Lee Sung Gu, Koh Hye Yeon, Han Se Jong, Park Heeyong, Na Deuk Chae, Kim Il-Chan, Lee Hong Kum, Yim Joung Han
Polar BioCenter, Korea Polar Research Institute, Incheon 406-840, South Korea.
Protein Expr Purif. 2010 May;71(1):108-14. doi: 10.1016/j.pep.2010.01.017. Epub 2010 Jan 25.
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.
一种内切几丁质酶先前已被纯化，其基因是从嗜冷南极细菌南极血杆菌（KCTC 13143）中克隆得到的。在本研究中，使用酵母表达系统（巴斯德毕赤酵母）并通过密码子优化来表达重组内切几丁质酶rChi21702。表达的rChi21702通过苯基琼脂糖柱色谱法进行纯化。最佳表达从1升生物反应器培养物中产生了1毫克纯化酶。当使用对硝基苯基 - （N - 乙酰葡糖胺）2作为底物时，该酶的比活性测定为20U/mg。体外测定和薄层色谱表明，当以几丁寡糖、胶体几丁质和发色底物对硝基苯基 - （N - 乙酰葡糖胺）2为底物时，该重组酶具有内切几丁质酶活性，其主要终产物为二乙酰壳二糖。rChi21702在37℃和pH 7.6时观察到最佳活性。有趣的是，rChi21702在10℃时表现出63%的最佳活性，在0℃时表现出44%的活性。综上所述，结果表明即使在酵母细胞中重组表达后，rChi21702仍具有耐低温内切几丁质酶活性。
