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Optimization of organic solvent-tolerant lipase production by Acinetobacter sp. UBT1 using deoiled castor seed cake.

ABSTRACT: Organic solvent-tolerant lipase-producing microorganisms were isolated from petrol spilled soil. From ten morphologically distinct lipase-producing bacterial isolates, highest amount of lipase-producing isolate UBT1 was identified as Acinetobacter sp. using 16S rRNA gene sequencing (NCBI Accession No: MH879815). An increase in lipase production from 42 U/mL to 243 U/mL was obtained when different deoiled seed cakes were supplemented instead of olive oil in the medium. Further optimization of media components by the statistical approach assisted in discerning the main influencing media components and their optimum concentrations. Nine components glucose, castor seedcake, potassium nitrate, gum arabic, calcium chloride, magnesium sulphate, potassium di-hydrogen phosphate, dipotassium hydrogen phosphate, and ferric chloride were selected for Plackett-Burman design. The optimum concentrations of three significant selected components for the lipase production were found to be 0.025 gm% glucose, 0.002 gm% calcium chloride, and 0.2 gm% potassium di-hydrogen phosphate as determined by Response Surface Methodology. Increase in lipase production with 292.29 U/mL was achieved in the media containing optimized components and 2 gm% deoiled castor seed cake. Purification studies with ammonium sulphate precipitation, dialysis, and gel permeation chromatography resulted in 77.54% recovery with 5.77-fold partially purified lipase. The residual activity of lipase in 50 and 75% concentration of n-hexane among other solvents after 24 h was 105.05 and 90.42%, respectively, indicating its solvent tolerance. The present study reports the isolation of organic solvent-tolerant lipase-producing Acinetobacter sp. UBT1, optimization of the culture media for lipase production using the deoiled castor seed cake, and its partial purification.

SUBMITTER: Patel R

PROVIDER: S-EPMC7644738 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarcticaIMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.

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Optimization of organic solvent-tolerant lipase production by Acinetobacter sp. UBT1 using deoiled castor seed cake.

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