Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus

doi:10.1007/s12010-015-1864-z

	Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus
	Ma, Jun-Yan 2,3,4; Cao, Hai-Long 4; Tan, Hai-Dong 4; Hu, Xue-Jun 2; Liu, Wu-Jun 4; Du, Yu-Guang 1,4; Yin, Heng 4
刊名	APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
	2016
卷号	178 期号:1 页码:144-158
关键词	Exoinulinase Kcinu1 Pichia Pastoris X-33 Site-directed Mutagenesis Zymogram Analysis
ISSN号	0273-2289
DOI	10.1007/s12010-015-1864-z
文献子类	Article
英文摘要	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.
WOS关键词	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
语种	英语
出版者	HUMANA PRESS INC
WOS记录号	WOS:000368686200011
内容类型	期刊论文
源URL	[http://cas-ir.dicp.ac.cn/handle/321008/171426]
专题	大连化学物理研究所_中国科学院大连化学物理研究所
通讯作者	Du, Yu-Guang; Yin, Heng
作者单位	1.Chinese Acad Sci, Inst Proc Engn, Beijing 100190, 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, Dalian Inst Chem Phys, Liaoning Prov Key Lab Carbohydrates, Nat Prod & Glycobiotechnol Res Grp, Dalian 116023, Peoples R China
推荐引用方式 GB/T 7714	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.