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食品与发酵工业  2019, Vol. 45 Issue (1): 29-35    DOI: 10.13995/j.cnki.11-1802/ts.017756
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
黑曲霉内切β-1,4-半乳聚糖酶AghA的分子克隆与特征解析
蔡可1,王太康1,王君1,董自星2*,田康明2,金鹏2,刘晓光2,王正祥1, 2*
1(天津科技大学 生物工程学院,天津,300457)
2(天津科技大学 化工与材料学院,天津,300457)
Molecular cloning and biochemical characterization of endo-beta-1,4-galactanase AghA from Aspergillus niger
CAI Ke1,WANG Taikang1,WANG Jun1,DONG Zixing2*, TIAN Kangming2,JIN Peng2,LIU Xiaoguang2,WANG Zhengxiang1, 2*
1(College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457, China)
2(College of Chemical Engineering and Materials Science,Tianjin University of Science & Technology,Tianjin 300457, China)
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摘要 利用内切β-1,4-半乳聚糖酶(endo-β-1,4-galactanase,EC 3.2.1.89)水解土豆浆制备低聚半乳糖,有望解决产品回收率低的问题。为此,该研究通过分子克隆技术,将黑曲霉β-1,4-半乳聚糖酶基因aghA在毕赤酵母中进行克隆表达,构建获得了重组菌GS115(pPIC-aghA)。摇瓶培养条件下,重组酶AghA的酶活为130 U/mL,高于大多数已报道的半乳聚糖酶的酶活。酶学性质的研究表明,该酶的最适反应pH值和温度分别为4.5和45 ℃,且在pH 4.0~6.0或30~50 ℃具有较好稳定性。大部分金属离子和EDTA对重组酶AghA的酶活没有显著影响;Fe3+对其活性有强烈的抑制作用;而Hg2+则可使AghA几乎完全失活。该酶对半乳聚糖的最大反应速率Vmax和米氏常数Km分别为400 mg/(mL·min)和0.08 mg/mL。此外,重组酶AghA还可以水解土豆浆,产生半乳二糖、半乳三糖和少量半乳四糖等低聚半乳糖。
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蔡可
王太康
王君
董自星
田康明
金鹏
刘晓光
王正祥
关键词:  内切β-1,4-半乳聚糖酶  低聚半乳糖  黑曲霉  分子克隆  酶学性质    
Abstract: High yields of galactooligosaccharides may be obtained by enzymatic hydrolysis of potato pulp using endo-β-1,4-galactanase (EC 3.2.1.89). In present study, the gene aghA encoding endo-β-1,4-galactanase from Aspergillus niger CICIM F0510 was thus cloned and expressed in Pichia pastoris, which generated the recombinant strain GS115 (pPIC-aghA). In shaking flask experiments, the activity of recombinant enzyme AghA was 130 U/mL. The optimal pH and temperature of AghA were 4.5 and 45 ℃, respectively, and it was stable at pH 4.0-6.0 or at 30-50 ℃. Most of chemical ions and EDTA had no significant effects on the activity of AghA, while it was strongly inhibited by Fe3+ and nearly completely lost in the presence of Hg2+. The Vmax and Km of AghA towards galactan (potato) were determined to be 400 mg/(mL·min) and 0.08 mg/mL, respectively. Besides, potato pulp was hydrolyzed by AghA into galactobiose, galactotrioses and a small amount of galactotetraose. The results obtained paved the way for large scale production of galactanase AghA and its applications in preparing galactooligosaccharides from potato pulp.
Key words:  endo-β-1,4-galactanase    galactooligosaccharides    Aspergillus niger    molecular cloning    enzymatic properties
收稿日期:  2018-05-12                出版日期:  2019-01-15      发布日期:  2019-02-01      期的出版日期:  2019-01-15
基金资助: 天津市高等学校创新团队培养计划“酶与生物催化关键技术”(TD12-5002)
作者简介:  硕士研究生(董自星博士和王正祥教授为共同通讯作者,E-mail:dzx@tust.edu.cn,zxwang0519@tust.edu.cn)。
引用本文:    
蔡可,王太康,王君,等. 黑曲霉内切β-1,4-半乳聚糖酶AghA的分子克隆与特征解析[J]. 食品与发酵工业, 2019, 45(1): 29-35.
CAI Ke,WANG Taikang,WANG Jun,et al. Molecular cloning and biochemical characterization of endo-beta-1,4-galactanase AghA from Aspergillus niger[J]. Food and Fermentation Industries, 2019, 45(1): 29-35.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.017756  或          http://sf1970.cnif.cn/CN/Y2019/V45/I1/29
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