title: Expression of recombinant endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 in Pichia pastoris by codon optimization.
abstract: An endochitinase was previously purified and the gene was cloned from the 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 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 20U/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 when chitooligomers, colloidal chitin, and the chromogenic p-NP-(GlcNAc)(2) are used as substrates. Optimal activity for rChi21702 was observed at 37 degrees C and a pH of 7.6. Interestingly, rChi21702 exhibited 63% of optimal activity at 10 degrees C and 44% activity at 0 degrees C. Taken together, the results indicate that rChi21702 has psychrotolerant endochitinase activity even after recombinant expression in yeast cells.
authors.affiliation: Polar BioCenter, Korea Polar Research Institute, Incheon 406-840, South Korea.
authors.forename: Sung Gu
authors.initials: SG
authors.lastname: Lee
authors.forename: Hye Yeon
authors.initials: HY
authors.lastname: Koh
authors.forename: Se Jong
authors.initials: SJ
authors.lastname: Han
authors.forename: Heeyong
authors.initials: H
authors.lastname: Park
authors.forename: Deuk Chae
authors.initials: DC
authors.lastname: Na
authors.forename: Il-Chan
authors.initials: IC
authors.lastname: Kim
authors.forename: Hong Kum
authors.initials: HK
authors.lastname: Lee
authors.forename: Joung Han
authors.initials: JH
authors.lastname: Yim
mesh_terms: D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology*; D001483:Base Sequence; D019149:Bioreactors / Q000382:microbiology; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003062:Codon / Q000235:genetics*; D005821:Genetic Techniques; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D011995:Recombination, Genetic / Q000187:drug effects; D013696:Temperature
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D002688:Chitinases; D000432:Methanol