CHAPTER ILITERATURE REVIEW 1.1 L-PHENYLACETYLCARBINOL Phenylacetylcarbinol (PAC) has two forms of enantiomer; one is the R-configuration and another is the S-configuration. (R)-PAC is known as L-phenylacetylcarbinol (L-PAC) for its laevo-rotary chiral form or by the IUPAC designated name of 1-hydroxyl-phenylpropan-2-one. It is a neutral organic compound of aromatic category due to the presence of the cyclic delocalization. L-PAC is widely used as an intermediate in the synthesis of Lephedrine and D-pseudoephedrine, two important pharmaceuticals with nasal decongestant properties (Oliver et al. 1997). Figure 1.1 below shows the chemical structure for L-PAC with the chemical structure is C9H10O2. Figure 1.1 Chemical structure of L-phenylacetylcarbinol Source: Pubchem 2013 1.L-PAC is transformed biologically through the action of pyruvate decarboxylase (PDC EC 4.1 Physical and chemical properties of L-PAC Properties CAS No. Shukla & Kulkarni 2000).17 g mol-1 1.865 kJ mol-1 -375. Alternatively. H (6. Table 1.2.1. it can be synthesized chemically from cyanohydrins but the biotransformation route remains the preferred method for the industry (Shukla & Kulkarni 2000). O (21. Today.019 oC 3.2 SELECTION OF MICROORGANISMS .542 240 hours -20oC freezer.71%).8o 1. 1997). under inert atmosphere Sources: Hussain 2009. The formation of this optically-active PAC by using brewer yeast and cell-free yeast extracts was first reported in 1921 by Neuberg & Liberman (Cheetham 2000.31%) 150. fermentation process to produce L-PAC can also be achieved by using various types of bacteria and yeasts. The biosynthesis pathway of L-PAC inside the yeast cells will be discussed in Section 1. ChemSpider 2012 1.969 x 104 mg/L (at 25 oC) 52.126 g cm-3 172 oC or 445 K 253 oC or 526 K 109.1 below lists some of the physical and chemical properties of L-PAC.119 – 1.1) which mediates condensation of added benzaldehyde with acetaldehyde generated metabolically from feedstock sugars via pyruvate (Oliver et al.98%). IUPAC name Appearance Molecular formula Elementary composition Molecular weight Density Melting point Boiling point Flash point Solubility Enthalpy of vaporization Special optical rotation Index of refraction Half life Storage Values or Descriptions 53439-91-1 1--hydroxy-1phenyl-2-propanone Powder C9H10O2 C (71. Table 1. 33 g/L and 3.33 2.75 60.80 Bioconversion (%) 25.2 in the following page shows the comparison of L-PAC concentration and bioconversion when several different yeast species were used for production using molasses and sugar cane juice as the raw materials.A few microorganisms have been associated with the production of L- phenylacetylcarbinol (L-PAC) in the industry.43 37. cerevisiae C. 2012). Meanwhile.80 g/L after incubation for 24 hours in the laboratory. pseudointermedia Issatchankia orientalis S.00 23. 2006). cerevisiae GCU36 a S. Several yeast species are commonly linked with the production of L-PAC.58 1. 2006 & Hussain 2009 a .47 2. pseudointermedia Issatchankia orientalis Molasses Molasses Molasses Molasses Sugarcane juice Sugarcane juice Sugarcane juice concentration (gL-1) 1. orientalis produces the highest L-PAC concentration of 2.00 23.47 28. Certain bacteria strains like Zymomonas mobilis and Escherichia coli (Shukla & Kulkarni 2000) are also shown to have potential for production in the industry scale. Candida pseudointermedia.2 Comparison of types of yeast in L-PAC production Name of organism Medium used L-PAC S.58 g/L and 1. Kluyveromyces marxianus (Miguez et al.84 1. Table 1. cerevisiae produces only about 1.61 Source: Kumar et al.49 3.84 g/L with using the same conditions (Kumar et al. These species include Saccharomyces cerevisiae. cerevisiae C. S.16 33.58 1. using molasses and sugarcane juice. 2006). Torulaspora delbrueckii (Shukla & Kulkarni 2002). respectively. Issatchankia orientalis and Candida utilis (Kumar et al. The results show that I. Table 1. 1. most industrial processes have relied on the use of either C. It is able to break down the food through aerobic and anaerobic respiration. cerevisiae (Hagel et al.3 The taxanomy classification for S.Nevertheless.2. cerevisiae Source: Ballesta & Larsen 2010 Table 1. S.2 Scanning electron micrograph showing the morphology of a typical S.3. Figure 1.2 below shows its morphology while the hierarchy of taxonomy is shown in Table 1. The cell wall lacks of peptidoglycan while its lipid components are ester linked.1 Saccharomyces cerevisiae Saccharomyces cerevisiae is a type of yeast. 2002). cerevisiae Kingdom Fungi . Figure 1. while also able to survive in an oxygen deficient environment for a period of time (Prescott et al. It has a cell wall made of chitin. utilis or S. 2012). has round globular to ovoid in shape yellow-green in colour and about 5 to 10 micrometer in diameter and reproduces by budding (Ballesta & Larsen 2010). cerevisiae is classified as saprotroph facultative anaerobe. It is also known as Baker’s yeast. commonly used in baking and brewing. 5 while the acceptable pH value for the growth is between 2. cerevisiae Source: Ballesta & Larsen 2010 It is also important to note that S. cerevisiae is not normally pathogenic to human. cerevisiae in human tissue can cause any diseases (Ballesta & Larsen 2010). 1. PDC then catalyzes the condensation of acetaldehyde and pyruvate to form acetoin. and by analog also causes condensation of added benzaldehyde and acetaldehyde to produce L-PAC. It is rarely reported that the colonization of S. Pyruvate (Pyruvic acid) is the end product of glycolysis (also known as Embden-Meyerhof-Parnas pathway) from the conversion and reduction of sugar and is allowed to accumulate exogenously during the exponential phase of yeast growth. the bioprocess itself is divided into two stages – first is to let the yeasts to grow and followed by a .Phylum Class Order Family Genus Species Ascomycota Saccharomycetes Saccharomycetales Saccharomycetaceae Saccharomyces S. It is seen that from the perspective of this project production. 2002).2. S.6. an enzyme which catalyzes reaction of benzaldehyde into benzyl alcohol.4 and 8. a by-product of the biosynthesis. The optimum level for S. cerevisiae can tolerate up to 40°C of temperature (Prescott et al.2 Biosynthesis Pathway of L-PAC The biosynthesis begins with the action of pyruvate decarboxylase (PDC) under anaerobic condition which catalyzes the conversion of pyruvate to acetaldehyde with the resultant loss of a molecule of CO2.Risk Group 1’ under the (NIH 2011). S. This reaction requires the co-factors thiamine pyrophosphate (TPP) and magnesium ion. cerevisiae is considered to be safe for usage in the industry as it is categorized under the United States Food and Drug Administration (FDA) designation list as ‘Generally recognized as safe’ (FDA 2011) and under National Institutes of Health (NIH) Guidelines for Research as an ‘agent that is not associated with disease in healthy human adults . cerevisiae is at 4. Yeast also contains alcohol dehydrogenase. the important raw materials used are glucose and benzaldehyde. 2009 1.3 Biosynthesis of L-phenylacetylcarbinol Source: Cox et al.3 SELECTION OF RAW MATERIALS In the industrial production of L-PAC.3. Benzaldehyde will be added into the process to facilitate in the formation of L-PAC.3 below.bioconversion stage where benzaldehyde is added (Oliver et al. 1.1 Glucose . 1997). Figure 1. The biosynthesis is illustrated in Figure 1. Glucose will be used in the production as the main carbon source. The suppression of alcohol dehydrogenase is critical in reducing the by-product formation. 0-52.0-12. In this production project.0-7. sweet sorghum and sago. It is a valuable raw material in animal feed industry.5 Density (80% DS) 1400kg/m3 Source: Asadi 2007 . Figure 1. This carbon source can exist in the form of carbohydrates such as starch and lignocelluloses or simple sugars like beet molasses.4 in the next page shows the quality standards for components and properties of molasses. and pharmaceutical industry (Asadi 2007).0% Protein 8. Figure 1.0% Ash 10.4 Quality Standards for Nonfood-Grade Molasses Quality Standards for Nonfood-Grade Molasses Sucrose 46.0-10. Beet molasses is a by-product of beet sugar refining which contains up to 60% sucrose and is categorized as one of the high sugar-content compounds. yeast. beet molasses is the suggested raw material.4 Beet molasses Source: Harini Ethimax 2012 Table 1.0-6. citric acid.0% Water 18.0-20.4 below shows the typical beet molasses while Table 1. alcohol production.0% pH 7.Glucose (C6H12O6) is an important raw material for fermentation can be obtained from variety of sources.0% Betain 4. Besides having higher yield of sugar content. Alibaba 2013 b. Global Seeds & Spices Enterprise is the company in Malaysia which deals with the import and supply of beet molasses. beet molasses is an easily obtained and an economical raw material in Malaysia. Table 1.3 that benzaldehyde is added into the process to bind with acetaldehyde produced from the EMP to produce L-PAC. as shown in Table 1. lower invert sugar content and lower suspended solids (Asadi 2007).5 below. . Table 1. Welltop Price in RM per metric tonne 530 720 900 400 Source: Tradekey News 2012 a.3. beet molasses contains higher sucrose content.6 below shows the chemical structure for benzaldehyde.2 Benzaldehyde Benzaldehyde (C7H6O) is a colourless liquid organic compound consisting of a benzene ring with a formyl substituent and probably one of the most industrially useful chemicals.6 in the next page lists some of the physical and chemical properties for benzaldehyde.The selection of beet molasses as the raw material is based on several factors. Inc. Maryland Department of Transportation 2012 c 1. Unlike cane molasses. As a recap.5 Market prices of beet molasses depending on geography locations Geography location Germany a Latvia b United States c Ukraine b Company or supplier name Hildemsheim/Braunschweig Avento SNI Solutions. it is shown in Figure 1. The market prices for beet molasses depend on the geography origin of the beet molasses. Figure 1. oxidized into benzaldehyde using cromyl chloride .8 kJ mol-1 1. Appearance Odour Molecular formula Molecular weight Density Melting point Boiling point Flash point Solubility Enthalpy of formation Index of refraction Values or Descriptions 100-52-7 Colourless liquid Bitter almond-like C7H6O 106.12 g mol-1 1.6 Physical and chemical properties of benzaldehyde Properties CAS No.0415 g cm-3 -26 oC or 247 K 179 oC or 452 K 62 oC Slightly soluble in cold water -36.5 Chemical structure of benzaldehyde Source: Pubchem 2013 Table 1.Figure 1.545 Sources: ChemSpider 2012 Commercial benzaldehyde can be obtained by the following industrial processes: 1. Étard reaction of toluene. followed by hydrolysis to form benzaldehyde 3. Ltd in China are recognized as the region supplier of benzaldehyde.. Benzaldehyde is an expensive chemical with the current market price can reach RM 305 per kilogram. Chlorination of toluene into benzal chloride. Orchid Chemical Supplies Ltd and Briture Co.2.4 SUMMARY . 1.