CHAPTER I

LITERATURE 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) in its laevo-rotary chiral form or by the IUPAC designated name of 1-hydroxyl-

phenyl-propan-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 L-ephedrine and D-pseudoephedrine, two pharmaceuticals with nasal

decongestant properties (Oliver et al. 1997). Table 1.1 below lists some of the

physical and chemical properties of L-PAC whereas Figure 1.1 shows the chemical

formula structure for L-PAC.

Table 1.1 Physical and chemical properties of L-PAC

Properties Values or Descriptions

CAS No. 53439-91-1IUPAC Name 1--hydroxy-1phenyl-2-propanoneAppearance PowderMolecular formula C9H10O2

Elementary composition C (71.98%), H (6.71%), O (21.31%)Molecular weight 150.17 g mol-1

Density 1.119 – 1.126 g cm-3

Melting point 172 oC or 445 KBoiling point 253 oC or 526 KFlash point 109.019 oCSolubility 3.969 x 104 mg/L (at 25 oC)Enthalpy of Vaporization 52.865 kJ mol-1

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Special optical rotation -375.8o

Index of Refraction 1.542Half life 240 hoursStorage -20oC Freezer, under inert atmosphere

Sources: Hussain 2009; ChemSpider 2012

Figure 1.1 Chemical structure of L-phenylacetylcarbinol

Source: Chikusa et al. 2001

L-PAC is transformed biologically through the pyruvate decarboxylase (PDC)-

mediated condensation of added benzaldehyde with acetaldehyde generated

metabolically from feedstock sugars via pyruvate (Oliver et al. 1997). The

fermentation to produce L-PAC can be achieved by using various types of bacteria and

yeasts. Alternatively, it can be synthesized chemically from cyanohydrins but the

biotransformation remains as the preferred route for the industry (Shukla & Kulkarni

2000). The biosynthesis pathway of L-PAC in yeast will be discussed in Section 1.3.

1.2 PRODUCT USAGE OF L-PAC

Generally, most L-PAC contributes as an intermediate for the production of L-

ephedrine hydrochloride, a well known bronchodilator (Chandrakant et al. 1997). This

pharmaceutical has the same effect as ‘Ma Huang’ in China, have been used for

several thousand years as folk remedies for inducing sweat, soothing breath and

easing excretion of urine (Rogers et al 1997). Due to its similarity with epinephrine

and cardiovascular effects, some claimed that it has the function in control obesity

(Astrup et al. 1992). However, due to the control of L-ephedrine by North America,

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illegal drug trafficking syndicate of Brazil and Canada targeted L-PAC as the

processing of amphetamine (United Nation 2009, Li et al 2009).

1.3 SELECTION OF MICROORGANISM

There are a few microorganisms which has been associated with the production of L-

phenylacetylcarbinol (L-PAC). Yeast has been commonly linked with high production

of L-PAC. Among the species of yeast which are capable of producing L-PAC are

Saccharomyces cerevisiae and Candida pseudointermedi (Kumar et al. 2006). Some

bacteria strains like Zymomonas mobilis (Shukla & Kulkarni 2000) are also used in

the production. Table 1.2 shows the comparison when these yeast species have been

used for L-PAC production using molasses as the raw material. Molasses has been

chosen as the raw material for our production considering its high potential yield

which will be discussed in Section 1.4.

Table 1.2 Comparison of types of yeast in L-PAC production

Name of organism Medium used

L-PAC concentration (gL-1)

Bioconversion (%)

S. cerevisiae Molasses 1.58 25.00C. pseudointermedi Molasses 1.47 23.43S. cerevisiae GCU36 Molasses 2.58 33.47

Source: Kumar et al. 2006 & Hussain 2009

Therefore, S. cerevisiae GCU36 has been selected considering its high

concentration of product and bioconversion.

1.3.1 Saccharomyces cerevisiae

Saccharomyces cerevisiae is a type of yeast and commonly used in baking and

brewing. It is known as Baker’s yeast. Being the most commonly studied, it is often

used in common fermentations. It has a cell wall made of chitin, has round globular to

ovoid in shape yellow-green in colour and about 5 to 10 micrometer in diameter and

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reproduces by budding (Ballesta & Larsen 2010). The cell wall lacks of peptidoglycan

while its lipid components are ester linked.

S. cerevisiae is classified as saprotroph facultative anaerobe. It is able to break

down the food through aerobic and anaerobic respiration; while also able to survive in

an oxygen deficient environment for a period of time (Prescott et al. 2002). Figure 1.2

below shows its morphology while the hierarchy of taxonomy is shown in Table 1.3.

Figure 1.2 Scanning electron micrograph showing the morphology of a typical S.

cerevisiae

Table 1.3 The taxanomy classification for S. cerevisiae

Kingdom FungiPhylum AscomycotaClass SaccharomycetesOrder SaccharomycetalesFamily SaccharomycetaceaeGenus SaccharomycesSpecies S. cerevisiae

Source: Ballesta & Larsen 2010

It is also important to note that S. cerevisiae is not normally pathogenic to

human. It is rarely reported that the colonization of S. cerevisiae in human tissue can

cause any diseases (Ballesta & Larsen 2010). S. cerevisiae is considered as safe under

the USFDA designation list as GRAS (FDA 2011). It is also safe for use (Agents that

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are not associated with disease in healthy human adults or Risk Group 1) under the

NIH Guidelines for Research (NIH 2011). The optimum level for S. cerevisiae is at

4.5 while the acceptable pH value for the growth is between 2.4 and 8.6. S. cerevisiae

can tolerate up to 40°C of temperature (Prescott et al. 2002). The mutation of

Saccharomyces cerevisiae can be performed by using ultraviolet radiations and nitrous

acid. Saccharomyces cerevisiae GCU36 was chosen considering the high L-PAC

which can be economical for our production (Hussain 2009).

1.3.2 Biosynthesis Pathway of L-PAC

The biosynthesis begins with the action of pyruvate decarboxylase (PDC) which

catalyzes the conversion of pyruvate to acetaldehyde with the resultant loss of a

molecule of CO2. Pyruvate is the end product of glycolysis, from the conversion of

sugar and is allowed to accumulate exogenously during the exponential phase of yeast

growth. This reaction requires the co-factors thiamine pyrophosphate (TPP) and

magnesium ion. PDC then catalyzes the condensation of acetaldehyde and pyruvate to

form acetoin, and by analog also causes condensation of added benzaldehyde to

produce L-PAC. It is seen that the process is itself divided into two stages – first is to

let the yeast grow and followed by a bioconversion stage where benzaldehyde is

added (Oliver et al. 1997). The biosynthesis is illustrated in Figure below.

Figure 1.3 Biosynthesis of L-phenylacetylcarbinol

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1.4 SOURCE OF RAW MATERIALS

Beet molasses is a byproduct of beet sugar refining. It contains about 50% sucrose and

categorized as one of the high sugar-content compounds. Other than that, it is a

valuable raw material in animal feed industry, yeast, citric acid, alcohol production,

and pharmaceutical industry (Asadi 2007). Figure 1.4 below shows the image of beet

molasses.

Figure 1.4 Beet Molasses

Source: HariniEthimax 2012

In production of L-phenylacetylcarbide, beet molasses is chosen as a raw

material with Saccharomyces Cerevisae GCU36 as the combination of this produce a

relatively high yield of L-phenylacetylcarbide. Based on Table 1.2, the yield of L-

phenylacetylcarbide is 2.58g/L. Besides producing high yield, beet molasses is an

easily obtained and relatively economic raw material in Malaysia. Table 1.4 shows the

quality standards for components and properties of molasses.

Table 1.4 Quality Standards for Nonfood-Grade Molasses

Quality Standards for Nonfood-Grade MolassesSucrose 46.0-52.0%Ash 10.0-12.0%Protein 8.0-10.0%Betain 4.0-6.0%Water 18.0-20.0%pH 7.0-7.5%Density (80% DS) 1400kg/m3

Source: Asadi 2007

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