Expression and optimized production of laminarinase RbLam16 from Rhodohalobacter barkolensis
ZHOU Haiyan1,2, ZHOU Jianbao1,2, YI Xiaonan1,2, LI Mian3, LIU Zhiqiang1,2*
1(Key Laboratory of Bioorganic Synthesis of Zhejiang Province (Zhejiang University of Technology), Hangzhou 310014, China); 2(National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals (Zhejiang University of Technology), Hangzhou 310014, China); 3(Zhejiang Huakang Pharmaceutical Co., Ltd., Kaihua 324302, China)
Abstract: For heterologous expression of the laminarinase RbLam16 from Rhodohalobacter barkolensis, the codon optimized and synthesized gene rblam16 was cloned into pPIC9K and transformed into Pichia pastoris GS115. The recombinant RbLam16 was prepared, purified and biochemically characterized. The results showed that, with laminarin from Laminaria japonica as the substrate, the recombinant RbLam16 exhibited the optimal activities at 55℃ and pH 7.0. It showed high thermostability (above 90% residual activity) at 55°C after incubation for 30 min and pH stability (up to 90% residual activity) after incubation at pH 7.0) and 4℃ for 24 h. The activity of RbLam16 was improved by Mn2+ and Co2+; but greatly inhibited by Cu2+, EDTA and SDS . After 120 h cultivation with initial pH 6.0, at 28℃, with the methanol addition volume of 1.5% every 24 h, the activity of RbLam16 reached 27.42 U/mL, i.e. 26.42% higher than that of the control. The high catalytic activity, remarkable thermal stability and pH tolerance provide more possibilities for its application in food and energy fields.
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