PMID- 26227111 OWN - NLM STAT- MEDLINE DCOM- 20160310 LR - 20210405 IS - 1472-6750 (Electronic) IS - 1472-6750 (Linking) VI - 15 DP - 2015 Jul 31 TI - High-level expression of human arginase I in Pichia pastoris and its immobilization on chitosan to produce L-ornithine. PG - 66 LID - 10.1186/s12896-015-0184-2 [doi] LID - 66 AB - BACKGROUND: L-ornithine (L-Orn), is an intermediate metabolite in the urea cycle that plays a significant role in humans. L-Orn can be obtained from the catalysis of L-arginine (L-Arg) by arginase. The Pichia pastoris expression system offers the possibility of generating a large amount of recombinant protein. The immobilized enzyme technology can overcome the difficulties in recovery, recycling and long-term stability that result from the use of free enzyme. METHODS: The recombinant human arginase I (ARG I) was obtained using an optimized method with the Pichia pastoris GS115 as the host strain. Chitosan paticles were cross-linked with glutaraldehyde and rinsed exhaustively. Then the expressed ARG I was immobilized on the crosslinked chitosan particles, and the enzymatic properties of both the free and immobilized enzymes were evaluated. At last, the immobilized ARG I was employed to catalyze L-Arg to L-Orn. RESULTS: The results indicated that these two states both exhibited optimal activity under the same condition of pH10 at 40 degrees C. However, the immobilized ARG I exhibited the remarkable thermal and long-term stability as well as broad adaptability to pH, suggesting its potential for wide application in future industry. After a careful analysis of its catalytic conditions, immobilized ARG I was employed to catalyze the conversion of L-Arg to L-Orn under optimal condition of 1 % glutaraldehyde, 1 mM Mn(2+), 40 degrees C, pH10 and an L-arginine (L-Arg) concentration of 200 g/L, achieving a highly converted content of 149.g/L L-Orn. CONCLUSIONS: In this work, ARG Iota was abundantly expressed, and an efficient, facile and repeatable method was developed to synthesize high-quality L-Orn. This method not only solved the problem of obtaining a large amount of arginase, but also provided a promising alternative for the future industrial production of L-Orn. FAU - Zhang, Xue AU - Zhang X AD - Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China. snowprincess1989@163.com. FAU - Liu, Jin AU - Liu J AD - Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China. 664617377@qq.com. FAU - Yu, Xianhong AU - Yu X AD - Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China. kukuyu2012@hotmail.com. FAU - Wang, Fei AU - Wang F AD - Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China. 448244821@qq.com. FAU - Yi, Li AU - Yi L AD - Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China. li-yi@hotmail.com. FAU - Li, Zhezhe AU - Li Z AD - Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China. 598392746@qq.com. FAU - Liu, Yunyun AU - Liu Y AD - Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China. 514944228@qq.com. FAU - Ma, Lixin AU - Ma L AD - Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China. malixing@hubu.edu.cn. LA - eng PT - Journal Article DEP - 20150731 PL - England TA - BMC Biotechnol JT - BMC biotechnology JID - 101088663 RN - 0 (Enzymes, Immobilized) RN - 0 (Recombinant Proteins) RN - 9012-76-4 (Chitosan) RN - 94ZLA3W45F (Arginine) RN - E524N2IXA3 (Ornithine) RN - EC 3.5.3.1 (ARG1 protein, human) RN - EC 3.5.3.1 (Arginase) SB - IM MH - Arginase/*biosynthesis/genetics MH - Arginine/metabolism MH - Chitosan/chemistry MH - Enzymes, Immobilized/chemistry/genetics MH - Gene Expression Regulation, Enzymologic MH - Humans MH - Ornithine/*biosynthesis MH - Pichia/genetics MH - Recombinant Proteins/*biosynthesis/genetics PMC - PMC4521451 EDAT- 2015/08/01 06:00 MHDA- 2016/03/11 06:00 PMCR- 2015/07/31 CRDT- 2015/08/01 06:00 PHST- 2014/12/05 00:00 [received] PHST- 2015/07/24 00:00 [accepted] PHST- 2015/08/01 06:00 [entrez] PHST- 2015/08/01 06:00 [pubmed] PHST- 2016/03/11 06:00 [medline] PHST- 2015/07/31 00:00 [pmc-release] AID - 10.1186/s12896-015-0184-2 [pii] AID - 184 [pii] AID - 10.1186/s12896-015-0184-2 [doi] PST - epublish SO - BMC Biotechnol. 2015 Jul 31;15:66. doi: 10.1186/s12896-015-0184-2.