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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
Source PublicationAPPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
ISSN0273-2289
Volume178Issue:1Pages:144-158
AbstractInulinase 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.
KeywordExoinulinase Kcinu1 Pichia Pastoris X-33 Site-directed Mutagenesis Zymogram Analysis
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
DOI10.1007/s12010-015-1864-z
Indexed BySCI
Language英语
WOS KeywordCRYPTOCOCCUS-AUREUS G7A ; INULIN HYDROLYSIS ; EXTRACELLULAR INULINASE ; ASPERGILLUS-FICUUM ; SCHWANNIOMYCES-OCCIDENTALIS ; PURIFIED INULINASE ; CRUDE INULINASE ; ENDO-INULINASE ; EXO-INULINASE ; PURIFICATION
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS SubjectBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
Funding OrganizationChinese 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 IDWOS:000368686200011
Citation statistics
Cited Times:10[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/20062
Collection研究所(批量导入)
Affiliation1.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
Recommended Citation
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.
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