Cloning, heterologous expression and characterization of β-glucosidase deriving from Moniliophthora perniciosa (Stahel) Aime and Phillips Mora.

IF 2.6 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY 3 Biotech Pub Date : 2024-11-01 DOI:10.1007/s13205-024-04128-x

Alison Borges Vitor, Keilane Silva Farias, Geise Camila Araújo Ribeiro, Carlos Priminho Pirovani, Raquel Guimarães Benevides, Gonçalo Amarante Guimarães Pereira, Sandra Aparecida de Assis

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Abstract

Β-glucosidase (BGLs) act synergistically with endoglucanases and exoglucanases and then are of great interest for biomass conversion into bioethanol. Thus, the aim of the current study is to produce a recombinant β-glycosidase from Moniliophtora perniciosa expressed in Escherichia coli cells. Enzyme coding sequence expression was confirmed through Sanger sequencing after using wheat bran (WB) and carboxymethylcellulose (CMC) as fungal growth media. Synthetic gene betaglyc-GH1 with optimized codons for E. coli expression was cloned in pET-28a. β-glucosidase recombinant (GH1chimera) was purified using a nickel column and its identity was confirmed through mass spectrometry. The recombinant enzyme presented an apparent molecular mass of 53.23 kDa on SDS-PAGE. Recombinant β-glucosidase has shown hydrolytic activity using p-nitrophenyl-β-D-glycopyranoside (pNPG) as substrate and maximum activity at pH 4.6 and 65 °C. Thus, the results indicate that the application of the GH1chimera in the hydrolysis of lignocellulosic materials to obtain glucose monomers can be efficient.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-024-04128-x.

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源自 Moniliophthora perniciosa (Stahel) Aime 和 Phillips Mora 的 β-葡萄糖苷酶的克隆、异源表达和特征描述。

Β-葡萄糖苷酶（BGLs）与内切葡聚糖酶和外切葡聚糖酶具有协同作用，因此在将生物质转化为生物乙醇方面具有重大意义。因此，本研究的目的是从大肠杆菌细胞中表达的钝顶莫尼利藻（Moniliophtora perniciosa）中产生一种重组β-糖苷酶。在使用麦麸（WB）和羧甲基纤维素（CMC）作为真菌生长培养基后，通过桑格测序确认了酶编码序列的表达。合成基因 betaglyc-GH1 已克隆到 pET-28a 中，并优化了大肠杆菌表达的密码子。用镍柱纯化了重组β-葡萄糖苷酶（GH1chimera），并通过质谱法确认了其身份。重组酶在 SDS-PAGE 上的表观分子质量为 53.23 kDa。以对硝基苯基-β-D-吡喃葡萄糖苷（pNPG）为底物，重组β-葡萄糖苷酶显示出水解活性，并在 pH 值为 4.6、温度为 65 ℃ 时活性最高。因此，研究结果表明，在水解木质纤维素材料以获得葡萄糖单体的过程中应用 GH1chimera 是有效的：在线版本包含补充材料，可查阅 10.1007/s13205-024-04128-x。

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来源期刊

3 Biotech Agricultural and Biological Sciences-Agricultural and Biological Sciences (miscellaneous)

CiteScore

6.00

自引率

0.00%

发文量

314

期刊介绍： 3 Biotech publishes the results of the latest research related to the study and application of biotechnology to: - Medicine and Biomedical Sciences - Agriculture - The Environment The focus on these three technology sectors recognizes that complete Biotechnology applications often require a combination of techniques. 3 Biotech not only presents the latest developments in biotechnology but also addresses the problems and benefits of integrating a variety of techniques for a particular application. 3 Biotech will appeal to scientists and engineers in both academia and industry focused on the safe and efficient application of Biotechnology to Medicine, Agriculture and the Environment.