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A statistical approach for optimizing the protocol for overexpressing lipase KV1 in Escherichia coli: purification and characterization

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journal contribution
posted on 24.11.2017 by Kalaivani Batumalaie, Elham Khalili, Naji Arafat Mahat, Fahrul Zaman Huyop, Roswanira Abdul Wahab

Lipase 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.


This work was supported by the Fundamental Research Grant Scheme [grant number FRGS R.J130000.7826.4F649]; Research University Grant Scheme [grant number Q.J130000.2626.13H09] from the Universiti Teknologi Malaysia, Johor.