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毕赤酵母中米曲霉木聚糖酶 xAor 的异源表达。

Heterologous Expression of Xylanase xAor from Aspergillus oryzae in Komagataella phaffii T07.

机构信息

Laboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, Russia.

Kurchatov Genomic Center of the Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, Russia.

出版信息

Int J Mol Sci. 2022 Aug 5;23(15):8741. doi: 10.3390/ijms23158741.

Abstract

Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.

摘要

木聚糖酶(EC 3.2.1.8)水解植物细胞壁的半纤维素。木聚糖酶用于食品和造纸工业,并用于木质纤维素向生物燃料的生物转化。在这项工作中,带有四个 xAor 木聚糖酶基因副本的生产菌株被组织成两个串联副本,以在 T07 酵母中进行最佳表达。从培养基中通过 Sephadex G-25 凝胶过滤和 DEAE-Sepharose 6HF 阴离子交换色谱法从培养物中纯化分泌的 35 kDa 木聚糖酶。通过液相色谱-串联质谱法分析重组酶的肽,其中氨基酸序列与来自 RIB40 的内切 1,4-β-木聚糖酶的蛋白质访问号 # O94163 相对应。在生物反应器中生产重组木聚糖酶,分泌的酶以 258240 IU/mL 的活性水解燕麦木聚糖。在培养基中具有高活性表明木聚糖酶可以在不进行纯化或浓缩的情况下用于工业应用。木聚糖酶的最适 pH 为 7.5,但酶在 pH 2.5 至 pH 10 之间具有活性。木聚糖酶在 35°C 至 85°C 的温度下具有活性,在 60°C 时活性最高。总之,该方案产生适合工业规模生产的可溶性、分泌型木聚糖酶。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/bcfc/9369408/d3f1d3a9c2e1/ijms-23-08741-g001.jpg

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