Expression of recombinant endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 in Pichia pastoris by codon optimization.
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.
Lee
Sung Gu
SG
Polar BioCenter, Korea Polar Research Institute, Incheon 406-840, South Korea.
Koh
Hye Yeon
HY
Han
Se Jong
SJ
Park
Heeyong
H
Na
Deuk Chae
DC
Kim
Il-Chan
IC
Lee
Hong Kum
HK
Yim
Joung Han
JH
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
D003062:Codon; D011994:Recombinant Proteins; D002688:Chitinases; D000432:Methanol