Recombinant expression and characterization of cold-adapted chitinase genes in Kluyveromyces lactis

Abstract:

[Objective] We aimed to construct recombinant Kluyveromyces lactis (K. lactis) strains to produce cold-adapted chitinases (chitinase A, chiA; chitinase C, chiC) of Pseudoalteromonas sp. DL-6. We purified and characterized the recombinant proteins. [Methods] We synthesized optimized-codons chitinase genes. The recombinant K. lactis expression plasmids (pKLAC1-chiA, pKLAC1-chiC) were constructed successfully and transformed into K. lactis by electric field pulses to achieve soluble expression of cold-adapted chitinases. The recombinant proteins were further purified using Ni-NTA chromatography. [Results] The recombinant K. lactis producing cold-adapted chitinases was successfully constructed and obtained high purity. The purified recombinant proteins, ChiA and ChiC, appeared as a single band of approximately 110 kDa and 90 kDa by SDS-PAGE, respectively. The activities of ChiA and ChiC were 51.45 U/mg and 108.56 U/mg measured by Schales' method. The optimum temperature was 20℃ and 30℃, and the optimum pH was 8.0 and 9.0, respectively. ChiA and ChiC were stable at pH 8.0-12.0 and below 40℃. ChiA and ChiC have obvious degradation activity to colloidal chitin and powdered chitin including α-chitin and β-chitin. Besides, they acted synergistically to efficiently degrade chitin substrates. [Conclusion] The expression, purification, enzymatic properties and degradation products of cold-adapted chitinases in K. lactis were achieved for the first time. These research results provide reference for the study of other cold-adapted chitinases.

Key words:cold-adapted chitinase;Kluyveromyces lactis;purification;enzymological characterization