title: Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display.
abstract: To develop a novel yeast whole-cell biocatalyst by yeast surface display technology that can hydrolyze chitin, the chitinaseC gene from Serratia marcescens AS1.1652 strain was cloned and subcloned into the yeast surface display plasmid pYD1, and the recombinant plasmid pYD1/SmchiC was electroporated into Saccharomyces cerevisiae EBY100 cell. Aga2p-SmChiC fusion protein was expressed and anchored on the yeast cell surface by induction with galactose, which was verified by indirect immunofluorescence and Western blotting. The chitinolytic activity of the yeast whole-cell biocatalyst or partially purified enzyme was detected by agar plate clear zone test, SDS-PAGE zymography and dinitrosalicylic acid method. The results showed that the chitinaseC gene from S. marcescens AS1.1652 strain was successfully cloned and expressed on the yeast cell surface, Aga2p-SmChiC fusion protein with molecular weight (67 kDa) was determined. Tests on the effect of temperature and pH on enzyme activity and stability revealed that the yeast whole-cell biocatalyst and partially purified enzyme possessed both thermal stability and activity, and even maintained some activity under acidic and weakly alkaline conditions. The optimum reaction temperature and pH value were set at 52 °C and 5.0, respectively. Yeast surface display technology succeeded in preparing a yeast whole-cell biocatalyst with chitinolytic activity, and the utilization of chitin could benefit from this process of enzyme preparation.
authors.affiliation: School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515, People's Republic of China, gylxb2001@gmail.com.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Li
authors.forename: Xiaobao
authors.initials: X
authors.lastname: Jin
authors.forename: Xuemei
authors.initials: X
authors.lastname: Lu
authors.forename: Fujiang
authors.initials: F
authors.lastname: Chu
authors.forename: Juan
authors.initials: J
authors.lastname: Shen
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.forename: Manyu
authors.initials: M
authors.lastname: Liu
authors.forename: Jiayong
authors.initials: J
authors.lastname: Zhu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002473:Cell Wall / Q000378:metabolism; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D018274:Electroporation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012706:Serratia marcescens / Q000201:enzymology*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D002688:Chitinases; C119825:chitinase C