INTRODUCTION:

Bio catalysis is the use of natural catalysts, such as protein enzymes, to perform chemical transformations on organic compounds. Both, enzymes that have been more or less isolated or enzymes still residing inside living cells are employed for this task.

Advantages of bio catalysis

The key word for organic synthesis is selectivity which is necessary to obtain a high yield of a specific product. There are a large range of selective organic reactions available for most synthetic needs. However, there is still one area where organic chemists are struggling, and that is when chirality is involved, although considerable progress in chiral synthesis has been achieved in recent years.

Enzymes display three major types of selectivity’s:

Chemo selectivity: Since the purpose of an enzyme is to act on a single type of functional group, other sensitive functionalities, which would normally react to a certain extent under chemical catalysis, survive. As a result, biocatalytic reactions tend to be "cleaner" and laborious purification of product(s) from impurities emerging through side-reactions can largely be omitted.

Regioselectivity and Diastereoselectivity: Due to their complex three-dimensional structure, enzymes may distinguish between functional groups which are chemically situated in different regions of the substrate molecule.

Enantioselectivity: Since almost all enzymes are made from L-amino acids, enzymes are chiral catalysts. As a consequence, any type of chirality present in the substrate molecule is "recognized" upon the formation of the enzyme-substrate complex. Thus a prochiral substrate may be transformed into an optically active product and both enantiomers of a racemic substrate may react at different rates.

These reasons and especially the latter are the major reasons why synthetic chemists have become interested in biocatalysis. This interest in turn is mainly due to the need to synthesize enantiopure compounds as chiral building blocks for drugs and agrochemicals. Another important advantage of biocatalysts are that they are environmentally acceptable, being completely degraded in the environment. Furthermore the enzymes act under mild conditions, which minimizes problems of undesired side-reactions such as decomposition, isomerization, racemization and rearrangement, which often plague traditional methodology.

Literature survey:

1) Natural methods of protein stabilization: Thermostable biocatalysts: ²

In this the authors used the approach to study i.e. to learn how nature has managed to stabilize these proteins using a detailed knowledge of their biochemical properties and three-dimensional structures. This is illustrated with several different classes of enzyme that have been studied at Exeter. They used alcohol dehydrogenase, aminoacylase, pyroglutamyl carboxypeptidase, γ-lactamase, dehalogenase and lysophospholipase. Enzymes that are naturally found in thermophilic and hyperthermophilic organisms are being used as robust biocatalysts in the fine chemical and pharmaceutical industries. They have important use in these industries due to their increased stability which is often required during commercial reaction conditions.

2) Biocatalyst activity in non aqueous environments correlates with centisecond-range protein motions: ³

In this recent studies explore the relationship between enzymatic catalysis and protein dynamics in the aqueous phase have yielded evidence that dynamics and enzyme activity are strongly correlated. Given that protein dynamics are significantly attenuated in organic solvents and that proteins exhibit a wide range of motions depending on the specific solvent environment, the non aqueous milieu provides a unique opportunity to examine the role of protein dynamics in enzyme activity. Thus Variable-temperature kinetic measurements, X-band electron spin resonance spectroscopy, 1H NMR relaxation, and 19FNMR spectroscopy experiments were performed on subtilisin Carlsberg colyophilized with several inorganic salts and suspended in organic solvents. The results indicate that salt activation induces a greater degree of transition-state flexibility, reflected by a more positive ΔΔS†, for the more active biocatalyst preparations in organic solvents. In contrast, ΔΔH† was negligible regardless of salt type or salt content. As a result 19F chemical shift measurements and hyperfine tensor measurements of biocatalyst formulations inhibited with 4-fluorobenzenesulfonyl fluoride and 4-ethoxyfluorophosphinyl-oxy-TEMPO, respectively, suggest that enzyme activation was only weakly affected by changes in active-site polarity.

3) Cytochrome c as a biocatalyst: ⁴

The present study shown that Type c cytochromes, which are involved in the electron transport system, are also able to catalyze peroxidase-like reactions in the presence of an electron acceptor, such as hydrogen peroxide or an organic hydroperoxide. This work reviews the catalytic activity of cytochrome c, and the potential design by site-directed mutagenesis and chemical modification of new biocatalysts for environmental purposes.

4) Site-directed mutagenesis improves the biocatalytic activity of iso-1-cytochrome c in polycyclic hydrocarbon oxidation: ⁵

In this iso-1-Cytochrome c from Saccharomyces cerevisiae is able to oxidize polycyclic aromatic hydrocarbons (PAH) in the presence of hydrogen peroxide. Anthracene and pyrene are oxidized by yeast cytochrome c to form anthraquinone and 1,8-pyrenedione, respectively. Iso-1-cytochrome c from S. cerevisiae was modified by site-directed mutagenesis of Phe82 and Cys102. The Phe82 substitution significantly altered the kinetic behavior of the protein; Cys102 modification affected neither the kinetic nor the stability constant. The Gly82; Thr102 variant was 10 times more active and showed a catalytic efficiency 10-fold greater than the wild-type iso-1-cytochrome c. However, Phe82 variants showed lower stability against inactivation by hydrogen peroxide than the wild-type protein. These site-directed mutations did not significantly alter the stability and activity of the hemoprotein in increasing concentrations of tetrahydrofuran.

5) De novo design of biocatalysts: ⁶

The challenging field of de novo enzyme design has begun to yield proteins with impressive catalytic efficiency. However, the current methods are not sufficient to design efficient enzymes for many reactions.

6) Intermediates of thiamine catalysis immobilized on silica surface as active biocatalysts for α-ketoacid decarboxylation: ⁷

The authors used two ‘active aldehyde’ intermediates of thiamine catalysis immobilized on a silica surface by a convenient method via their phosphate groups. These bio-composite materials have been evaluated as catalysts for pyruvate and benzoyl-formate decarboxylation in either the presence or not of an aldehyde additive. They are stable and very effective catalysts for the production of 2-hydroxy-ketones, acetoine and benzoin.

7) Solubilization of cytochrome c in organic media with fluoroalkyl end-capped N-(1,1-dimethyl-3-oxobutyl) acrylamide oligomer: a new approach to fluorinated biocatalyst in organic media: ⁸

The authors suggested self-assembled molecular aggregates of fluoroalkyl end-capped N-(1,1-dimethyl-3-oxobutyl)acrylamide oligomer can solubilize cytochrome c in organic media such as methanol, although the corresponding non-fluorinated polymer cannot solubilize cytochrome c in organic media. Interestingly, the resulting fluorinated oligomer–cytochrome c aggregate was found to act effectively as a new fluorinated biocatalyst for the oxidation of pinacyanol chloride with hydrogen peroxide in the non-aqueous methanol.

8) Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids:⁹

The authors introduced the hydantoinase process as an economically attractive method for the production of many unnatural chiral amino acids, which are components of potential pharmaceuticals.

9) Lipases as practical biocatalysts:¹⁰

The authors were states that novel developments in the study of lipases, ubiquitous enzymes used in numerous industrial processes, offer exciting opportunities leading to further practical applications of lipases in synthetic organic chemistry.

10) Metagenomic, gene discovery and the deal biocatalyst: ¹¹

In this optimum condition for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the ‘ideal biocatalyst’ for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of ‘sequence space’ that is not addressed by traditional screening.

11) Biocatalysis- Biological systems for the reduction of chemicals :¹²

Authors described biocatalysis harnesses the catalytic potential of enzymes to produce building blocks and end-products for the pharmaceutical and chemical industry. Located at the interface between fermentation processes and petrol-based chemistry, biotransformation processes broaden the toolbox for bioconversion of organic compounds to functionalized products.

12) Fusion protein of Vitreoscilla hemoglobin with D-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C :¹³

An artificial flavohemoprotein was constructed by fusing Vitreoscilla hemoglobin (VHb) with D-amino acid oxidase (DAO) of Rhodotorula gracilis to determine whether bacterial hemoglobin can be used as an oxygen donor to immobilized flavoenzyme. This chimeric enzyme significantly enhanced DAO activity and stability in the bioconversion process of cephalosporin C. In a 200-mL bioreactor, the catalytic efficiency of immobilized VHb-DAO against cephalosporin C was 12.5-fold higher than that of immobilized DAO, and the operational stability of the immobilized VHb-DAO was approximately threefold better than that of the immobilized DAO. In the scaled-up bioprocess with a 5-L bioreactor, immobilized VHb-DAO (2500 U/L) resulted in 99% bioconversion of 120 mM cephalosporin C within 60 min at an oxygen flow rate of 0.2 (v/v) × min. Ninety percent of the initial activity of immobilized VHb-DAO could be maintained at up to 50 cycles of the enzymatic reaction without exogenous addition of H2O2 and flavin adenine dinucleotide (FAD). The purity of the final product, glutaryl-7-aminocephalosporanic acid, was confirmed to be 99.77% by HPLC analysis. Relative specificity of immobilized VHb-DAO on D-aminoadipic acid, a precursor in cephalosporin C biosynthesis, increased twofold, compared with that of immobilized DAO, suggesting that conformational modification of the VHb-DAO fusion protein may be altered in favor of cephalosporin C.

13) The search for the ideal biocatalyst :¹⁴

While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.

14) Biocatalysts for clean industrial products and processes:¹⁵

Biocatalysis inherently offers the prospect of clean industrial processing and has become an accepted technology throughout most sectors. The convergence of biology and chemistry has enabled a plethora of industrial opportunities to be targeted, while discoveries in biodiversity and the impact of molecular biology and computational science are extending the range of natural and engineered biocatalysts that can be customized for clean industrial requirements.

15) Protein engineering of oxygenases for biocatalysis :¹⁶

In this authors explain oxygenase enzymes have limited practical applications because of their complexity, poor stabilities, and often low catalytic rates. However, their ability to perform difficult chemistry with high selectivity and specificity has kept oxygenases at the forefront of engineering efforts. Growing understanding of structure–function relationships and improved protein engineering methods are paving the way for applications of oxygenases in chemical synthesis and bioremediation.

16) Selection of mutations for increased protein stability: ¹⁷

There are many ways to select mutations that increase the stability of proteins, including rational design, functional screening of randomly generated mutant libraries, and comparison of naturally occurring homologous proteins. The protein engineer's toolbox is expanding and the number of successful examples of engineered protein stability is increasing. Still, the selection of thermostable mutations is not a standard process. Selection is complicated by lack of knowledge of the process that leads to thermal inactivation and by the fact that proteins employ a large variety of structural tricks to achieve stability.

17) Resolution and synthesis of (S)-1-(2-naphthyl)ethanol with immobilized pea protein: as a new biocatalyst: ¹⁸

The authors synthesized (S)-1-(2-Naphthyl)ethanol by immobilized pea (Pisum sativum L.) protein (IPP) from (R, S) 2-naphthyl ethanol (> 99% ee, yield; about 50%), in which the (R)-enantiomer was selectively oxidized to 2-acetonaphthone. IPP could be reused consecutively at least three times without any decrease of yield and optical purity.

18) Identification of Candida tenuis xylose reductase as highly selective biocatalyst for the synthesis of aromatic alpha-hydroxy esters and improvement of its efficiency by protein engineering:¹⁹

In this wild-type Candida tenuis xylose reductase and two Trp-23 mutants thereof catalyze NADH-dependent reduction of a homologous series of aromatic alpha-keto esters with absolute pseudo re-face stereoselectivity and broad tolerance for the substituent on the aromatic ring, giving the corresponding R-alcohols in high yield.

19) Application of immobilized bovine enterokinase in repetitive fusion protein cleavage for the production of mucin 1 :²⁰

Bovine enterokinase is a serine protease that catalyzes the hydrolysis of peptide bonds and plays a key role in mammalian metabolism. Because of its high specificity towards the amino acid sequence (Asp)(4)-Lys, enterokinase is a potential tool for the cleavage of fusion proteins, which are gaining more importance in biopharmaceutical production. A candidate for adaptive cancer immunotherapy is mucin 1, which is produced recombinantly as a fusion protein in CHO cells. Here, we present the first repetitive application of immobilized enterokinase for the cleavage of the mucin fusion protein. The immobilization enables a facile biocatalytic process due to simplified separation of the biocatalyst and the target protein. Immobilized enterokinase was applied in a maximum of 18 repetitive reactions. The enzyme utilization (total turnover number) was increased significantly 419-fold compared to unbound enzyme by both immobilization and optimization of process conditions. Slight enzyme inactivation throughout the reaction cycles was observed, but was compensated by adjusting the process time accordingly. Thus, complete fusion protein cleavage was achieved. Furthermore, they obtained isolated mucin 1 with a purity of more than 90% by applying a simple and efficient purification process. The presented results demonstrate enterokinase to be an attractive tool for fusion protein cleavage.

20) Enzyme-Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique Biocatalysts: ²¹

The authors here represent the improvement of catalytic efficiency of immobilized enzymes via materials engineering was demonstrated through the preparation of bioactive nanofibers. Bioactive polystyrene (PS) nanofibers with a typical diameter of 120 nm were prepared and examined for catalytic efficiency for biotransformations. The nanofibers were produced by electrospinning functionalized PS, followed by the chemical attachment of a model enzyme, R-chymotrypsin. The observed enzyme loading as determined by active site titration was up to 1.4% (wt/wt), corresponding to over 27.4% monolayer coverage of the external surface of nano fibers. Furthermore, nano fibrous R-chymotrypsin exhibited a much-improved non aqueous activity that was over 3 orders of magnitude higher than that of its native counterpart suspended in organic solvents including hexane and isooctane. It appeared that the covalent binding also improved the enzyme’s stability against structural denaturation, such that the half-life of the nano fibrous enzyme in methanol was 18-fold longer than that of the native enzyme.

21) Non-Aqueous Biocatalysis in Heterogeneous Solvent Systems: ²²

Biocatalysis has become a useful alternative to chemical transformations for the production of a range of compounds with applications in the food, feed, chemical and pharmaceutical industries. However, it is not necessarily an easy task to obtain the desired levels of performance in terms of rate, yield and selectivity of the reaction. One strategy for optimizing biocatalyst performance is to use non-conventional media, such as non-aqueous heterogeneous systems. In this article, author highlights some of the current trends in biocatalysis in such systems, focusing on reverse micelles, supercritical fluids and ionic liquids.

22) Biocatalytic synthesis of novel electronic and photovoltaic materials: ²³

In this article a new class of ruthenium complex-based macrodye and a dinuclear complex were synthesized via a biocatalytic route employing hematin as an efficient biocatalyst. The photovoltaic overall efficiency of the dinuclear complex was found to be 2.1 % and higher than the polymeric complex (0.33 %). Furthermore, authors developed an environmentally benign methodology for the synthesis of novel pegylated polyphenolics. The reaction conditions used do not require any organic solvents, and all the reactions were performed in aqueous media. The synthesized polymers were soluble in both organic and aqueous media, and provide further opportunity to tailor the properties. Finally, a novel biomimetic method for the synthesis of a conducting molecular complex of polypyrrole and of thiophene substitute in the presence of a polyelectrolyte, such as polystyrene sulfonate (SPS), is presented. A synthetic enzyme based on hematin was used to catalyze the polymerization of pyrrole (PYR) and 3,4-ethylene di-oxy thiophene (EDOT) in the presence of SPS. Copolymers of EDOT and PYR have also been synthesized, and these novel materials have been shown to exhibit high electrical conductivity.

23) Cutinase structure, function and biocatalytic applications: ²⁴

This review analyses the role of cutinases in nature and their potential biotechnological applications. The cloning and expression of a fungal cutinase from Fusarium solani f. pisi, in Escherichia coli and Saccharomyces cerevisiae hosts are described. The three dimensional structure of this cutinase is also analysed and its function as a lipase discussed and compared with other lipases. The bio catalytic applications of cutinase are described taking into account the preparation of different cutinase forms and the media where the different types of enzymatic reactions have been performed, namely hydrolysis, esterification, transesterification and resolution of racemic mixtures. The stability of cutinase preparations is discussed, particularly in anionic reversed micelles considering the role of hexanol as substrate, co-surfactant and stabilizer. Process development based on the operation of cutinase reactors is also reviewed.

24) Deracemization of (±)-2,3-disubstituted oxiranes via biocatalytic hydrolysis using bacterial epoxide hydrolases: kinetics of an enantioconvergent process :²⁵

Asymmetric biocatalytic hydrolysis of (±)-2,3-disubstituted oxiranes leading to the formation of vicinal diols in up to 97% ee at 100% conversion was accomplished by using the epoxide hydrolase activity of various bacterial strains. The mechanism of this deracemization was elucidated by 18OH2-labelling experiments using a partially purified epoxide hydrolase from Nocardia EH1. The reaction was shown to proceed in an enantioconvergent fashion by attack of OH2 at the (S)-configured oxirane carbon atom with concomitant inversion of configuration.

25) Biopolymers for Biocatalysis: Structure and Catalytic Mechanism of Hydroxynitrile Lyases: ²⁶

Here the authors discussed use of hydroxynitrile lyases which catalyze the reversible cleavage of α-cyanohydrins to yield hydrocyanic acid and the corresponding aldehyde or ketone. Besides its biological interest, this class of enzymes is also of relevance in industrial biocatalysis for the enantioselective condensation of HCN with a variety of aldehydes and ketones. Several distinctly different types of hydroxynitrile lyases (HNLs) are known, which must have originated through convergent evolution from different ancestral proteins. Insights into the reaction mechanisms emerged from a combination of structural, enzyme kinetic, spectroscopic, and molecular modeling data. For all three types of HNLs, mechanisms involving acid-base catalysis were proposed. In members belonging to the α,β-hydrolase type, the amino acid residues of the catalytic triad presumably act as general acid/base, whereas for flavine adenine dinucleotide (FAD)-dependent HNLs a single histidine residue fulfills this function. In the third type of HNL; which is related to carboxypeptidase; acid-base catalysis involves the carboxylate of the C-terminal residue. The catalytic relevance of a positive electrostatic potential in the active site was suggested in some of the mechanistic proposals.

26) Model Development for Fermentation and Biocatalysis Process: ²⁷

A production process based on fermentation or biocatalysis is typically raw material cost intensive. Therefore, strain optimization and process optimization is a key to success. Optimization can be effectively achieved with the understanding of the physiology and mechanism of the micro-organism or biocatalyst. This knowledge can be represented in the form of mathematical models that can simulate the behavior of the bioprocess. A popular modeling strategy involves modeling of the process dynamics around the rate limiting process, which usually is a key enzymatic reaction(s). These rates depend both on the substrate / inhibitor concentrations and enzyme concentrations. Recently, successful models have been developed based on Michelis-Menten type of kinetic forms along with cybernetic principles. The kinetic form accounts for the effects of substrate concentration including the rate limiting substrate / inhibitor and substrate catabolite repression. Cybernetic principles on the other hand simulate the dynamic profiles of the enzyme concentrations, which may depend on some kind of optimality criteria. This workshop will discuss the modeling strategies with some case studies of industrially relevant bioprocesses. While the models may be presented in the context of a specific process, the strategy would be generic and can be applied toward the model development of any bioprocess.

CONCLUSION:

All these reviews explains the methods of protein stabilization, use of Cytochrome c as a biocatalyst, De novo design of biocatalysts, Biocatalysts for clean industrial products and processes. Biocatalysis is the use of natural catalysts, such as protein enzymes, to perform chemical transformations on organic compounds. Either enzymes that have been more or less isolated or enzymes still residing inside living cells are employed for this task. Site-directed mutagenesis improves the biocatalytic activity of iso-1-cytochrome c in polycyclic hydrocarbon oxidation. So the various biocatalysts were used in variety of reactions to give predominant product, high yield less reaction time with ecofriendly reaction.

ACKNOWLEDGEMENT:

The authors are grateful to Mrs. Fatma Rafiq Zakaria, the Chairman, Maulana Azad Educational Trust for providing the necessary facility and encouragement. The authors are thankful to Dr. M. H. Dehghan, Principal, and Y.B. Chavan College of Pharmacy for his keen interest and moral support.

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27. Workshop by Professor Pramod Wangikar, Department of Chemical Engineering, Indian Institute of Technology, Bombay. Venue: - Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833.

Received on 25.06.2011 Modified on 10.07.2011

Asian J. Research Chem. 4(9): Sept, 2011; Page 1355-1360