Abstract
ABSTRACTLipase is one of the most important industrial enzymes, widely used in the preparation of food additives, cosmetics and pharmaceuticals. In order to obtain a large amount of lipase, in the present study, a gene encoding intracellular lipase was cloned from Acinetobacter haemolyticus. The recombinant lipase KV1 containing a His-tag was expressed in Esherichia coli BL21 (DE3) cells, using pET-30a as the expression vector. Using the central composite design, screening and optimization of induction conditions (cell density before induction, IPTG (isopropyl β-D-1-thiogalactopyranoside) concentration, post-induction temperature and post-induction time) were made. All parameters significantly (P < 0.05) influenced the expression of lipase KV1, rendering a 70% increase in enzyme production at optimum induction conditions (OD600 before induction: 0.6, IPTG concentration: 0.5 mmol/L, post-induction temperature: 40 °C, post-induction time: 16 h). The expressed recombinant lipase KV1 was purified using Ni-affinity chromatography, affording ∼3.1-fold of the enzyme with an estimated relative molecular mass of 39 kDa. The recombinant lipase KV1 exhibited its maximum activity at 40 °C and pH 8.0. Beneficially, the recombinant lipase KV1 retained its relative activities (>80%) even up to 24 h between pH 7−12; suggesting that the recombinant lipase KV1 may be suitable for a wide range of industrial applications.
Highlights
There is a growing demand for industrial enzymes, especially of microbial origin, owing to their wide versatility
Bacterial lipases are highly conserved within the sequences of the open reading frame (ORF); more variations can be observed above and below the ORF [23]
The primary screening of positive clones on agar plates was carried out using tributyrin-Luria– Bertani (LB) agar (100 mg/mL ampicillin) (Supplementary Figure S1 (A,B)), the simplest triglyceride occurring in natural fats and oils
Summary
There is a growing demand for industrial enzymes, especially of microbial origin, owing to their wide versatility. More industrial players are switching over to the use of enzymes for an array of applications on an industrial scale [2]. Among such enzymes, lipases are preferred in organic chemistry, the pharmaceutical industry, biophysics, biochemical and process engineering, biotechnology, microbiology, biochemistry, etc. Due to high demand for functional bacterial lipases showing neutral or alkaline pH optimum [3,4], overexpressing the wild-type lipase KV1 protein in a suitable bacterial system would increase its quantity for characterization and development for prospective manufacturing uses, as well as biodegradation and bioremediation purposes
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