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.

Improved catalytic and antifungal activities ofBacillus thuringiensiscells with surface display of Chi9602ΔSP

“…After being displayed on the B. thuringiensis MB333 cell surface and cultured for 24 h, most cells were vegetative cells and the pH tolerance, thermostability and UV radiation resistance of Chi9602DSP-relative chitinase-specific activities were slightly improved compared with, which is similar to, the results observed by Chen et al (2012) (Chen et al 2012) and Li et al (2014) (Li et al 2014). In addition, the B. thuringiensis MB333 whole-cell chitinase activity is probably ascribed to fusion enzymes in the cell wall because they are exposed to the cell surface and more likely to come into contact with substrates.…”

“…() (Li et al . ). In addition, the B. thuringiensis MB333 whole‐cell chitinase activity is probably ascribed to fusion enzymes in the cell wall because they are exposed to the cell surface and more likely to come into contact with substrates.…”

Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H

“…Each gel lane was loaded with 10 µL of the mixture and the samples were separated as above. The gel was immersed in 50 mL 100 mM sodium acetate buffer (pH 6) containing 1% Triton X-100 and incubated overnight at 37°C, with gentle agitation, before staining with Coomassie brilliant blue R250, followed by destaining [31]. A SDS-PAGE under reducing conditions was run using glycosylated chitinase.…”

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications

“…It has been reported that biochemical and catalytic properties can be improved by immobilizing a protein on the yeast cell surface ( Shiraga et al, 2005 ; Li et al, 2014 ; Moura et al, 2015 ). Since the first YSD system was developed by Boder and Wittrup (1997) , this platform has been employed for the directed evolution of antibodies, peptides and proteins.…”