Gene Expression, Biochemical Characterization of a sn-1, 3 Extracellular Lipase From Aspergillus niger GZUF36 and Its Model-Structure Analysis.
Gene Expression, Biochemical Characterization of a sn-1, 3 Extracellular Lipase From Aspergillus niger GZUF36 and Its Model-Structure Analysis.
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黑曲霉 GZUF36 sn-1, 3 胞外脂肪酶的基因表达、生化特征及其模型结构分析
DOI：
10.3389/fmicb.2021.633489
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发表时间：
2021
影响因子：
5.2
通讯作者：
He L
中科院分区：
文献类型：
--
作者：
Xing S;Zhu R;Cheng K;Cai Y;Hu Y;Li C;Zeng X;Zhu Q;He L
在本研究中，黑曲霉GZUF36胞外脂肪酶(PEXANL1)在巴斯德毕赤酵母中表达、鉴定，并对预测的结构模型进行了分析。优化后的培养条件表明，当甲醇浓度为1%，诱导时间为96h时，产酶活性最高，为66.5±1.4U/mL(P<；0.05)。纯化后的PEXANL1在pH为4.0、温度为40℃时活性最高，在大多数有机溶剂(20%v/v浓度)中活性保持不变。Triton X-100、Tween20、Tween80和浓度为0.01%(w/v)的十二烷基硫酸钠对纯化的PEXANL有促进作用，而所有被测的金属离子都对纯化的PEXANL有抑制作用。超声辅助PEXANL1催化合成1，3-二甘油三酯的结果表明，在超声作用25min时，1，3-二甘油酯的含量迅速增加到36.90%(P<；0.05)，当超声作用35min时，1，3-二甘油酯的含量下降到19.93%。比较模拟结果表明，PEXANL1的结构为α/β水解酶折叠。结构叠加和分子对接结果证实PEXANL1的Ser162、His274和Asp217残基参与了催化反应。小角X射线散射分析表明PEXANL1在溶液中具有单体性质。PEXANL1的从头计算模型与其模型结构重叠。本工作基于同源模型和小角X射线散射，提出了一个可靠的黑曲霉脂肪酶结构模型。此外，本研究的数据将为今后合理设计合适的晶体脂肪酶变异体提供依据。
In this study, a sn-1, 3 extracellular lipases from Aspergillus niger GZUF36 (PEXANL1) was expressed in Pichia pastoris, characterized, and the predicted structural model was analyzed. The optimized culture conditions of P. pastoris showed that the highest lipase activity of 66.5 ± 1.4 U/mL (P < 0.05) could be attained with 1% methanol and 96 h induction time. The purified PEXANL1 exhibited the highest activity at pH 4.0 and 40°C temperature, and its original activity remained unaltered in the majority of the organic solvents (20% v/v concentration). Triton X-100, Tween 20, Tween 80, and SDS at a concentration of 0.01% (w/v) enhanced, and all the metal ions tested inhibited activity of purified PEXANL. The results of ultrasound-assisted PEXANL1 catalyzed synthesis of 1,3-diaglycerides showed that the content of 1,3-diglycerides was rapidly increased to 36.90% with 25 min of ultrasound duration (P < 0.05) and later decreased to 19.93% with 35 min of ultrasound duration. The modeled structure of PEXANL1 by comparative modeling showed α/β hydrolase fold. Structural superposition and molecular docking results validated that Ser162, His274, and Asp217 residues of PEXANL1 were involved in the catalysis. Small-angle X-ray scattering analysis indicated the monomer properties of PEXANL1 in solution. The ab initio model of PEXANL1 overlapped with its modeling structure. This work presents a reliable structural model of A. niger lipase based on homology modeling and small-angle X-ray scattering. Besides, the data from this study will benefit the rational design of suitable crystalline lipase variants in the future.
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影响因子：
8
作者：
COLOVOS, C;YEATES, TO
通讯作者：
YEATES, TO
影响因子：
3.9
作者：
Antonetti, Claudia;Gori, Samuele;Galletti, Anna Maria Raspolli
通讯作者：
Galletti, Anna Maria Raspolli
影响因子：
6.1
作者：
Konarev, PV;Volkov, VV;Svergun, DI
通讯作者：
Svergun, DI
影响因子：
3.2
作者：
Dittrich, Nickel;Berrocal-Almanza, Luis C.;Conrad, Melanie L.
通讯作者：
Conrad, Melanie L.
影响因子：
6.1
作者：
Kozin, MB;Svergun, DI
通讯作者：
Svergun, DI