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) for

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 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

L-PAC is transformed biologically through the action of pyruvate decarboxylase

(PDC EC 4.1.1.1) which mediates condensation of added benzaldehyde with

acetaldehyde generated metabolically from feedstock sugars via pyruvate (Oliver et al.

1997). 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,

Shukla & Kulkarni 2000). Today, fermentation process to produce L-PAC can also be

achieved by using various types of bacteria and yeasts. Alternatively, it can be

synthesized chemically from cyanohydrins but the biotransformation route remains the

preferred method for the industry (Shukla & Kulkarni 2000). The biosynthesis pathway of

L-PAC inside the yeast cells will be discussed in Section 1.2. Table 1.1 below lists some

of the physical and chemical properties of 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

Special optical rotation -375.8o

Index of refraction 1.542Half life 240 hoursStorage -20oC freezer, under inert atmosphere

Sources: Hussain 2009; ChemSpider 2012

1.2 SELECTION OF MICROORGANISMS

A few microorganisms have been associated with the production of L-

phenylacetylcarbinol (L-PAC) in the industry. Several yeast species are commonly linked

with the production of L-PAC. These species include Saccharomyces cerevisiae,

Kluyveromyces marxianus (Miguez et al. 2012), Torulaspora delbrueckii (Shukla &

Kulkarni 2002), Candida pseudointermedia, Issatchankia orientalis and Candida utilis

(Kumar et al. 2006). Certain bacteria strains like Zymomonas mobilis and Escherichia coli

(Shukla & Kulkarni 2000) are also shown to have potential for production in the industry

scale.

Table 1.2 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. The results show that I. orientalis

produces the highest L-PAC concentration of 2.33 g/L and 3.80 g/L after incubation for

24 hours in the laboratory, using molasses and sugarcane juice, respectively. Meanwhile,

S. cerevisiae produces only about 1.58 g/L and 1.84 g/L with using the same conditions

(Kumar et al. 2006).

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. pseudointermedia Molasses 1.47 23.43Issatchankia orientalis Molasses 2.33 37.16S. cerevisiae GCU36 a Molasses 2.58 33.47

S. cerevisiae Sugarcane juice 1.84 28.00C. pseudointermedia Sugarcane juice 1.49 23.75Issatchankia orientalis Sugarcane juice 3.80 60.61

Source: Kumar et al. 2006 & Hussain 2009 a

Nevertheless, most industrial processes have relied on the use of either C. utilis or

S. cerevisiae (Hagel et al. 2012).

1.2.1 Saccharomyces cerevisiae

Saccharomyces cerevisiae is a type of yeast, commonly used in baking and brewing. It is

also known as Baker’s yeast. 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 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

Source: Ballesta & Larsen 2010

Table 1.3 The taxanomy classification for S. cerevisiae

Kingdom Fungi

Phylum 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 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 - Risk Group 1’ under the (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).

1.2.2 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. 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. 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

and acetaldehyde to produce L-PAC. Yeast also contains alcohol dehydrogenase, an

enzyme which catalyzes reaction of benzaldehyde into benzyl alcohol, a by-product of the

biosynthesis. It is seen that from the perspective of this project production, the bioprocess

itself is divided into two stages – first is to let the yeasts to grow and followed by a

bioconversion stage where benzaldehyde is added (Oliver et al. 1997). The suppression of

alcohol dehydrogenase is critical in reducing the by-product formation. The biosynthesis

is illustrated in Figure 1.3 below.

Figure 1.3 Biosynthesis of L-phenylacetylcarbinol

Source: Cox et al. 2009

1.3 SELECTION OF RAW MATERIALS

In the industrial production of L-PAC, the important raw materials used are glucose and

benzaldehyde. Glucose will be used in the production as the main carbon source.

Benzaldehyde will be added into the process to facilitate in the formation of L-PAC.

1.3.1 Glucose

Glucose (C6H12O6) is an important raw material for fermentation can be obtained from

variety of sources. This carbon source can exist in the form of carbohydrates such as

starch and lignocelluloses or simple sugars like beet molasses, sweet sorghum and sago.

In this production project, beet molasses is the suggested raw material. 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. 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 typical beet molasses while Table 1.4 in the

next page shows the quality standards for components and properties of molasses.

Figure 1.4 Beet molasses

Source: Harini Ethimax 2012

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 7.0-7.5Density (80% DS) 1400kg/m3

Source: Asadi 2007

The selection of beet molasses as the raw material is based on several factors.

Besides having higher yield of sugar content, beet molasses is an easily obtained and an

economical raw material in Malaysia. Unlike cane molasses, beet molasses contains

higher sucrose content, lower invert sugar content and lower suspended solids (Asadi

2007). The market prices for beet molasses depend on the geography origin of the beet

molasses, as shown in Table 1.5 below. Global Seeds & Spices Enterprise is the company

in Malaysia which deals with the import and supply of beet molasses.

Table 1.5 Market prices of beet molasses depending on geography locations

Geography location Company or supplier name

Price in RM per metric tonne

Germany a Hildemsheim/Braunschweig 530Latvia b Avento 720United States c SNI Solutions, Inc. 900Ukraine b Welltop 400

Source: Tradekey News 2012 a, Alibaba 2013 b, Maryland Department of Transportation 2012 c

1.3.2 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.

As a recap, it is shown in Figure 1.3 that benzaldehyde is added into the process to bind

with acetaldehyde produced from the EMP to produce L-PAC. Figure 1.6 below shows

the chemical structure for benzaldehyde. Table 1.6 in the next page lists some of the

physical and chemical properties for benzaldehyde.

Figure 1.5 Chemical structure of benzaldehyde

Source: Pubchem 2013

Table 1.6 Physical and chemical properties of benzaldehyde

Properties Values or Descriptions

CAS No. 100-52-7Appearance Colourless liquidOdour Bitter almond-likeMolecular formula C7H6OMolecular weight 106.12 g mol-1

Density 1.0415 g cm-3

Melting point -26 oC or 247 KBoiling point 179 oC or 452 KFlash point 62 oCSolubility Slightly soluble in cold waterEnthalpy of formation -36.8 kJ mol-1

Index of refraction 1.545

Sources: ChemSpider 2012

Commercial benzaldehyde can be obtained by the following industrial processes:

1. Étard reaction of toluene, oxidized into benzaldehyde using cromyl chloride

2. Chlorination of toluene into benzal chloride, followed by hydrolysis to form

benzaldehyde

3.

Benzaldehyde is an expensive chemical with the current market price can reach RM 305

per kilogram. Orchid Chemical Supplies Ltd and Briture Co., Ltd in China are recognized

as the region supplier of benzaldehyde.

1.4 SUMMARY