• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1502-1510
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• 2018

Organic solvent-tolerant (OST) enzymes are widely applied in various industries for their activity and stability in organic solvents, for their higher substrate solubility, and for their greater stero-selectivity. However, the criteria for identifying OST enzymes largely remain undefined. In this study, we compared the amino acid composition of 19 OST esterases with that of 19 non OST esterases. OST esterases have increased the ratio of Ala and Arg residues and decreased the ratio of Asn, Ile, Tyr, Lys, and Phe residues. Based on our amino acid composition analysis, we cloned a carboxylesterase (EstSP2) from a psychrophilic bacterium, Sphingomonas glacialis PAMC 26605, and characterized its recombinant protein. EstSP2 is a substrate specific to p-nitrophenyl acetate and hydrolyzed aspirin, with optimal activity at $40^{\circ}C$; at $4^{\circ}C$, the activity is approximately 50% of its maximum. As expected, EstSP2 showed tolerance in up to 40% concentration of polar organic solvents, including dimethyl sulfoxide, methanol, and ethanol. The results of this study suggest that selecting OST esterases based on their amino acid composition could be a novel approach to identifying OST esterases produced from bacterial genomes.

• Journal of Life Science
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• v.27 no.11
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• pp.1381-1392
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• 2017

This review described solvent-tolerant lipases and their potential industrial, biotechnological and environmental impacts. Although organic solvent-tolerant lipase was first reported in organic solvent-tolerant bacterium, many organic solvent-tolerant lipases are in not only solvent-tolerant bacteria but also solvent-intolerant bacterial and fungal strains, such as the well-known Bacillus, Pseudomonas, Streptomyces and Aspergillus strains. As these lipases are not easily inactivated in organic solvents, there is no need to immobilize them in order to prevent an enzyme inactivation by solvents. Therefore, the solvent-tolerant lipases have the potential to be used in many biotechnological and biotransformation processes. With the solvent-tolerant lipases, a large number insoluble substrates become soluble, various chemical reactions that are initially impossible in water systems become practical, synthesis reactions (instead of hydrolysis) are possible, side reactions caused by water are suppressed, and the possibility of chemoselective, regioselective and enantioselective transformations in solvent and non-aqueous systems is increased. Furthermore, the recovery and reuse of enzymes is possible without immobilization, and the stabilities of the lipases improve in solvent and non-aqueous systems. Therefore, lipases with organic-solvent tolerances have attracted much attention in regards to applying them as biocatalysts to biotransformation processes using solvent and non-aqueous systems.

• Journal of Life Science
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• v.21 no.5
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• pp.700-705
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• 2011

Using enrichment procedures, we isolated organic solvent-tolerant Bacillus sp. BCNU 5005 from waste water and soil in the Ulsan industrial plant region. BCNU 5005 had a maximum similarity of 98% with B. subtilis and was designated as B. subtilis based on phylogenetic analyses using 16S rDNA sequences. Generally, most bacteria and their enzymes are destroyed or inactivated in the presence of high concentrations of organic solvents. However, the lipase activity of B. subtilis BCNU 5005 was very stable in the presence of various kinds of solvents (25%, v/v) except chloroform, ethylbenzene and decane. Furthermore, BCNU 5005 was determined to have a degradative ability towards organic solvents. This organic solvent tolerant Bacillus sp. BCNU 5005 could be used as a new potential resource for biotransformation and bioremediation.

• Journal of Life Science
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• v.23 no.10
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• pp.1246-1251
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• 2013

An organic solvent-tolerant lipase of Pseudomonas sp. BCNU 154 that was isolated from wastewater in the industrial complex region had optimal activity at $37^{\circ}C$ and pH 8. This crude extracellular lipase from BCNU 154 exhibited maximum stability in toluene, retaining about 6.01 U/ml (117.53%) activity for 2 h. $Ca^{2+}$, $Mg^{2+}$, $NH_4{^+}$, and $Na^+$ ions and triton X-100 activated the enzymes, whereas $Ba^{2+}$, $Hg^{2+}$, and $Zn^{2+}$ ions inhibited their activity. Pseudomonas sp. BCNU 154 lipase revealed stable activity comparable to that of the commercial immobilized Novozym 435. Thus, this organic solvent-tolerant lipase could have potential as a whole cell biocatalyst in industrial chemical processes without the use of immobilization.

• Journal of Life Science
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• v.25 no.3
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• pp.345-348
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• 2015

An organic solvent stable lipase from solvent-tolerant Pseudomonas sp. BCNU 171 had an optimal pH of 8 and an optimal temperature of 37℃. This crude extracellular lipase from BCNU 171 exhibited increased stability in the presence of various types of solvents at high concentrations (25%, v/v). The lipase stability was found to be highest in the presence of xylene (137%), followed by toluene (131%), octane (130%), and butanol (104%). Overall, BCNU 171 lipase tended to be more stable than immobilized commercial lipase (Novozyme435) in the presence of organic solvents. Furthermore, BCNU 171 lipase maintained about 90% of its enzyme original activity in the presence of NH₄⁺, Na⁺, Ba²⁺, Hg²⁺, Ni²⁺, Cu²⁺, and Ca²⁺ion and significantly increased its enzyme activity in the presence of various emulsifying agents. Thus, the organic solvent stable lipase from Pseudomonas sp. BCNU 171 could be usable as a potential whole cell biocatalyst and for synthetic applications of enzymes for industrial chemical processes in organic solvents without using immobilization.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1159-1165
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• 2011

Biodiesel is methyl and ethyl esters of long-chain fatty acids produced from vegetable oils or animal fats. Lipase enzymes have occasionally been used for the production of this biofuel. Recently, biodiesel production using immobilized lipase has received increased attention. Through enhanced stability and reusability, immobilized lipase can contribute to the reduction of the costs inherent to biodiesel production. In this study, methanol-tolerant lipase M37 from Photobacterium lipolyticum was immobilized using the cross-linked enzyme aggregate (CLEA) method. Lipase M37 has a high lysine content (9.7%) in its protein sequence. Most lysine residues are located evenly over the surface of the protein, except for the lid structure region, which makes the CLEA preparation yield quite high (~93%). CLEA M37 evidences an optimal temperature of $30^{\circ}C$, and an optimal pH of 9-10. It was stable up to $50^{\circ}C$ and in a pH range of 4.0-11.0. Both soluble M37 and CLEA M37 were stable in the presence of high concentrations of methanol, ethanol, 1-propanol, and n-butanol. That is, their activities were maintained at solvent concentrations above 10% (v/v). CLEA M37 could produce biodiesel from olive oil and alcohols such as methanol and ethanol. Additionally, CLEA M37 generated biodiesel via both 2-step methanol feeding procedures. Considering its physical stability and reusability, CLEA M37 may potentially be used as a catalyst in organic synthesis, including the biodiesel production reaction.