Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production
Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production
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臭豆腐盐水解淀粉芽孢杆菌新型碱性脂肪酶的功能表达及生物柴油生产固定化酶的开发
DOI：
10.1007/s10482-014-0274-5
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发表时间：
2014-11-01
影响因子：
2.6
通讯作者：
Wei, Wei
中科院分区：
文献类型：
--
作者：
Cai, Xianghai;Ma, Jing;Wei, Wei
采用富集方法，从臭豆腐卤水中分离到一株解脂芽孢杆菌（Bacillus amyloliquefaciens nsic8），经形态学、生理、生化及16S rDNA序列分析鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。同时，通过同源性分析，从Nsic-8中获得了参与酯代谢的关键酶基因lipBA。该基因具有645 bp的开放阅读框，编码214个氨基酸的脂肪酶（LipBA）。所得氨基酸序列与B. amyloliquefaciens IT-45 （NCBI数据库）的脂肪酶同源性最高，属于三酰基甘油脂肪酶家族（EC 3.1.1.3）。脂肪酶基因在大肠杆菌BL21（DE3）中以pET-28a质粒表达。酶活和比活分别为250±16 U/ml和1750±153 U/mg。重组酶最适pH为9.0℃，最适温度为40℃。LipBA在碱性条件下具有较高的稳定性，在pH 7.0 ~ 11.0范围内稳定。以4-硝基苯基棕榈酸酯为底物的LipBA的Km和Vmax分别为1.04±0.06 mM和119.05±7.16 μmol/(ml min)。纯化后的重组脂肪酶在最佳条件下（30℃，固定化时间3 h，酶回收率92%）进行固定化，并应用于生物柴油生产。初步研究表明，酯交换反应在生物柴油生产中具有潜在的应用价值。
Using enrichment procedures, a lipolytic strain was isolated from a stinky tofu brine and was identified as Bacillus amyloliquefaciens (named B. amyloliquefaciens Nsic-8) by morphological, physiological, biochemical tests and 16S rDNA sequence analysis. Meanwhile, the key enzyme gene (named lipBA) involved in ester metabolism was obtained from Nsic-8 with the assistance of homology analysis. The novel gene has an open reading frame of 645 bp, and encodes a 214-amino-acid lipase (LipBA). The deduced amino acid sequence shows the highest identity with the lipase from B. amyloliquefaciens IT-45 (NCBI database) and belongs to the family of triacylglycerol lipase (EC 3.1.1.3). The lipase gene was expressed in Escherichia coli BL21(DE3) using plasmid pET-28a. The enzyme activity and specific activity were 250 ± 16 U/ml and 1750 ± 153 U/mg, respectively. The optimum pH and temperature of the recombinant enzyme were 9.0 and 40 °C respectively. LipBA showed much higher stability under alkaline conditions and was stable at pH 7.0–11.0. The Km and Vmax values of purified LipBA using 4-nitrophenyl palmitate as the substrate were 1.04 ± 0.06 mM and 119.05 ± 7.16 μmol/(ml min), respectively. After purification, recombinant lipase was immobilized with the optimal conditions (immobilization time 3 h at 30 °C, with 92 % enzyme recovery) and the immobilized enzyme was applied in biodiesel production. Also the preliminary study of the transesterification shows the potential value in biodiesel production applications.