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Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus
Ma, Jun-Yan1,2,3; Cao, Hai-Long1; Tan, Hai-Dong1; Hu, Xue-Jun2; Liu, Wu-Jun1; Du, Yu-Guang1,4; Yin, Heng1
2016
发表期刊APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
ISSN0273-2289
卷号178期号:1页码:144-158
摘要Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 +/- 0.6 U mL(-1) of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 A degrees C and an optimum pH of 4.5. It had a K (m) of 0.322 mM and a V (max) of 4317 mu M min(-1) mg(-1) protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 A degrees C for 1 h or at pH ranging from 3.0 to 6.0 at 4 A degrees C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase.
关键词Exoinulinase Kcinu1 Pichia Pastoris X-33 Site-directed Mutagenesis Zymogram Analysis
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine
DOI10.1007/s12010-015-1864-z
收录类别SCI
语种英语
关键词[WOS]CRYPTOCOCCUS-AUREUS G7A ; INULIN HYDROLYSIS ; EXTRACELLULAR INULINASE ; ASPERGILLUS-FICUUM ; SCHWANNIOMYCES-OCCIDENTALIS ; PURIFIED INULINASE ; CRUDE INULINASE ; ENDO-INULINASE ; EXO-INULINASE ; PURIFICATION
WOS研究方向Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS类目Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
项目资助者Chinese High-tech Research and Development program(2011AA10A205 ; National Key Laboratory of Biochemical Engineering(2012KF-06) ; Youth Innovation Promotion Association of Chinese Academy of Sciences ; 2014AA093511)
WOS记录号WOS:000368686200011
引用统计
被引频次:7[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://ir.ipe.ac.cn/handle/122111/20062
专题研究所(批量导入)
作者单位1.Chinese Acad Sci, Dalian Inst Chem Phys, Liaoning Prov Key Lab Carbohydrates, Nat Prod & Glycobiotechnol Res Grp, Dalian 116023, Peoples R China
2.Dalian Univ, Coll Med, Dalian 116022, Peoples R China
3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
4.Chinese Acad Sci, Inst Proc Engn, Beijing 100190, Peoples R China
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Ma, Jun-Yan,Cao, Hai-Long,Tan, Hai-Dong,et al. Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus[J]. APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY,2016,178(1):144-158.
APA Ma, Jun-Yan.,Cao, Hai-Long.,Tan, Hai-Dong.,Hu, Xue-Jun.,Liu, Wu-Jun.,...&Yin, Heng.(2016).Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus.APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY,178(1),144-158.
MLA Ma, Jun-Yan,et al."Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus".APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 178.1(2016):144-158.
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