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

• Main Authors: Kalaivani Batumalaie, Elham Khalili, Naji Arafat Mahat, Fahrul Zaman Huyop, Roswanira Abdul Wahab
• Format: Article
• Language: English
• Published: Taylor & Francis Group 2018-01-01
• Series: Biotechnology & Biotechnological Equipment
• Subjects: Acinetobacter haemolyticus; central composite design; lipase; protein overexpression; response surface methodology
• Online Access: http://dx.doi.org/10.1080/13102818.2017.1407670

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.