Monday, January 27, 2020
Overview Of Using Enzymes In Biocatalysis Green Chemistry Biology Essay
Overview Of Using Enzymes In Biocatalysis Green Chemistry Biology Essay ABSTRACT Aminotransaminases are a group of enzymes that play a key role in the amino acid metabolism catalysing the transfer of amino groups into keto acids resulting in the production of amino derivatives. It is PLP-dependent.they have many applications as biocatalysts because of their ability to introduce amino into ketone with good enantio- and regioselectivity. Aminotransaminases have been classified by three groups on the basis of substrate specifity, PLP-fold similarity and structural similarities. One of the important sub-groups of transaminases is omega transaminase (à †°-AT) which is capable of transferring amino group from a primary amine that does not contain a carboxyl group. There are many available à †°-ATs but the first crystal structure of this type of enzyme identified from Chromobacterium violaceum. Keywords: Aminotransaminase; PLP-dependent; regioselectivity; chromobacterium violaceum. INTRODUCTION Transaminases or aminotransferases are the group of the transferase enzymes which are involved in the reversible transfer of amino groups from amino acid to ÃŽÂ ±-keto acids. The enzyme uses pyridoxal-5-phosphate (PLP) in the reaction, therefore, it has been classified under PLP-dependent enzymes (Mehta et al., 1993). In current decades the importance of transaminases have significantly increased as a result of their tremendous potential for the production of both natural and unnatural amino acids and enantiomerically pure chiral amines which are important particularly for pharmaceutical industry (Shin et al., 2000). This review will mainly focus on the structure, mechanism and biotechnological application of omega transaminase (à †°-AT) enzymes from different sources. The first section will give a general overview of using enzyme in white biotechnology. The second part will give general overview of transaminases with different classes of transaminases. In the following part, general reaction mechanism of transaminase and structure of the chromobacterial omega transaminase will be explained in detail. Under the last subheading biotechnological application of omega transaminase will be discussed. Finally it will give the project aims and conclusion. Overview of using enzymes in biocatalysis/green chemistry Enzymes are fundamental catalysts which are capable of acting on a wide range of complex compounds as substrates. They are exquisitely selective catalysing reaction leading to production enantio- and regio-selective intermediates (Schmid et al., 2001). Isolated enzymes and whole cell biocatalysts are commonly used to produce optically pure compounds. Isolated enzymes are generally used for the aim of the catalysis of hydrolytic and isomerisation reactions; whereas; whole cells are typically used for synthetic reactions (Schmid et al., 2001). Both of them have some advantages and disadvantages. The disadvantages of using whole cells in the biocatalysis reactions are varied. For instance, substrate molecule might be toxic and results to death of cell; or the size of substrate molecule might be so huge that cannot pass through membrane; or there may be other enzymes in the cell that acts on the same substrate and cause to the production more than one compound (Wubbolts et al., 1994). In spite of these disadvantages, no requirement for recycling process of co-enzyme makes them good candidate to be used in biocatalysis because of the economic factors. The rapid increase in the development of research area of protein engineering, including molecular evolution, and enzyme engineering, has resulted in rapid growth of biocatalysis. The protein engineering yields molecule with modified structure, function and selectivity, in aqueous environment; whereas, the enzyme engineering leads to remarkable improvement particularly in organic solvent. Using organic solvent provides many advantages such as higher substrate solubility, modified enzyme specifity that results in the new and higher enzymatic activity that previously were only feasible using genetic modifications or complex reaction pathways inside the cell. As a consequence, applications of biocatalysis in organic environment vary from chiral resolution of pharmaceutical intermediates, chemical compounds to enantio- and regioselective polymerisation (Schmid et al., 2001). The use of biocatalysis in the industry for the synthesis of synthetic compounds has been significantly increased as the use of biocatalytic process for producing industrial intermediate has become easier. Biocatalytic reactions can be performed in the organic solvents and also water. This allows selective and efficient conversion of both water soluble and apolar organic molecules using biocatalytically active cells or molecules. The production of optically active substances is an area of growing demand in the fine chemical industry and biocatalysis has developed from a niche technology to a commonly used manufacturing method. The selectivity and cushy operational conditions of biocatalysists are increasingly applied in industry to modify complex target molecules. (Panke et al., 2004). General Overview of Transaminases Different classes of transaminases -classification Aminotransferases (EC 2.6.1.X) are the group of enzymes that take a significant role in the transamination reactions. They involve in the exchange of oxygen from alfa keto acid and amine from an amino acid, thus, they remove the amino group from the amino acid and transfer it to alfa keto acid and converting it into amino acid (Mehta et al., 1993). Using aminotransferases in the biocatalysis provides many advantages over other group enzymes for the production of chiral compounds. The reasons which make them so attractive are being able to act on wide range of substrate, having rapid reaction rates, no necessity for cofactor recycling (Taylor et al., 1998). Their relaxed substrate specificity, rapid reaction rates and no requirement for external cofactor regeneration makes transaminase enzymes attractive biocatalysts compared with chemical methods for the production of chiral amines A considerable number of à †°-transaminases have been identified until recently. Diamine-ketoglutaric TA is the first identified enzyme that converts the compounds bearing no carboxylic acid (Kim, 1964). It is classified as à †°-AT and is a member of sub-class 2 aminotransferases (Mehta et al., 1993). à †°-TA enzymes are known as ÃŽÂ ²-Ala:pyruvate TAs (EC 2.6.1.18) because they use pyruvate as the amine acceptor (Kaulmann et al., 2007). One of the best identified members of this group enzymes are isolated from V. fluvialis JS17. This enzyme does not show any activity towards ÃŽÂ ²-Ala but it shows broad substrate specifity towards particularly aromatic amines and (S)-enantiomers (Shin et al, 2002). It was purified and its enzymatic properties were characterised. Its molecular mass was determined to be 100 kDa and subunit mas determined to be 50 kDa. Its optiumum pH is 9.2 and optimum temperature is 37 oC. Its activity increased with pyruvate and PLP but it is inactivated with (S)-ÃŽÂ ±-methylbenzylamine. The result indicates that this is an amine: pyruvate transaminase (Shin et al., 2003). The à †°-AT isolated from Bacillus thuringiensis JS64 is highly enantioselective towards ÃŽÂ ±-methylbenzylamine (Shin and Kim, 1998). The à †°-AT Ä °isolated from Pseudomonas sp. F-126 is an isologous alpha 4 tetramer. The subunit is rich in secondary structure and consists of two domains. PLP is located in the large domain. It shows high homology with AspAT. This consequence reveals that these enzymes have common evolutionary features (Watanabe et al., 1989). In contrast to ÃŽÂ ±-transaminase catalysed reactions to produce ÃŽÂ ±-amino acids, à †°-transaminase reactions are not limited by a low equilibrium constant during the kinetic resolution (Shin and Kim, 1998). The enzymatic properties of three à †°-TAs from Klebsiella pneumonia JS2F, Bacillus thuringiensis JS64 and Vibrio fluvialis JS17 were compared to understand their mechanism and application towards production of chiral amines. All enzymes showed high enantioselectivity towards (S)-ÃŽÂ ±-MBA and broad specifity for arylic and aliphatic chiral amines. In addition to pyruvate, aldehydes showed high amino acceptor activities. All enzymes were inhibited by substrate, (S)-ÃŽÂ ±-MBA, above 200mM concentration. Only Vibrio fluvialis JS17 à †°-TA was inhibited by pyruvate above 10mM. The enzyme was not only inhibited by substrate but also inhibited by product. In the product inhibition case acetophenone and alanine are the main inhibitors but acetophenone is much more effective than alanine (Shin and Kim, 2001). Aminotransferases have been classified by Mehta and co-workers into four sub-groups according to their primary structure similarity (Table 1) (Mehta et al., 1993). Table 1. Classification of aminotransferases on the basis of structural similarities (Mehta et al., 1993) C:UsersadnanDesktopsub-groups of enzymes.png The members of subgroup 1 aminotransferases are Aspartate aminotransferase (AspAT), Alanin aminotransferase (AlaAT), aromatic amino acid transferase and histidine aminotransferase. Subgroup 1 aminotransferases are demonstrated to be the most versatile ones among the all subgroup hence they are able to react with alanine, dicarboxylic and aromatic amino acids. In one of the studies it was shown that the substrate specifity of AspAT and tyrosine aminotransferase overlap. This finding is based on the research which demonstrated the mitochondrial and cytosolic isoenzymes of aspartate aminotransferase from chicken heart accept L-phenylalanine, L-tyrosine and L-tryptophan as substrates (Mavrides and Christen, 1978). Group 2 aminotransferases include ornithine AT, à †°-AT, 4-aminobutyrate AT (GABA-AT,). They are all known as omega transaminase because of the location of the amine group. In these enzymes amino group is in a distal position from the carboxylic acid group on the amine donor substrate. (Sayer, 2009; PhD thesis). Both of the ornithine aminotransferase (Orn-AT) and 4-aminobutyrate aminotransferase (GABA-AT) are pyridoxal-phosphate (PLP)-dependent enzymes that have been identified in human, plants and animals until recently. Both enzymes catalyse a wide range of reactions on amino acids (Storici et al., 1999; Markova et al., 2005). Each enzyme catalyses the transamination reactions by a ping-pong bi-bi mechanism. The mechanism comprises two-half reactions. The half-reaction converting ketoglutarate to glutamate is the same for all transaminases. Therefore, the change in substrate specifity is resulted from the second half reaction in which an amino group is transferred distant from the ÃŽÂ ±-carbon. As a result of this, these enzymes have been identified as omega transaminases (Markova et al., 2005). GABA aminotransaminase is a PLP dependent and Fe-S cluster containing enzyme which involves in regulation of the concentration of major inhibitory neuro-transmitter GABA. This enzyme degrades GABA to succinic semialdehyde (Storici et al., 1999). Subgroup 3 aminotransaminases are BcaaAT and D-alanine aminotransferase (DaAT). The amino acid substrates of the two members of subgroup 3 enzymes have different chirality but they share the same oxo-acid as substrate. Subgroup 4 aminotransferases include SerAT and pSerAT. The two members of this group act on structurally and biosynthetically related substrates (Mehta et al., 1993). Grishin and co-workers further classified all PLP-dependent enzymes and aminotransferases categorizing them according to their PLP folds (Grishin et al., 1995). Similar outcomes were obtained with Mehtas classification. One of the classifications has been performed by John Ward in which he classified à †°-ATs in four groups according to their substrate specifity. 1. ÃŽÂ ²-alanine:ÃŽÂ ±-ketoglutarate aminotransferase, highly specific substrate activity. 2. MBA (ÃŽÂ ²-alanine):pyruvate aminotransferase, broad substrate specificity. 3. MBA:pyruvate aminotransferase, broad substrate specificity but inactive on ÃŽÂ ²-alanine. 4. ÃŽÂ ²-alanine (MBA):pyruvate aminotransferase, broad substrate specificity.(Sayer, 2009; PhD thesis). The à †°-AT from Chromobacterium violaceum belongs to the sub-group 3. The research which was conducted by Kaulmann and co-workers demonstrated that his enzyme does not show any activity towards ÃŽÂ ²-alanine where as it has comparatively broad substrate specifity against aromatic, aliphatic amines and amino-alcohols. It has a molecular weight of 51 kDa and shows 38% sequence identity to the à †°-AT from V. fluvialis JS17 (Kaulmann et al., 2007). PLP-DEPENDENT ENZYMES PLP is an important cofactor for amino acid metabolism. PLP-dependent enzymes catalyse a wide range of reactions such as transamination, decarboxylation, racemisation, aldol condensation, ÃŽÂ ±,ÃŽÂ ²-elimination and ÃŽÂ ²,ÃŽÂ ³-elimination of amino acids, and amine oxidation (Soda et al., 2001). PLP forms a covalent bound with the substrate molecule and performs as an electrophilic catalyst (Percudani and Peracchi, 2003). The mechanistic studies revealed two key chemical characteristics of the cofactor; an imine is formed between aldehyde group of PLP and amino group of substrates. The other basic characteristic of the cofactor is being able to perform as electron sink and withdrawing electron from substrate compounds (John, 1995). In 1974, it was hypothesised that the complete family of PLP-dependent enzymes had evolved from a common ancestor. They proposed this hypothesis the result of the investigating of the mechanism of the seven PLP-dependent enzymes. It was observed that protonation of the C4 carbon of the coenzyme proceeds stereospecifically with the same stereo-face in all different enzymes. This result is is explained as proof for the evolution of complete family of PLP-dependent enzymes from a common ancestorial protein (Dunathan and Voet, 1974). REACTION MECHANISM OF TRANSAMINASES The aminated form of PLP, pyridoxamine 5à ¢Ã¢â€š ¬Ã‚ ²-phosphate (PMP), appears only in the transamination reactions (Fig. 1). Transamination reactions basically comprise of two half reactions. In the first step, the aldimine is deprotonated to be converted into a quinoid intermediate, which in turn accepts a proton at a different position to form a ketimine. The resulting ketimine is hydrolysed leaving PMP behind which performs as an amine donor in the second half-reaction. PLP is subsequently recycled. PMP interacts with the apoenzyme via only non-covalent interactions, whereas, PLP is covalently bound to the active site lysine residue of the enzyme molecule. Apotransaminases have been identified to bind PMP about 100-fold less tightly than PLP. PMP can be displaced from the enzyme with high concentrations of sulphate or phosphate ions (Schell et al., 2009). C:UsersadnanDesktop.gif Scheme 1. The first half reaction mechanism of aminotransferases (Adapted from Schell et al., 2009). STRUCTURE OF the Chromobacterial OMEGA TRANSAMINASE The C. violaceum à †°-AT is the first enzyme among à †°-aminene pyruvate AT whose structure was investigated in detail using X-ray. The C. violaceum à †°-AT protomer is folded into two domains similar to other class II aminotransferases that were classified by Mehta and co-workers based on their primary structure similarity. The enzyme comprises of a large domain which includes residues 62-343 and a relatively small domain containing the N and C-terminal parts of the polypeptide chain residues between 6-61 and 344-456. The large domain has a typical ÃŽÂ ±/ÃŽÂ ²/ÃŽÂ ± sandwich fold constituted a central seven stranded ÃŽÂ ²-sheet and helix-loop-helix segment. The small domain is constituted largely of the C-terminus which is comprises of ÃŽÂ ²-sheet that is packed against helices. The overall protein fold is represented in figure 1 (Sayer, 2009; PhD thesis). Figure 1. The tertiary structure of the C. violaceum à †°-AT promoter. The ÃŽÂ ²-strands are tagged as S, ÃŽÂ ±-helices are marked as H (Sayer, 2009). As it was mentioned previously the aminotransferases require PLP as a cofactor. The enzyme was crystallised with PLP to investigate the binding interactions of PLP with active site residues. The cofactor is linked to the active site lysine residue through covalent bond forming lysine-pyridoxal-5-phosphate in all four sub-units. The PLP binding site is demonstrated to be situated between small and large domains at the interface of the two sub-units. The oxygen atoms of phosphate moiety interact with the main-chain amides of Gly120, Ser121 and the side chain of Ser121. The carboxyl group of Asp259 is located within hydrogen bond distance to the pyridine nitrogen of PLP. Aspartic acid is interacted with Val261 and Histidine154. The interactions are demonstrated in figure 2 (ibid). plp_contacts Figure 2. Stereo diagram of the C. violaceum haloenzyme active site. Stick model represents the lysine-PLP Schiff base. Yellow dot lines represent hydrogen bonds and the interacting residues are shown as lines. Neighbouring subunit residues are demonstrated by *. The structure of C. violaceum à †°-AT gabaculine complex is also solved. Gabaculine is a naturally occurring inhibitor and first isolated from Streptomyces toyacaensis as an inhibitor of GABA-AT (Kobayashi et al., 1977). Gabaculine interacts to the aminotransferase forming Schiff base with PLP as the m-carboxyphenylpyridoxamine phosphate (mCPP) ligand in the subunit A. The inhibitor molecule is interacted to the enzyme on the re face of the cofactor at the bottom of the active site. A salt bridge is formed between carboxyl group of gabaculine and side chain of Arg416. Despite the fact that à †°-AT act on the substrates that do not contain carboxyl groups, amine derivatives that bear a carboxyl group will be orientated by Arg416. The gabaculine is surrounded by Trp60, Ala231, Ile262, Leu59 and His318 from the neighbouring subunit to the bound cofactor. The hydrophobic pocked is formed through this interaction.. The interaction between active site residues and m CCP is in dicated in figure 3. gaba_lines_stereo Figure 3. Stereoview of the C. violaceum à †°-AT bound with gabaculine in the presence mCPP ligand (represented with stick) and amino acid residues within 4.5 Ã…. Residues from the neighbouring subunit to the bound cofactor are demonstrates as *. The structure of C. violaceum à †°-AT with pyruvate-PLP complex was solved to investigate active site pyruvate binding pocket (Figure 4). The carboxyl group of pyruvate forms a salt bridge with Arg416 and a hydrogen bond to the indole nitrogen of Trp60. The overall pocket is hydrophobic made up by the residues Tyr168, Phe22, Phe88, Leu59, Ala231 and Ile262. pyr_lines Figure 4. Stereoview of the binding interaction of the C. violaceum à †°-AT with pyruvate-PLP complex is demonstrated as stick model. Hydrogen bonds are represented in yellow colour, * shows residues from the adjacent subunit to the bound cofactor. BIOTECHNOLOGICAL APPLICATION OF OMEGA TRANSAMINASES A number of important parameter such as enantioselectivity, reaction equilibrium stability of enzyme, effect of inhibitors, and product separation must be taken into account in order to perform successful kinetic resolution and asymmetric synthesis for the synthesis of enantiomerically pure amines (Kim et al., 2003). Enantiomerically pure amines can be synthesized using two fundamental methods that employ à †°-ATs. One of them is kinetic resolution that performs on racemic amines; the other one is asymmetric synthesis starting with prochiral ketones that together correspond to the transamination reaction run forward and in reverse subsequently. Amines with opposite conformation are attainable if the same omega transaminase in kinetic resolution or asymmetric synthesis. For instance, if (S)-enantiomer is observed during asymmetric synthesis, the same enzyme will produce the (R)-enantiomer in the kinetic resolution (Koszelewski et al., 2010). Despite the fact that, asymmetric synthesis permits a 100% yield of demanded optically pure compound, it has been preferred less in recent time due to the difficulties related to reaction equilibrium and stereoselectivity. The stereoselectivity of the enzyme molecule to produce amines must be high with ee value of >99%, but it is almost impossible for the available à †°-transaminase (ibid). Kinetic Resolution In some reaction an enzymatic reaction is performed between a chiral molecule and a racemic acid mixture. In this case kinetic resolution occurs resulting to a kinetic preference, for one of the enantiomer over the other enantiomer (Novasep, 2010). The significance of kinetic resolution has remarkably increased as the importance of optically pure amines has increased. One group of enzymes that have resulted in the production of optically active compounds are transaminases. One of the simplest techniques for investigating kinetic resolution of chiral primary amines involves the employment of a stoichiometric equivalent of the amino acceptor. In this approach, the thermodynamic equilibrium is on the product side and comprises the enantiomerically rich amine, ketone and amino acids. The main positive side of this method is that it needs only à †°-AT (Koszelewski et al., 2010). As it is mentioned previously this method has been used commonly but it has two main drawbacks that ketone product and starting pyruvate molecule cause an inhibitory effect on the à †°-TAs enzymes (Yun et al., 2005). Different strategies have been developed to overcome these problems. The à †°-TA from Vibrio  ¬Ã¢â‚¬Å¡uvialis JS17 has been identified to show high enantioselectivity for the (S) enantiomers of various chiral amines, such as ÃŽÂ ±-MBA and sec-butylamine, with remarkable stability and a high reaction rate (Shin and Kim, 1998). Nevertheless, production of ketone may result in the inhibition of the enzyme preventing it to show its activity (Shin and Kim, 1997). A restricted solution to this issue was using an extractive biphasic reaction system that reduced the inhibitory acetophenone concentration in the aqueous phase (ibid). However, in this system the organic phase interacts with aqueous phase and acts as extractant of acetophenone. To keep the concentration of acetophenone in the aqueous phase at low levels, the aqua phase was unavoidable because the exchange the organic extractant need to control the aqueous pH with acid to neutralise basic ÃŽÂ ±-MBA diffused from the organic phase also made the process complex. An enzyme-membrane rea ctor (EMR) coupled with hollow-fiber membrane contractor was employed for the production of chiral amines and to get over the problems in a two-liquid phase reaction system. In the EMR system, to preserve the extraction capacity, a simple exchange of solvent in the organic reservoir and pH control are necessary to transfer only acetophenone through the contractor. Other advantage of this system over two-liquid phase reaction system is confining the enzyme in the reactor with ultrafiltration membrane eases reuse of enzyme. The main problem with this system could possibly be the economics of the availability of purified enzyme because a sufficiently high-circulation rate of sunstrate solution is desirable to residence time and minimize the product inhibition (Shin et al., 2001). An effective synthesis of enantiopure (S)-amino acids and chiral (R)-amines was carried out using ÃŽÂ ±/à †°-AT coupling reaction and à †°-AT was found to be inhibited by ketone product. To remove inhibitory reaction product a two-liquid phase reaction system in which dioctylpthalate was selected as solvent to achieve the best system. One of the most important advantages of ÃŽÂ ±/à †°-AT coupling reaction over aqueous phase is that; it can be carried out at high substrate concentrations to fulfil industrial large scale production of chiral amine and amino acid compounds (Cho et al., 2003). A concept has been developed to improve rate and enantioselectivity in à †°-AT-catalysed kinetic resolution using a protection group. For this purpose the kinetic resolution of 3-aminopyrrolidine and 3-aminopiperidine with à †°-AT was expedited using a protective group. 1-N-Cbz-protected group. Upon application of protective group the reaction rate was 50-fold higher. Enantioselectivity was also considerably increased upon carbamate protection in comparision with the unprotected compound (86 vs.99 ee%). However, benzyl protection of former substrate did not affect enantioselectivity because of the difference in the flexibility of the benzyl- or carbamate-protected 3-aminopyrrolidine. Despite of 50% yield limitation in kinetic resolution, this strategy is an efficient way to synthesise enantiopure 3-aminopyrrolidines (Hà ¶hne et al., 2008). The other approach to overcome product inhibition by aliphatic ketones is using an enrichment culture in combination with random mutagenesis for production and purification of mutant à †°-TA. This technique is mainly based on using 2-aminoheptane as amine donor and nitrogen source in minimal medium, and 2-butanone as an inhibitory ketone. Consequently, the higher growth rates of mutants resistant to inhibition allow them to be enriched in culture reducing the number of colonies that needs to be screened. A mutant enzyme, à †°-TAmla, which shows significantly reduced product inhibition by ketone, was determined. Using this mutant enzyme 2-aminoheptanone was resolved to (R)-2-aminoheptane with ee value>99, 53% conversion and enantioselectivity of >100 (Yun et al., 2005). b) Asymmetric synthesis Performing an asymmetric synthesis reaction is not as easy as kinetic resolution reactions because of unconvenient equilibrium and product inhibition. The main advantage of asymmetric synthesis over kinetic resolution is resulting 100% yield in the production of desired optically pure amine. However, side products may affect the enzyme catalysed reaction; therefore, these problems must be overcome to apply successful asymmetric synthesis (Koszelewski et al., 2010). One of the easiest methods for amination involves applying an excess of amine donor due to the necessity of only a single transaminase. Nevertheless, the issue here is the reaction equilibrium and potential inhibition by co-product and excess of starting an amine. In one of the studies, alanine was applied in 16-fold excess for the amination of 4-methoxyphenylacetone with 94% conversion (Nakamichi et al., 1990; Koszelewski et al., 2010). To overcome pyruvate inhibition problem two enzyme system has been used. One of the commonly used method involves Lactate Dehydrogenase (LDH)-reduction of pyruvate. One-pot, two-step dereacemisation cascade reaction was employed to lead to the production of optically pure pharmaceutical intermediates through kinetic resolution and following stereoselective amination. The main advantage of this cascade reaction is circumventing the restriction of kinetic resolution (50% conversion) leading high yield of optically pure amines. In the second step, side product pyruvate was removed using lactate dehydrogenase to shift the equilibrium to the product side. The disadvantage of this system is requirement for coenzyme recycling (Koszelewski et al., 2009). The use of whole cells is hindered by the reason that undesired side reaction such as the reduction of alcohol to ketone products. In one of the studies it was shown that the equilibrium can be shifted using pyruvate decarboxylase (PDC). Dec arboxylation of pyruvate to produce acetaldehyde and CO2 with PDC is more advantageous than LDH-catalysed reduction of pyruvate owing to no requirement for cofactor recycling (Hà ¶hne et al., 2008). One of the important examples of asymmetric synthesis is the amination reaction of acetophenone with alanine for the objective of producing (S)-ÃŽÂ ±-MBA. The equilibrium constant of the reaction is 8.8110-4 and (S)-ÃŽÂ ±-MBA and pyruvate are more reactive substrates than acetophenone and alanine (Shin and Kim, 1999). The stereoselectivity of C. violaceum TA-mediated amination of an ÃŽÂ ±,ÃŽÂ ±-dihydroxyketone, 1.3-dihydroxy-1-phenylpropane-2-one, was investigated. It was shown that the enzyme is not enantioselective towards the racemic 1.3-dihydroxy-1-phenylpropane-2-one, whereas; it is highly stereoselective for the (2S)-2-amino-1-phenyl-1,3-propanediols in 99% ee (Smithies et al., 2009). CONCLUSION AND AIMS OF PROJECT After drawing various analyses, the general overview, structure, mechanism and biotechnological applications of à †°-transaminases were reviewed in order to shed some lights on the characteristics of the à †°-transaminases. Transaminases have been used broadly owing to its capacity to produce amino acids and chiral compounds which are important for pharmaceutical industries. Transaminases have been divided to sub-groups according to their substrate specifities and structure. Transaminases, under sub-group 2 are known as à †°-AT because the distal amino group of the substrate undergoes the reaction and include OrnTA, GABA-TA and à †°-amino acid:pyruvate AT. Among them the most important one is à †°-amino acid:pyruvate AT due to the reason that only this enzyme shows catalytic activity towards primary and aliphatic amines bearing no carboxyl group. The other advantages of à †°-TAs are having broad substrate specifity, high enantioselectivity an d no requirement for the cofactor recycling. Two main biotechnological applications of à †°-ATs are asymmetric synthesis and kinetic resolution. They have both advantage and disadvantage over each other. The disadvantages of kinetic resolution are being subjected to pyruvate and ketone inhibition and having 50% yield. The asymmetric synthesis results in 100% yield for the manufacturing of desired optically pure amine. However, product may cause to the inhibition of the enzyme. To overcome these problems some approaches have been developed including biphasic reaction system, using enzyme-membrane reactor (EMR) coupled with hollow-fiber membrane contractor, using protective groups. The aim of this project is to purify and crystallise the à †°-AT enzymes and characterise their subsrate specifity.
Sunday, January 19, 2020
Gay and Lesbian Advertising
Advertising â€Å"Out†of the Box In 1994, Ikea created a wave of controversy when they became one of the first companies to market to the gay community on mainstream daytime television. Despite the initial backlash; this ad eventually sparked countless other companies jump on the â€Å"Gay and Lesbian Advertising Bandwagon†. But what is the motive behind advertising specifically to this community, do the businesses take a genuine interest in the gay community or is it strictly for expanding business?Additionally, once a business decides to advertise using gay content, what is the appropriate way to go about doing so, and how does their strategy differ between gays and lesbians? Only 4 to 10% of the population identifies themselves as gay or lesbian. (Okenfull, 50) So why are businesses so concerned with gaining the support of this demographic? One of the biggest reasons being their buying power. While they don’t make more than heterosexual couples, a large major ity are not raising children, so they have more disposable income.Thus, they have even been referred to as the â€Å"Dream Market†with a potential buying power of $641 billion annually (Okenfull, 49). Because this is the foremost reason why advertisers go after the gay community, this makes us question whether they have a genuine interest in the gay community. â€Å"We market to gays and lesbians for business reasons because we want to sell out product to consumers. It doesn’t get more complicated than that. â€Å" -Miller beer spokeswoman (Sender, 2) It is common knowledge that the only point of advertisement is to drive in more business.However, analyzing the overall impact of advertising on society, it seems as if there is more that comes into play than just a business decision. In fact, regardless of the company's motive to produce advertisements that have gay content, the overall impact of infiltrating gay content into mainstream media has actually helped advan ce and liberate the gay community. Big corporations not only reflect society’s current values, but they are also largely responsible for shaping society's values. If there are more gay and lesbian imagery in the media, society will adapt he perception that they are everywhere and normalizes gayness. Also, having large corporations back a minority-based cause, will make the majority more accepting and make the people in opposition to the cause seem more ignorant. Essentially, even just the facade of a company publicly declaring their support for gayness makes a powerful statement to society. Not only is appearing to be in support of gay rights is something that will boost your business, but to have any anti-gay statement or claims can detrimentally hurt your company as well. In 1977, Coors was accused of firing gays, along with several other minority groups. Journal of Community Research) Not long after much controversy, chairman Pete Coors adopted an Gay-Friendly policy and a lso extended benefits to same-sex couples. In 2000, Coors hiked up their spending on gay advertising, putting them in the number two spot for most money spent on gay advertisements. (Chura, 1) After a company becomes seen as anti-gay, it is evident that they must fight desperately to overcome this stigma just to stay in the game. Beer is a prime example of a product that's advertisements have saturated the gay market to the extent of which if you do not participate, then you’re stance on gay rights is questioned.There is a very positive outcome for the many companies that show their support to the gay community as well as a negative outcome for companies that don’t. Despite this; it still leaves many consumers skeptical of their motives. Many advertisements, in a failed attempt to show acceptance, have become the target of gay critics. The harshest of backlash of these ads are in ones in which they depict obvious and offensive gay stereotypes. It is evident that advert isers must handle these types of ads with care, depicting gay stereotypes can lead to further segregation rather than promoting equality.Gay consumers are very skeptical to how a company presents gay advertisements, but perhaps the biggest obstacle is to still keep heterosexual consumers that are turned off by or blatantly opposed to homosexuality. When Ikea first ran their ad on daytime television, there was a positive response from gay consumers. However, during this time period it still created enough controversy that outweighed the benefits of appealing to that market. This is when agencies quickly realized that limiting their advertisements to the confines of strictly gay and lesbian print media such as OUT magazine was a safer route for the time being.Although the initial reaction by heterosexuals to this first commercial was negative and controversial, this was also a time in history where the attitude around gay media was at a turning point . Gay shows became less of a niche market, and more mainstream. â€Å"More recently, gay-oriented shows such as the L Word, Will and Grace, Ellen, Queer as Folk, and Queer Eye for a Straight Guy have increasingly pushed the boundaries of cultural accepatnce of homosexuality and have diminished the risk of backlash for firms who are percieved to be â€Å"gay friendly. †(Okenfull, 50)Shows dealing with this content presumably have made the hetereosexual community more understanding to this type of lifestyle and generationally speaking, the current generation became much more likely to support the gay rights movement. Thus, integrating gay ads into mainstream television and print media was back on the table. Because the decision for advertisers to target the gay community is still a very controversial subject for both straight and gay consumers, it is imperative to acknowledge the vast differences in how to effectively target this audience.First, it is important to recognize gay male consumers and lesbian fema le consumers as a very different audience. Additionally, it is important to assess how strong their gender identity is. The strength of one’s gender identity is based on several different factors, such as how strong their involvement and sense of belonging is to the gay community. Gay males that are identified as having a high degree of sexual identity, are more attracted to ads that display explicit gay content. (Okenfull, 54) To illustrate an ad that is â€Å"explicitly†gay, (refer to picture 1), I chose Virgin Mobiles â€Å"Hook up Fearlessly†.The ad almost positively insinuates that the two males depicted in the ad are gay, if not gay, then extremely turned on by the a gay act. This type of explicitness would be most attractive to males who have a high gay identity rather than low. There is a large power construct in this advertisement. The male with wings is forcefully holding down the other male to kiss him while the male on bottom, while he does seem sub missive, he is still enjoying the act taking place. I personally like this ad; mostly because of the actual relevance to the buying process.Consumers always have an apprehension to buying products, let alone a phone contract. This ad is conveying to the potential consumer to just give in and try it. While the male on bottom might have been apprehensive about the kiss, just as â€Å"You’ll Love Us†guarantee gives you the freedom to chose without â€Å"strings attached†. It is a â€Å"Just try it! †ad, and while hooking up in an office space is taboo; doing it with with a gay angel is even more controversial and daring, and you might just â€Å"Love It†. I like this ad because it communicates the point fluidly, and replaces any uncertainty with a sense of empowerment and fearlessness.While ads like these are very effective in communicating with the demographic who have a strong sense of gender identity, low identity gays and lesbians are more respo nsive to a less explicit type of advertising. Generally, gays and lesbians who have a low sense of gender identity want to define themselves by traits other than just their sexual preference. By being less explicit, they get the opportunity to read between the lines, not feel alienated by their gender identity, and not have their identity to be oversexualized. To target this audience, advertisers indicates gayness without actually using a lesbian or gay couple.Instead they use implicit gay imagery which uses symbols and phrases that indicates gayness such as rainbows, pride, and being â€Å"out†. (Okenfull, 55) To illustrate an add that does this, I chose Chevy’s electric car ad (refer to ad 2). There are a mother and father car facing their child car. The caption says â€Å"Mom, Dad, I’m electric. †The bottom of the page uses a thin bar that is intended to look like a rainbow. The copy at the bottom is â€Å"So, whatever revs your engine, we support yo u 100%, Happy Motor City Pride from the entire Chevrolet family. †This is another ad that I think is done beautifully.They used a social norm of what society knows as the â€Å"coming out scene†. The electric car is declaring that it is not just any old car, just as a gay or lesbian would have to come out to their parents that they are not just another heterosexual. What also intrigues me about that ad is the italics on â€Å"I’m electric†. This is presumably a play off the upbeat â€Å"It’s electric†song. The car is excited to tell its parents that he is gay, conveying a sense of empowerment and excitement, instead of how we typically think of this moment, which can often be a shameful and intimidating moment for many children.I also really like the ad for it’s relevance to the gay community. According to a recent survey, only ? of hetereosexuals admitted to being concerned about the environment while 55% of gays and lesbians identi fied that they are more likely to â€Å"go green†. (Koretzky). It is targeting consumers that are interested in their message, while also encouraging acceptance and becoming empowered. While even high identity lesbians are most attracted to implicit gay imagery, their liking towards explicit lesbian imagery comes secondary (before explicit gay imagery).But interestingly enough, lesbian imagery is extremely underrepresented. It was reported that in one of leading gay and lesbian magazines, The Advocate, lesbian-targeted imagery accounted for only 3% of the magazine advertising. Why is this? (Okenfull, 65) Not only do lesbians have typically just as much buying power as gay males, but heterosexual consumers are even more comfortable with lesbian imagery over gay imagery, which lowers the risk of heterosexuals being offended or turned off from a product. In general, lesbians are less discriminated than gays. his is something that shows up in the workplace; according to a recent study, gay males make 23% less than straight males, lesbians make about the same as straight women. (Williams Institute) Perhaps society taking a particular favoritism to lesbianism is the very reason that advertisements that depicting lesbians couples in ads targeted towards lesbians are more underrepresented. There is a oversexualization attenuated by the heterosexual community of how lesbians are viewed. I chose an ad that was apart of a campaign for a swiss Italian ski resort (picture 3).A website was commenting on the success of this ad with the title of the Article â€Å"Lesbians Good for Ski Business. †the comment on this ad stated, â€Å"Seems some brilliant ad agency came up with a campaign to promote the Swiss Italian ski resort Airolo by featuring two women about to kiss (pictured above). We’re going to hazard a guess that the intended demographic was not lesbians. They were probably targeting lager louts looking for a good stag party getaway now that Prag ue has kicked them out. †(Get Outdoor Blog) The imagery does not have any clever tagline, or anything to empower lesbains.In fact, one of the main centerpieces of the point of this ad is the male in the background completely enamored and excited by the thought of two girls kissing. The comment on the article even states that â€Å"the intended demographic was not lesbains. †and makes the point that this ad was to promote a party scene. In fact, most viewers probably would not even assume the two girls in the ad as lesbians. By no means does the ad seem to support the gay community, it rather just sexualizes the fantasy of watching two women make out.A sexual lesbian experience can be arousing to straight men and women, conveying a â€Å"party†type of experience while if the couple was actually a gay couple about to engage in a kiss, this ad would most likely be only arousing to gay men, and convey the message that â€Å"We are a gay friendly place†rath er than a â€Å"Party Place†. Another advertisement that depicts the sexual objectification of women is the Nikon ad. There is a hand holding up a camera in which he captures the image of two women in lingerie on top of each other. While the gender of the person taking the photo is not identified, it appears to be a male.Because of the interest in the (presumed) male figure, this ad makes it very evident that the relationship between the two women is not a committed lesbian relationship. It is portrayed to be just a sexual one with an objective to please the male. The copy at the bottom states that the Nikon S60 detects up to 12 faces. When you look closer at the ad, you can spot four males in the adjacent apartment building watching the girl-on-girl action. Many consumers were very upset with this ad, believing it to be both sexist and delegitimizing to lesbian relationships. They made the argument that, the advertisement is sexist relies heavily on the notion that the lesbi ans are unwitting, innocent subjects violated by a voyeuristic male glaze. †(Turnbell) The writer refutes this argument by stating that the two women made the conscious decision to objectify themselves by leaving the curtains open, as well letting another male to capture their (not-so) intimate moment on a camera. Additionally, the ad is relevant to the product because it successfully communicates in a clever way that that even though the faces are so far away, the camera is advanced enough to still detect the voyeurs faces.Personally, I completely agree with the writer's argument. However, I would also argue that these types of ads do present a problem to the lesbian community. How can advertisements tastefully depict a lesbian encounter that meaningfully supports and empowers the lesbian community? Also, how can you conveys that the two women are legitimately homosexual, rather than a sexualizing their experience? This objectification of the lesbian relationship is likely w hy many lesbian advertisements are underrepresented.In general, they are most comfortable to advertisements that use implicit hints of sexuality rather than involving males or females (such as the car commercial). Some argue that â€Å"lesbians are female versions of male sexuality†, but this argument is scrutinized because lesbians describe their experience to be completely different. Typically, lesbians are more socially accepted than gay males are. However, they have to fight harder to convince society that their identity is more than just a sexual desirability of a female. One company that has successfully done so is JC Penny’s.In 2012, they released their â€Å"Freedom of Expression†ad (See Picture 5) that features two mothers and their daughter. While a group called â€Å"One Million Mothers†presented backlash to this ad, the overall response was positive. There is no overt sexual nature in this picture, it is just depicting a family photo with tw o mothers. â€Å"As jcpenney focuses on becoming America’s favorite store, we want to be a store for all Americans. In celebration of Mother’s Day, we’re proud that our May book honors women from diverse backgrounds who all share the heartwarming experience of motherhood. †-Eric Bovin (ABC news)In conclusion; advertisers have recognized the great importance of tapping into the gay market. Not only is establishing your company as being in support of this cause positive, but in some markets; it is necessary. Overall, Advertising to the gay and lesbian community is not one-size-fits-all. It are also specific precautions that you must take in entering into this type of market. Not only are you going up against the criticisms of anti-gay groups, but you are also up against the scrutiny of whether your advertisement is legitimate or whether you are using the leverage of the gay community to seem more accepting and grow your consumer base.There are Advertising Agencies must take into account the way in which homosexuals identify themselves in terms of their involvement as well as the how males and females differ in what appeals to them. Works Cited Hilary Chura, â€Å"Coors hikes spending on gay ads, March 27, 2000 Katherine Sender, Business Not Politics: The Making of the Gay Market, New York: Columbia University Press, 2005, 331 pp. , â€Å"The Dynamics of Brand Legitimacy: An Interpretive Study in the Gay Men's Community (PDF)†.Journal Article, Journal of Consumer Research, University of Chicago Press. JSTOR 10. Going Green, Going Gay? Micheal Koretzky, Jan 13, 2011 The Williams Institute, â€Å"Documented Evidence of Employment Discrimination and Its Effects on LGBT People†(July 2011). Get Outdoors Blog, â€Å"Lesbians for Good Ski Business†(Decemebr 21,2007) James Turnbell, â€Å"A Sexist Advertisement? Lesbians and the Politics of the Male Glaze†(December 13, 2008) ABC News, â€Å"JC Penney Features Same Sex Couple in May Catalouge†, (May 12, 2012)
Friday, January 10, 2020
Financial Planner Essay
Among the many career fields to choose from, I’d like to pursue one in the financial planning industry. A financial planner’s job is diverse and could be in areas like management of cash flow, investment and retirement planning, tax and estate planning, insurance and risk management planning, among others. There are also job opportunities outside personal planning that provides more financial rewards. I believe this job to be interesting, challenging and beneficial to people. This isn’t like other jobs wherein you’re only there to perform a task  sometimes without knowing the end result or the product of your efforts. As a financial planner, a person is able to help other people in more significantly, even in a life-changing manner. It’s a fact that many people have great ideas and good intentions regarding their finances, but few have the willpower and the right know-how to execute their plans. That’s where I see myself helping. I’d like to see people to be more financially stable and create for themselves a nest egg for their future. People should be aware of financial planning at an early age. The earlier a person starts setting aside part of his income for a financial plan, the more secure is his future going to be. Another reason why I’d like to have a career in this field is because I get stimulated when dealing with numbers. Crafting plans after studying pages of figures and mathematical formulas is something that does not daunt me. According to journals and news articles that have been published regarding the financial planning field, the demand for financial planners are rising globally as more and more people are becoming aware of the importance of having tailored financial plans for themselves. Over the years, financial planners have gradually, but consistently, created a niche for themselves in the market. There are several factors that have contributed to the upswing of demand for financial planners. In the earlier years, people were happy with social security benefits and old age Planner 2 pensions provided by the government. But years later, people’s outlook has shifted and the idea of being more comfortable and secure in old age have dominated the minds not just of those who are near retirement but even those who are still relatively young. Awareness among young professionals is seen to continue within the next decade, making it reasonably safe to assume that demand for financial programs and financial planners will continue. Another reason why the financial planning business has risen is caused by uncertainties in the stock markets. People are now opting to seek professional advice, before investing in a particular bond or stock. There is also a growing demand for financial planners outside the personal planning arena. Financial planners have found themselves becoming more and more necessary in banks and other financial institutions. Their expertise is needed in determining risks involved in investment vehicles. The pay scale and benefits that a certified financial planner receives differ from company to company, experience, and type of certification. A person who holds a position in higher management, like a vice president or portfolio manager, will earn a six-digit figure. Likewise, a person who’s been a financial planner for more than twenty years will also receive a six-digit salary. Entry-level compensation is often in the range of $30,000 to $40,000. The banking industry and other financial institutions’ salaries are often higher compared to other industries. For instance, a job posting for a financial advisor in Connecticut offers an annual income of $50,000 to $80,000. Another example is an ad for looking for financial planning director, which offers at least $150,000 annual income. But what’s more attractive about being a financial planner is the opportunity to earn more than your basic salary. Commissions and incentives are given for every transaction. A person’s overall income is often measured by how well he networks and sells financial products.
Thursday, January 2, 2020
KOTTER MODEL OF CHANGE Free Essay Example, 1250 words
The challenge with the prescription of the best way to run a firm is that there may be very many suggestions on the way different people believe is the best duration for the school to be opened. I will thus create a clear vision regarding the operation of the institution that will easily be understandable to all groups of people. The vision will incorporate the key values upheld by the school in order to be relevant and thus enable all people to identify with it. After creating the vision together with the coalition team, I will need to come up with a summary of the strategy of executing the vision. The summary will be capable of being described to the interested parties within a short period of not more than five minutes. Communicating the vision The staff and parents may not all respond positively to the idea of increasing the opening hours the first time they hear of the proposal. This is because different people have different ways of reasoning which will result to differing believes on what should be done (Samson & Daft 2011, p. 421). We will write a custom essay sample on KOTTER MODEL OF CHANGE or any topic specifically for you Only $17.96 $11.86/pageorder now There is thus a need to keep communicating the vision frequently to the stakeholders in order to make them understand it well (Leonard & McGuire 2007, p. 48). Constant communication will also help in counteracting the conflicting views that the people face in their course of interaction with others and thus keep them aligned to the vision. I will use every opportunity that prevails itself to communicate the vision both directly and indirectly, for example when parents come to school because of some issues relating to their children I will make sure I communicate the vision. Furthermore, each decision I make will be controlled by the vision of extending the school opening hours. Empowering action At this stage, I will put in the structures for change by instituting and empowering all-important structures that will be necessary for the change to take place (Kumra, Manfredi & Vickers 2011 p. 90). This will involve empowering the change leaders to deliver the required change in the opening period of the school and in including meals and bed rest in the program. It will also include reviewing the job descriptions, remunerations and performance of the staff to ensure that they are in line with the vision. Any person who brings a positive change in line with the vision will be rewarded accordingly in order to encourage all people to adopt the change of the system. At this level I will also identify the people who may be resisting the change and therefore help them accordingly in order to be able to accept the change.
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