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-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
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 CAS No. 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 (71.98%), H (6.71%), O (21.31%) 150.17 g mol-1 1.119 – 1.126 g cm-3 172 oC or 445 K 253 oC or 526 K 109.019 oC 3.969 x 104 mg/L (at 25 oC) 52.865 kJ mol-1 -375.8o 1.542 240 hours -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
S. cerevisiae C. pseudointermedia Issatchankia orientalis S. cerevisiae GCU36 a
Molasses Molasses Molasses Molasses
concentration (gL-1) 1.58 1.47 2.33 2.58
S. cerevisiae C. pseudointermedia Issatchankia orientalis
Sugarcane juice Sugarcane juice Sugarcane juice
1.84 1.49 3.80
Source: Kumar et al. 2006 & Hussain 2009 a
Bioconversion (%) 25.00 23.43 37.16 33.47 28.00 23.75 60.61
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 Class Order Family Genus Species
Ascomycota Saccharomycetes Saccharomycetales Saccharomycetaceae Saccharomyces 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 Molasses Sucrose 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
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 Germany a Latvia b United States c Ukraine b
Company or supplier name Hildemsheim/Braunschweig Avento SNI Solutions, Inc. Welltop
Price in RM per metric tonne 530 720 900 400
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 CAS No. 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 1.0415 g cm-3 -26 oC or 247 K 179 oC or 452 K 62 oC Slightly soluble in cold water -36.8 kJ mol-1 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
