文献类型：期刊文献

英文题名：Protein engineering of a cold-adapted rhamnogalacturonan acetylesterase: In vivo functional expression and cinnamyl acetate synthesis

作者：Tang, Xu-dong[1];Dong, Feng-ying[1];Zhang, Qi-hao[1];Lin, Lin[2,3];Wang, Ping[4];Xu, Xiang-yang[5];Wei, Wei[1];Wei, Dong-zhi[1]

机构：[1]East China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Shanghai Univ Med & Hlth Sci, Shanghai Key Lab Mol Imaging, Shanghai 201318, Peoples R China;[3]Natl Engn Res Ctr Nanotechnol, Res Lab Funct Nanomat, Shanghai 200241, Peoples R China;[4]Weigao Shanghai R&D Ctr, Shanghai 201203, Peoples R China;[5]Zaozhuang Jienuo Enzyme Co Ltd, Zaozhuang 277100, Peoples R China

年份：2021

卷号：107

起止页码：129

外文期刊名：PROCESS BIOCHEMISTRY

收录：;EI(收录号：20212310454697);WOS:【SCI-EXPANDED(收录号:WOS:000661406800005)】；

语种：英文

外文关键词：Protein engineering; Rhamnogalacturonan acetylesterase; Paenibacillus polymyxa; Cold-adapted; Cinnamyl acetate

摘要：The rhamnogalacturonan acetylesterase gene (named pp1113), encoding a novel cold-adapted rhamnogalacturonan acetylesterase, was cloned from Paenibacillus polymyxa. pp1113 was functionally expressed in Escherichia coli by inducing expression at 20 degrees C for 16 h. The pH and temperature optimums for the recombinant enzyme were 8.5 and 30 degrees C, respectively. As an SGNH-type acetylesterase, pp1113 demonstrated high activity against acetyl ester substrates and could maintain approximately 60 % activity at 0 degrees C.The specific activity of purified pp1113 was 65.95 U/mg (C2). The Km and Vmax values of purified pp1113 were 3.89 mM and 81.07 & micro;mol/min, respectively. We obtained an R232L variant with improved thermal stability and catalytic activity through site-directed mutagenesis. T1/2 of R232L at 50 degrees C increased from less than 30 min to 6 h when compared to wild-type pp1113, and T1/2 extended to 8 h after immobilization. The R232L mutant improved the conversion rate of 0.1 M cinnamyl alcohol at 40 degrees C to 94 % in the reaction of synthesis of cinnamyl acetate. After purification, the R232L mutant was immobilized (pH 8.0 phosphate buffer 0.5 M, 20 h at 20 degrees C, protein loading reached 17 mg/g). This was the first report of the rhamnogalacturonan acetylesterase gene obtained from Paenibacillus polymyxa with detailed enzymatic properties.