Biotransformation Serves as an Alternative Tool to the Chemical Synthesis
Received Date: Jan 22, 2018 / Accepted Date: Jan 29, 2018 / Published Date: Jan 31, 2018
Proteases are hydrolytic enzymes with high selectivity which does not need any expensive cofactors and can be used as synthetic tools. Most of the synthetic reactions are carried out in presence of organic solvents. Proteases in organic solvents can catalyze reactions such as esterification and peptide synthesis. Unfortunately most of the protease loses their activity in the presence of organic solvents. Several protein engineering methods have been used to increase stability and activity of enzyme in organic solvents. If the enzyme is naturally organic solvent tolerant then no such modification is needed thus there is a continuous demand of microorganisms that can produce solvent tolerant enzymes.
Microorganisms are good source of enzymes because of short generation time, easy genetic modification which are useful for bulk production . First organic solvent tolerant enzyme has been reported from Pseudomonas aeruginosa was a lipolytic enzyme . Many extremophilic bacteria such as thermophiles and halophiles serve as a good source of organic solvent tolerant enzyme besides their counterpart mesophiles [3,4]. Halophiles are adapted to grow at high salt conditions thus the enzymes from halophiles require salt for activity and stability. High salt environments are low water environments thus halophiles are adapted to cope with low water activity.
New application of protease as antifouling agent needs organic solvent tolerance and high activity in saline sea water. Undesirable attachment and accumulation of phytoplankton zooplankton and other microorganisms on ship surface is termed as fouling. Microbial biofouling occur in many steps which firstly involves the formation of a conditional layer than unicellular microorganisms of marine ecosystem attached to it and lastly large multicellular organisms attached to it and cause biofouling . This process is very much similar to bacterial biofouling of implants, when an implant is placed in the body proteins and other macromolecules adsorbed on the surface of implant and forms a conditional layer, eventually this conditional layer is colonized by neutrophills and macrophages. Colonization is followed either by collagen encapsulation or bacterial infection (Figure 1). If bacterial infection takes place before encapsulation than it is impossible to cure infection .
Several antifouling strategies have been used to overcome such problems which include the preparation of anti-adhesion coatings by chemical or physical adsorption of hydrophilic polymer molecules that can work as a steric and/or hydration barrier between the underlying surface and the proteins and/or glycoproteins of the cells thus prevents the initial attachment. Several biomolecules such as Bovine Serum Albumin (BSA), dextran, hyaluronic acid, chitosan, alginate, and mannitol were used as anti- adhesive [7,8]. Besides these polymers such as Poly ethylene glycol (PEG), polyacrylamides were also used as anti-adhesive [9,10]. Several anti fouling coatings were used to stop fouling in marine industry which involves use of tributyltin self-polishing copolymer (TBT-SPC) in paints. Till 2008, Tri-n-butyl tin (TBT) has been extensively used as antibiofouling agent in marine paint industry. TBT has adverse effect on marine ecological diversity. Thus paints containing TBT has been banned. Haloarchaeal proteases serve as a better alternative in antifouling coating. As most of the coating materials are suspended in organic solvent, there is urgent need to have organic solvent compatible protease and other related enzymes. Organic solvents reduce water activity thus most of the salt stable halophilic enzyme remain active and stable in the presence of organic solvents. Besides conventional protease may also work suboptimally in saline water condition which is the most important criteria for application of enzyme as antifoulant. Archaea are also important to understand life as halites have been found from mars also .
TBT-SPC was an environmental threat thus it was completely banned from January 2008. Use of enzymes in paints has provided an alternative and environmental friendly way to overcome fouling. Enzymes present in the paint directly interact with glycoproteins of microorganism thus reduces attachment to the ship surface. Organic solvents are the essential component of paints thus it is mandatory to use an organic solvent stable enzyme in such preparations. Organic solvent tolerant and stable proteases from different bacteria sources have proved beneficial in marine industry to stop fouling . In industrial bio catalysis, cross-linked enzyme aggregates (CLEAs) are very beneficial in terms of economy and environment. CLEAs are easily obtained from crude enzyme thus economic over immobilization through protein engineering. General mode of preparation of CLEAs is given in Figure 2 glutaraldehyde is used as cross-linking agent for decades. Glutaraldehyde bring about inter and intramolecular aldol condensations reaction between the free amino groups of lysine residues, on the surface of neighbouring enzyme molecules which involves schiff's base formation and Michael-type 1,4 addition to a, β-unsaturated aldehyde moieties resulting the formation of CLEAs .
CLEAs can also be prepared by the cross- in the presence of a siloxane e.g. (MeO)4Si, resulting the formation of CLEA- silica composite . Major advantage of using CLEAs is that they can be recycled. Immobilizations of enzyme as CLEAs increase stability at high temperatures . Skovgard et al. stabilized subtilisins, from different bacterial sources, by converting them in cross-linked enzyme aggregates- CLEAs. Protease activity of CLEAs in artificial seawater (ASW) was tested to find out their stability towards marine conditions furthermore they incubated the CLEAs in xylene an important component of ship paint. They found that catalytic activity was increased as compared to the initial catalytic activity in ASW for 7 days. A possible explanation is that continuous hydration of paint increases in seawater which leads to an increased amount of molecules leaching from the paint surface. Silicates can be used as matrices for enzyme immobilization some organic solvent proteases have been summarized in Table 1.
|Source||Incubation Condition||Stability||Unstable in the presence||References|
|Pseudomonas aeruginosa K||37°C, 14 days||25% (v/v) Deccane, Octane||5 % (v/v) Benzene, Heptane, Xylene|||
|Pseudomonas aeruginosa PST-01||30°C, 15 days||25% (v/v) Ethanol, methanol, DMSO, Octanol, Butanol||25 % (v/v) Benzene, Haptane, Xylene|||
|Pseudomonas aeruginosa PseA||30°C, 72 h||25% (v/v) Benzene, Heptane, Hexane, Toluene||25 % (v/v) Butanol|||
|Pseudomonas aeruginosa PT121||30°C, 5 or 14 days||50% (v/v) Benzene, Heptane, Hexane, Toluene and DMSO||50 % (v/v) DMF, Ethanol,|||
|Pseudomonas aeruginosa san-ai||30°C, 10 days||25% (v/v) DMF||25 % (v/v) Hexane, Benzene, Acetone|||
|Bacillus sp. APR-4||4°C, 24 h||50% (v/v) Ethanol, Methanol Benzene, Butanol||50 % (v/v) Acetone|||
|Bacillus cereus BG1||30°C, 1–55 days||25% (v/v) DMSO, Ethanol, methanol||25 % (v/v) Acetonitrile|||
|gamma-Proteobacterium||30°C, 10 days||33% (v/v) Ethanol, Methanol Butanol DMSO, Xylene|||
|Natrialba magadii||30 o C, 24 h, 1.5 M NaCl||15% or 30% (v/v) DMSO||15 %(v/v) Acetone, Ethanol, Acetonitrile|||
|Halobacterium sp. SP1||20°C, 30 min||33% (v/v) Toluene, Xylene|||
|halophilic Bacillus sp.||30°C, 24 h||50% (v/v) Ethanol, Methanol||50 % (v/v) Benzene, Toluene|||
|Geomicrobium sp. EMB2||30°C, 24 h||50 (v/v) Toluene, Butanol, Heptane, Hexane, Benzene|||
|Staphylococcus aureus strain MSSA 476||Toluene, xylene and cyclohexane|||
Table 1: Organic solvent tolerant protease from different microorganisms.
One of the most promising approaches is the use of enzymes which can interact directly with microorganisms on the surface. For successful use in paints enzyme must possess solvent stability and should function in saline conditions. Most of the solvent stable protease show less activity in artificial sea water due to inhibition by NaCl, Mg+2 and Ca+2. Most halophilic proteins are not suitable for such environment because they require high sat concentration for activity.
A protease from a moderately halophilic Bacillus sp. strain isolated from sea water has maximum activity at pH optimum 9.0, t1/2 190 min at 60°C and 1% (w/v) NaCl. The protease shows stability in polar and nonpolar solvents at high concentrations . The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology thus there is a continuous demand of microorganisms that can produce solvent tolerant enzymes [20,30].
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Citation: Gupta M (2018) Biotransformation Serves as an Alternative Tool to the Chemical Synthesis. J Pharmacogenomics Pharmacoproteomics 9: e159. Doi: 10.4172/2153-0645.100e159
Copyright: ©2018 Gupta M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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