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通过环替换提高来自RN.1的果胶酸裂解酶的最佳pH值和比活性。

Improvement of optimum pH and specific activity of pectate lyase from RN.1 using loop replacement.

作者信息

Li Piwu, Wei Xiaofeng, Wang Yun, Liu Hui, Xu Yanpeng, Zhang Ziyang, Li Junlin, Wang Jianbin, Guo Chuanzhuang, Sui Songsen, Wang Junqing, Wang Ruiming

机构信息

State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, China.

Department of Biological Engineering, Qilu University of Technology, Jinan, Shandong, China.

出版信息

Front Bioeng Biotechnol. 2023 Jul 4;11:1242123. doi: 10.3389/fbioe.2023.1242123. eCollection 2023.

DOI:10.3389/fbioe.2023.1242123
PMID:37469444
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10352616/
Abstract

Alkaline pectate lyase plays an important role in papermaking, biological refining and wastewater treatment, but its industrial applications are largely limited owing to its low activity and poor alkali resistance. The alkaline pectate lyase BspPel from RN.1 was heterologously expressed in BL21 (DE3) and its activity and alkali resistance were improved by loop replacement. Simultaneously, the effect of R260 on enzyme alkaline tolerance was also explored. Recombinant pectate lyase (BspPel-th) showed the highest activity at 60°C and pH 11.0, and showed significant stability over a wide pH range (3.0-11.0). The specific enzyme activity after purification was 139.4 U/mg, which was 4.4 times higher than that of the wild-type enzyme. BspPel-th has good affinity for apple pectin, since the and were 29 μmol/min. mL and 0.46 mol/L, respectively. Molecular dynamics simulation results showed that the flexibility of the loop region of BspPel-th was improved. The modified BspPel-th has considerable potential for industrial applications with high pH processes.

摘要

碱性果胶酸裂解酶在造纸、生物精炼和废水处理中发挥着重要作用，但其工业应用在很大程度上受到其低活性和耐碱性差的限制。从RN.1中获得的碱性果胶酸裂解酶BspPel在BL21(DE3)中进行了异源表达，并通过环置换提高了其活性和耐碱性。同时，还探究了R260对酶耐碱性的影响。重组果胶酸裂解酶(BspPel-th)在60℃和pH 11.0时表现出最高活性，并且在较宽的pH范围(3.0 - 11.0)内表现出显著的稳定性。纯化后的比酶活为139.4 U/mg，比野生型酶高4.4倍。BspPel-th对苹果果胶具有良好的亲和力，其 和 分别为29 μmol/min·mL和0.46 mol/L。分子动力学模拟结果表明，BspPel-th环区的柔韧性得到了改善。修饰后的BspPel-th在高pH值工艺的工业应用中具有相当大的潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/2e9ce64900e1/fbioe-11-1242123-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/8acade572573/fbioe-11-1242123-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/aac304517803/fbioe-11-1242123-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/3dd2a91c631d/fbioe-11-1242123-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/42c394b01b44/fbioe-11-1242123-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/2e9ce64900e1/fbioe-11-1242123-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/8acade572573/fbioe-11-1242123-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/aac304517803/fbioe-11-1242123-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/3dd2a91c631d/fbioe-11-1242123-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/42c394b01b44/fbioe-11-1242123-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfe1/10352616/2e9ce64900e1/fbioe-11-1242123-g006.jpg

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3
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