Tailoring pullulanase PulAR from Anoxybacillus sp. AR-29 for enhanced catalytic performance by a structure-guided consensus approach,Bioresources and Bioprocessing

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Tailoring pullulanase PulAR from Anoxybacillus sp. AR-29 for enhanced catalytic performance by a structure-guided consensus approach
Bioresources and Bioprocessing ( IF 4.3 ) Pub Date : 2022-03-21 , DOI: 10.1186/s40643-022-00516-4
Shu-Fang Li _{1,

2,

3} , Shen-Yuan Xu _{1,

2,

3} , Ya-Jun Wang _{1,

2,

3} , Yu-Guo Zheng _{1,

2,

3}

Affiliation

Pullulanase is a well-known debranching enzyme that can specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability and catalytic efficiency under industrial conditions. In the present study, four residues (A365, V401, H499, and T504) lining the catalytic pocket of Anoxybacillus sp. AR-29 pullulanase (PulAR) were selected for site-directed mutagenesis (SDM) by using a structure-guided consensus approach. Five beneficial mutants (PulAR-A365V, PulAR-V401C, PulAR-A365/V401C, PulAR-A365V/V401C/T504V, and PulAR-A365V/V401C/T504V/H499A) were created, which showed enhanced thermostability, pH stability, and catalytic efficiency. Among them, the quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- and 9.6-fold higher catalytic efficiency toward pullulan at 60 ℃, pH 6.0 and 5.0, respectively. In addition, its thermostabilities at 60 ℃ and 65 ℃ were improved by 2.6- and 3.1-fold, respectively, compared to those of the wild-type (WT). Meanwhile, its pH stabilities at pH 4.5 and 5.0 were 1.6- and 1.8-fold higher than those of WT, respectively. In summary, the catalytic performance of PulAR was significantly enhanced by a structure-guided consensus approach. The resultant quadruple mutant PulAR-A365V/V401C/T504V/H499A demonstrated potential applications in the starch industry.

Graphical Abstract

中文翻译：

定制来自 Anoxybacillus sp. 的支链淀粉酶 PulAR。 AR-29 通过结构引导共识方法增强催化性能

普鲁兰酶是一种众所周知的脱支酶，可以特异性水解淀粉和寡糖中的α-1,6-糖苷键，但其在工业条件下稳定性和催化效率较低。在本研究中，四个残基（A365、V401、H499 和 T504）排列在Anoxybacillus sp 的催化袋中。通过使用结构引导的共识方法，选择 AR-29 支链淀粉酶 (PulAR) 进行定点诱变 (SDM)。创建了五个有益突变体（PulAR-A365V、PulAR-V401C、PulAR-A365/V401C、PulAR-A365V/V401C/T504V 和 PulAR-A365V/V401C/T504V/H499A），它们表现出增强的热稳定性、pH 稳定性和催化能力效率。其中，四重突变体PulAR-A365V/V401C/T504V/H499A在60℃、pH 6.0和5.0下对支链淀粉的催化效率分别提高了6.6倍和9.6倍。此外，与野生型（WT）相比，其在60℃和65℃的热稳定性分别提高了2.6倍和3.1倍。同时，其在pH 4.5和5.0时的pH稳定性分别比WT高1.6和1.8倍。总之，结构引导共识方法显着增强了 PulAR 的催化性能。由此产生的四重突变体 PulAR-A365V/V401C/T504V/H499A 展示了在淀粉工业中的潜在应用。

图解摘要

更新日期：2022-03-21

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