Endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 (KCTC 13143): Expression of codon-optimized recombinant enzyme in Pichia pastoris
- Endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 (KCTC 13143): Expression of codon-optimized recombinant enzyme in Pichia pastoris
- Other Titles
- 코돈 최적화에 의한 피키아 파스토리스를 이용한 남극 유래 상귀박터 KOPRI 21702에서 분리된 엔도카이티네이즈의 재조합 발현
- Koh, Hye Yeon
Lee, Sung Gu
Yim, Joung Han
Lee, Hong Kum
- Antarctica; Codon optimization; Endochitinases; GlcNAc; psychrophilic
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- Koh, Hye Yeon, et al. 2009. Endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 (KCTC 13143): Expression of codon-optimized recombinant enzyme in Pichia pastoris. SCAR. SCAR. 2009.07.28~.
- Chitinases are environmentally important biocatalysts for N-acetyl-D-glucosamine (GlcNAc) production from chitin, which is the second most abundant and renewable polymer on earth, next to cellulose. Endochitinases have an enzymatic activity in random cleavage at internal sites in chitin. An endochitinase was previously purified and the gene was cloned from a psychrophilic Antarctic bacterium, Sanguibacter antarcticus (KCTC 13143). In the present study, recombinant endochitinase, rChi21702, was expressed using a yeast expression system (Pichia pastoris) and codon optimization. The expressed rChi21702 was purified by a Phenyl-Sepharose column chromatography. Optimal expression yielded 1-mg purified enzyme from 1-L bioreactor culture. When p-NP-(GlcNAc)2 was used as a substrate, the specific activity of the enzyme was determined to be 20 U per mg. In vitro assays and thin-layer chromatography demonstrated that the recombinant enzyme has endochitinase activity that produces diacetyl-chitobiose as a dominant end product as determined using chitooligomers, colloidal chitin, and the chromogenic p-NP-(GlcNAc)2 as substrates. Optimal activity for rChi21702 was observed at 37°C and a pH of 7.6. Interestingly, rChi21702 exhibited 63% of optimal activity at 10°C and 44% activity at 0°C. Taken together, the results indicate that rChi21702 kept psychrotolerant endochtinase activity even after recombinant expression in yeast cells.. Supported by Korea Polar Research Institute (PE09050).
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