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林产化学与工业 ›› 2021, Vol. 41 ›› Issue (5): 15-22.doi: 10.3969/j.issn.0253-2417.2021.05.003

• 重点研发专栏 • 上一篇    下一篇

伴侣蛋白提高木糖苷酶的可溶性表达及其协同酶解木聚糖

李琦, 朱文慧, 姜云鹏, 赵林果()   

  1. 南京林业大学 化学工程学院; 江苏省林业资源高效加工利用协同创新中心, 江苏 南京 210037
  • 收稿日期:2021-03-18 出版日期:2021-10-28 发布日期:2021-11-04
  • 通讯作者: 赵林果 E-mail:njfu2304@163.com
  • 作者简介:赵林果, 教授, 研究领域为酶工程和天然活性物质生物催化转化; E-mail: njfu2304@163.com
    李琦(1988-), 女, 江苏镇江人, 实验师, 博士, 研究方向为酶工程
  • 基金资助:
    国家重点研发计划资助项目(2017YFD0601001);南京林业大学大学生创新训练计划项目(2020NFUSPITP0080)

Enhancement of Soluble Expression of β-Xylosidase by Molecular Chaperone and Its Synergistic Enzymatic Hydrolysis of Xylan

Qi LI, Wenhui ZHU, Yunpeng JIANG, Linguo ZHAO()   

  1. Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
  • Received:2021-03-18 Online:2021-10-28 Published:2021-11-04
  • Contact: Linguo ZHAO E-mail:njfu2304@163.com

摘要:

通过分子伴侣蛋白共表达的方式,对6种商业化的分子伴侣蛋白进行筛选以提高β-木糖苷酶(Xln-DT)的可溶性表达量。研究结果表明:构建诱导型质粒pG-KJE8和含有xln-DT基因的重组表达质粒pET-28a-xln-DT共表达系统后,Xln-DT的可溶性表达量为原酶初始表达量的1.31倍;以质量分数2.0%甘油为碳源,质量分数1.2%蛋白胨为氮源,添加0.01 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)、5 μg/L四环素、1.5 g/L L-阿拉伯糖,32℃下诱导60 h,重组酶的表达量可达11.9 U/mL,较原酶提高了4.22倍。此外,利用内切木聚糖酶(XynB-DT)和Xln-DT协同酶解木聚糖,在添加Xln-DT后,榉木、桦木、玉米芯、燕麦和甘蔗渣5种木聚糖的酶解率分别为84.4%、90.2%、79.6%、77.3%和64.7%,相较于仅添加XynB-DT的酶解率有了显著的提高。

关键词: β-木糖苷酶, 分子伴侣, 可溶性表达, 协同酶解, 木聚糖

Abstract:

Based on the purpose of improving the soluble expression of β-xylosidase(Xln-DT) from Dictyoglomus thermophilum, six commercial chaperone proteins were screened by co-expression of chaperone proteins. The results showed that the soluble expression of Xln-DT was increased to 1.31 times of initial expression of proenzyme after the co-expression system of inducible plasmid pG-KJE8 and recombinant expression plasmid pet-28a-xln-DT containing xln-DT gene were constructed. The terrific-broth medium was induced by adding 0.01 mmol/L isopropylthio-β-D-galactoside(IPTG), 5 μg/L tetracycline, 1.5 g/L L-arabinose with 2.0% glycerol and 1.2% peptone as carbon and nitrogen sources, respectively. Finally, enzyme activity of Xln-DTwas 11.9 U/mL at 32℃ for 60 h, which was 4.22 times higher than thatof the original enzyme in LB medium. In addition, the process and enzymatic mode of 5 kinds of xylan with endoxylanase XynB-DT and β-xylosidase Xln-DT were investigated. After adding Xln-DT, the enzymatic hydrolysis yield of fivekinds of xylans (beechwood xylan, birchwood xylan, corncob xylan, oat xylan and bagasse xylan) were 84.4%, 90.2%, 79.6%, 77.3% and 64.7%, respectively, which was significantly improved compared with that only adding XynB-DT. All the results could not only significantly improve the soluble expression of Xln-DT, but also had a good application prospect in the production of xylooligosaccharides and xylose by enzymatic hydrolysis of xylan.

Key words: β-xylosidase, molecular chaperone, soluble expression, synergistic enzymolysis, xylan

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