis SPT 27. African Journal of Biotechnology, Volume 3 (10), 519-522. Ednord, R. and K. Dietrich (1996). Kinetics of starch hydrolysis with Bacillus amyloliquefaciens alpha-amylase under high hydrostatic pressure. J. Biotechnol., Volume 48 (11-12), 409-414. Emanuilova, E. I. and K. Toda (1984). Alpha-amylase production in batch and continuous culture by Bacillus caldolyticus. Appl. Microbiol. Biotechnol., Volume 19, 301-305. Ekunsaumi, T. Laboratory Production and Assay of Amylase by Fungi and Bacteria. UWWashington country. Fukumoto, J., Yamamoto, T., and K. Ichikawa (1951). Crystallization of bacterial saccharogenic amylase and the properties of the cyrstalline amylase. Proc. Jap. Acad., Volume 27, 352-358. Fuwa, H. (1954). A new method for microdetermination of amylase activity by the use of amylose as the substrate. J. Biochem., Volume 41, 583-603. Harger, C., Sprada, D., and E. Hiratsuka (1982). Amilase Fungica. In: Bioquimica das Fermentacoes. 56. Haq, I., Ashraf, H., Zahara, R., and M. A. Qadeer (1998). Biosynthesis of alpha-amylase by Bacillus subtilis GCB-12 using agricultural by products as substrates. Biologia, Volume 44 (1&2), 154-163. Haq, I., Ashraf, H., Zahara, R., and M. A. Qadeer (2003). Production of alpha-amylase by Bacillus licheniformis using an economical medium. Bioresource Technology , Volume 87, 57-61. Haq, I., Ashraf, H., Iqbal, J.., and M. A. Qadeer (2005). Pearl millet, a source of alphaamylase production by Bacillus licheniformis. Bioresource Technology , Volume 96, 12011204. Hendrickx, M., Tobback, P., Avila, I., and S. Cordt (1994). DSC and protein-based timetemperature integrators: Case study of alpha-amylase stabilized by polyols and/or sugar. Biotechnol. Bioeng., Volume 4 (7), 859-865. 46 Ivanova, V., Yankov, D., Kabaivanova, L. and D. Pashkkoulov (2001). Simultaneous biosynthesis and purification of two extra cellular Bacillus hydrolases in aqueous two alphaamylase. J. Biochem. Eng., Volume 8 (1), 61-81. Kathiresan K. and S. Manivannan (2006). Alpha-amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. African Journal of Biotechnology Volume 5 (10), 829-832. Khajeh, K., Naderi, H., Ranjbar, B., Moosavi, A., and M. Nemat (2001). Chemical modification of lysine residues in Bacillus alpha-amylases: Effect on activity and stability. Enzyme. Microbiol. Technol., Volume 28 (6), 543-549. Krishnan, T. and A. K. Chandra (1982). Effect of oilseed cakes on alpha-amylase production by Bacillus licheniformis CUMC 305. Appl. Environ. Microbiol., Volume 44 (2), 270-274. Krishnan, T. and A. K. Chandra (1983). Correlation between alpha-amylase production and sporulation in Bacillus licheniformis CUMC 305 with respect to the effect of some carbohydrates and phenylmethylsulfonyl flouride treatment. Zentralbl Mikrobiol., Volume 138 (6), 475-485. Kumar, S., Singh, A. B., Abidi, A. B., Upadhyah, R. G., and A. Singh (1988). Proximate composition of jackfruit seeds. J. Food Sci. Tech, Volume 25, 308-309. Madigan, Michael T., Martinko, John M., and Jack Parker (2003). Brock Biology of Microorganisms. 10th Edition. Pearson Education, Inc. 980-981. Nickless, D. M., Sobieski, R. J., and S. S. Crupper (2001). Genetic Regulation of Amylase Expression in Bacillus. Bioscene, Volume 27 (4), 27-29. Niziolek S. (1998). Production of extracellular amylolytic enzymes by some species of the genus Bacillus. Acta. Microbiologica. Polonica., Volume 47 (1), 19-29. Nomura, M., Moruo, B., and S. Akabor (1956). Studies on amylase fermentation by Bacillus subtilis. Effect of high concentration of polyetylene glycol on amylase formation by Bacillus subtilis. J. Biochem., Volume 43, 143. Odoemalam, S. A. (2005). Functional Properties of Raw and Heat Processed Jackfruit (Artocarpus heterophyllus). Pakistan Journal of Nutrition, Volume 4 (6), 366-370. Onyeike, E. N., Olungwe, T., and A. A. Uwakwe (1995). Effect of heat treatment and defatting on the proximate composition of some Nigerian local soup thickeners. Food Chem, 53, 173-175. Padmanabhan, S., Ramakrishna, M., Lonsane, B.K., and M. M. Krishnaiah (1992). Enhanced leaching of product at elevated temperatures: Alpha-amylase produced by Bacillus 47 licheniformis M27 in solid state fermentation system. Lett. Appl. Microbiol., Volume 15 (6), 235-238. Pandey, A. (1990). Aspects of Fermenter Design for Solid State Fermentation, Process Biochemistry. Volume 26, 335-361. Pandey, A., Nigam P., Soccol C. R., Soccol, V. T., Singh D., and R. Mohan (2000). Advances in microbial amylases. Biotechnol. Appl. Biochem, Volume 31, 135-152. Pandey, A., Webb, C., Soccol, C. R., and C. Larroche (2005). Enzyme Technology. New Delhi: Asiatech Publishers, Inc. 197. Pretorius, S., De-Kock, M. J. Britz, T. J., Potgieter, H. J. and P. M. Lategan (1986). Numerical taxonomy of alpha-amylase producing strain of Bacillus species. J. Appl. Bacteriol., Volume 60 (4), 351-360. Pratima, B. and Umender, S. (1989). Production of alpha-amylase in a low cost medium by Bacillus licheniformis TCRC-B13. J. Ferment. Bio. Eng., Volume 67 (6), 422-423. Ramesh, M. V. and B. K. Lonsane (1990). Critical importance of moisture content of the medium in alpha-amylase production by Bacillus licheniformis M27 in a solid-state fermentation system. Appl. Microbiol, Biotechnol., Volume 33 (5), 501-505. Ramesh, M.V. and B. K. Lonsane (1991). Ability of solid state fermentation technique to significantly minimize catabolic repression of alpha-amylase production by Bacillus licheniformis M26. Appl. Microbiol. Biotechnol., Volume 35 (5), 591-593. Rehana, F., Venkatasubbaiah, P. and K. Nand (1989). Preliminary studies on the production of thermostable alpha-amylase by a mesophilic strain of Bacillus licheniformis. Chem. Mikrobiol. Technol. Lebensem., Volume 12 (1), 8-13. Rothstein, D. M., Devlin, P. E., and R. L. Cate (1986). Expression of alpha-amylase in Bacillus licheniformis. American Society for Microbiology, Volume 168 (2), 839-842. Sai Annapurna S. and D. Siva Prasad (1991). Purification of trypsin/ chymotrypsin inhibitor from jackfruit seeds. Journal of the Science of Food and Agriculture, Volume 54 (3), 399411. Saito N. and K. Yamamoto (1975). Regulatory factors affecting alpha-amylase production in Bacillus licheniformis. American Society for Microbiology, Volume 121 (3), 848-856. Shukla J. and R. Kar (2006). Potato peel as a solid substrate for thermostable alpha-amylase production by thermophilic Bacillus isolates. World Journal of Microbiology and Biotechnology, 417-422. 48 Smith, B. W. and J. H. Roe (1949). A photometric method for the determination of alphaamylase in blood and urine with the use of the starch-iodine color. J. Biol. Chem., Volume 179, 53-56. Spier, M. R., Vandenberghe L. P. D. S., Woiciehowski A. L., C. R. Soccol (2006). Production and Characterization of Amylases by Aspergillus niger under Solid State Fermentation Using Agro Industrial Products. International Journal of Food Engineering, Volume 2 (3), 6. Tulyathan V., Tananuwong K., Songjinda P., and N. Jaiboon (2002). Some Physicochemical Properties of Jackfruit (Artocarpus heterophyllus Lam) Seed Flour and Starch. Science Asia, Volume 28, 37-41. Vortruba, J., Emanuilova, E., Kaymakchiev, A. and J. Pazlarova (1984). Kinetics of alphaamylase production in a continuous culture of Bacillus licheniformis. Folia. Microbiol., Volume 29 (1), 19-22. Weemaes, C., De-Cordt, S., Goossens, K., Ludikhuyze, L., Hedrickx, M., Heremans, K., and P. Tobback (1996). High pressure, thermal, and combined pressure temperature stabilities of alpha-amylases from Bacillus species. Biotechnol. Bioeng. Volume 50 (1), 49-56. Wilson, J. J. and W. M. Ingledew (1982). Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes. Appl. Env. Microbiol., Volume 44, 301-307. 49 APPENDICES APPENDIX A Composition of HIMEDIA® M001 Table A.1 Composition of HIMEDIA® M001 Standard Formula Ingredients Concentrations (g/L) Peptic digest of animal tissue 5 Beef extract 1.5 Yeast extract 1.5 Sodium Chloride 5 Agar 15 Final pH (at 25°C) 7.4 ± 0.2 APPENDIX B Assay on Alpha-amylase Activity Table B.1 Alpha-amylase activity at pH 7 and 30°C Absorbance 0.0157 0.0174 0.0126 0.0138 0.0147 0.0145 0.0151 0.0139 0.0147 0.0135 Alpha-amylase activity (U/ml) -0.10828054 0.197452229 0.121019108 0.063694267 0.076433121 0.03821656 0.114649681 0.063694267 0.140127388 Average (U/ml) Substrate Control 10% Carbon, 20% Nitrogen Trial 1 Trial 2 Trial 3 15% Carbon, 20% Nitrogen Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 0.1592 0.059 20% Crabon, 20% Nitrogen 0.1061 Table B.2 Alpha-amylase activity at pH 7 and 37°C Absorbance Substrate Control 0.0434 10% Carbon, 20% Nitrogen Trial 1 0.0183 Trial 2 0.0155 Trial 3 0.0134 15% Carbon, 20% Nitrogen Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Ac − At × 40 D Ac Alpha-amylase activity (U/ml) 0.57831013 0.642857142 0.691244239 0.440092165 0.652073732 0.735023041 0.076036866 0.28110599 0.140552995 Average (U/ml) 0.6374 0.0243 0.0151 0.0115 0.0401 0.0312 0.0373 0.609 20% Carbon, 20% Nitrogen 0.1658 Computation: α -Amylase activity (U/ml)= Ac= absorbance of substrate control At= absorbance of test sample 40= 4.0 mg starch present in the reaction tube times 10 D= enzyme dilution factor APPENDIX C Assay on Protein Concentration BSA Standard Curve Table C.1 Tabulation of absorbance versus BSA concentration BSA concentration (ug/mL) 0 125 250 500 1000 1500 0.7 0.6 Absorbance 595 nm 0.5 0.4 0.3 0.2 0.1 0 0 200 400 600 800 1000 1200 1400 1600 BSA Concentration, ug/mL y = 0.0004x + 0.0123 R2 = 0.9921 Absorbance 0 0.0414418 0.1344418 0.2535418 0.4144418 0.6321418 Figure C.1 Plot of BSA standard curve Table C.2 Protein concentration at pH 7 and 30°C Dilution factor: 0 Protein Concentration Trial Absorbance (g/L) 10% Carbon, 20% Nitrogen 1 2 3 1.2459 1.308 1.2436 3084 3239.25 3078.25 Average (mg/ml) 3.1338 15% Carbon, 20% Nitrogen 1 2 3 1.3387 1.2407 1.4376 3316 3071 3563.25 3.3168 20% Carbon, 20% Nitrogen 1 2 3 1.2687 1.2767 1.2672 3141 3161 3137.25 3.1464 Table C.3 Protein concentration at pH 7 and 37°C Dilution factor: 0 Trial Absorbance 10% Carbon, 20% Nitrogen 1 2 3 1.1669 1.2175 1.1125 Protein Concentration (g/L) 2886.5 3013 2750.5 Average (mg/ml) 2.8833 15% Carbon, 20% Nitrogen 1 2 3 1.0205 1.1039 1.2623 2520.5 2729 3125 2.7915 20% Carbon, 20% Nitrogen 1 2 3 1.3225 1.3114 1.1222 3275.5 3247.75 2774.75 3.0993 APPENDIX D Alpha-amylase activity, Protein Concentration, and Biomass Concentration of Samples Taken at Regular Intervals Table D.1 Alpha-amylase activity of samples taken at regular intervals Absorbance 0.0455 0.0324 0.0311 0.0306 0.0271 0.0188 0.016 0.0155 0.0155 0.0152 0.0149 Alpha-amylase activity (U/ml) 0.287912087 0.316483516 0.327472527 0.404395604 0.586813186 0.648351648 0.659340659 0.659340659 0.665934065 0.672527472 Substrate Control Time (hour) 0 1 2 4 6 8 18 24 27 30 Alpha-amylase activity (U/ml) 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 Time (h) Figure D.1 Plot of Alpha-amylase activity of samples taken at regular intervals Table D.2 Protein concentration of samples taken at regular intervals Time (hour) 0 1 2 4 6 8 18 24 27 30 Protein concentration (mg/ml) 3.5 3 2.5 2 1.5 1 0.5 0 0 5 10 15 20 25 30 35 Time (h) Concentration Absorbance (g/L) 1.0918 2698.75 1.2021 2974.5 1.2305 3045.5 1.3278 3288.75 1.3438 3328.75 1.3019 3224 1.3253 3282.5 1.3312 3297.25 1.305 3231.75 1.3198 3268.75 Concentration (mg/ml) 2.6988 2.9745 3.0455 3.2888 3.3288 3.224 3.2825 3.2973 3.2318 3.2688 Figure D.2 Plot of Protein concentration of samples taken at regular intervals Table D.3 Biomass concentration of samples taken at regular intervals Time (h) 0 1 2 4 6 8 18 24 27 30 Preweighted micro test tube (g) 7.5 7.5 7.6 8.7 7.5 7.7 5.4 5.3 7.8 8.7 Dried sample in micro test tube Dry weight of biomass (g) (g) 8.5 1 8.8 1.3 8.6 1 10.5 1.8 9.4 1.9 9.2 1.5 6.5 1.1 6.9 1.6 8.4 0.6 10.6 1.9 Biomass concentration (mg/ml) 0.2 0.26 0.2 0.36 0.38 0.3 0.22 0.32 0.12 0.38 Dry weight of biomass (g/L) 200 260 200 360 380 300 220 320 120 380 Biomass concentration (mg/ml) 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 Time (h) Figure D.3 Plot of Biomass concentration of samples taken at regular intervals Table D.4 Specific enzyme activity (Yamylase/protein), Yp/x, and Productivity of samples taken at regular intervals Time (h) 0 1 2 4 6 8 18 24 27 30 Alpha-amylase activity (U/ml) 0.287912087 0.316483516 0.327472527 0.404395604 0.586813186 0.648351648 0.659340659 0.659340659 0.665934065 0.672527472 Yp/x 1.439560435 1.217244292 1.637362635 1.123321122 1.544245226 2.16117216 2.997002995 2.060439559 5.549450542 1.769809137 Protein Concentration (mg/ml) 2.69875 2.9745 3.0455 3.28875 3.32875 3.224 3.2825 3.29725 3.23175 3.26875 Productivit y 0.316483516 0.163736264 0.101098901 0.097802198 0.081043956 0.036630037 0.027472527 0.024664225 0.022417582 Biomass (mg/ml) 0.2 0.26 0.2 0.36 0.38 0.3 0.22 0.32 0.12 0.38 Specific enzyme activity 0.106683497 0.106398896 0.107526688 0.122963316 0.17628635 0.201101628 0.200865395 0.199966839 0.206059895 0.205744542