BMC Biotechnology | |
High-level expression of human arginase I in Pichia pastoris and its immobilization on chitosan to produce L-ornithine | |
Xue Zhang1  Jin Liu1  Xianhong Yu1  Fei Wang1  Li Yi1  Zhezhe Li1  Yunyun Liu1  Lixin Ma1  | |
[1] 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 | |
关键词: Transformation; L-arginine; Chitosan; Immobilization; Recombinant human arginase I; L-ornithine; | |
Others : 1223101 DOI : 10.1186/s12896-015-0184-2 |
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received in 2014-12-05, accepted in 2015-07-24, 发布年份 2015 | |
【 摘 要 】
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 °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 °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 Ι 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.
【 授权许可】
2015 Zhang et al.
【 预 览 】
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【 参考文献 】
- [1]Salvatore F, Cimino F, Maria C, Cittadioi D. Mechanism of the protection by L-ornithine-L-aspartate mixture and by L-arginine in ammonia intoxication. Arch Biochem Biophys. 1964;107:499–503.
- [2]Ruffio P. Amino acid products and technology. In: Business opportunity report Norwalk, Connecticut, USA. 1986.
- [3]Wan HG, Lu B, Cai H, Zhu QP, Wu QC. The preparation and application of L-ornithine. Chin J Bioprocess Eng. 2008;6:12–6.
- [4]Nesmeyanov AN, Freidlina RK, Petrova RG. Preparation of dl-proline and dl-ornithine from 1, 1, 1, 5-tetrachloropentane. Russ Chem Bull. 1957; 6:459-66.
- [5]Lee SY, Shin HS, Park JS, Kim YH, Min J. Proline reduces the binding of transcriptional regulator ArgR to upstream of argB in Corynebacterium glutamicum. Appl Microbiol Biotechnol. 2010;86:235-42
- [6]Bornscheuer UT. Immobilizing Enzymes: how to create more suitable biocatalysts. Angew Chem Int Ed. 2003; 42:3336-7.
- [7]Hanefeld U, Gardossib L, Magnerc E. Understanding enzyme immobilization. Chem Soc Rev. 2009; 38:453-68.
- [8]Beruter J, Colombo JP, Bachmann C. Purification and properties of arginase from human liver and erythrocytes. Biochem J. 1978; 175:449-54.
- [9]Bascur L, Cabello J, Veliz M, Gonzalez A. Molecular forms of human-liver arginase. Biochim Biophys Acta. 1966; 128:149-54.
- [10]Wheatley DN. Controlling cancer by restricting arginine availability-argininecatabolizing enzymes as anticancer agents. Anticancer Drugs. 2004; 15:825-33.
- [11]Dala E, Szajani B. Immobilization, characterization, and laboratory-scale application of bovine liver arginase. Appl Biochem Biotechnol. 1994; 49:203-15.
- [12]Patchett ML, Danie RM, Morgan HW. Characterisation of arginase from the extreme thermophile Bacillus caldovelox. Biochim Biophys Acta. 1991; 1077:291-8.
- [13]Soru E. Purification of bacterial arginase. J Chromatogr. 1965; 20:325-33.
- [14]McGee DJ, Zabaleta J, Viator RJ. Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily. Eur J Biochem. 2004; 271:1952-62.
- [15]Viator RJ, Rest RF, Hildebrandt E, McGee DJ. Characterization of Bacillus anthracis arginase: effects of pH, temperature, and cell viability on metal preference. BMC Biochem. 2008; 9:15. BioMed Central Full Text
- [16]Kanda M, Ohgishi K, Hanawa T, Saito Y. Arginase of Bacillus brevis Nagano: purification, properties, and implication in gramicidin S biosynthesis. Arch Biochem Biophys. 1997; 344:37-42.
- [17]Li PZ, Go XG, Arellano RO, Renugopalakrishnan V. Glycosylated and phosphorylated proteins—expression in yeast and oocytes of Xenopus: prospects and challenges—relevance to expression of thermostable proteins. Protein Express Purif. 2001; 22:369-80.
- [18]Woodley JM. Immobilized biocatalysts. Solid Supports Catal Org Synth. 1992;254–271.
- [19]Zeng J, Zheng LY. Studies on Penicillium sp.ZDZ1 chitosanase immobilized on chitin by cross-linking reaction. Process Biochem. 2008; 38:531-5.
- [20]Chen GQ, Choi I, Ramachandran B, Gouaux JE. Total gene synthesis: novel single step and convergent strategies applied to the construction of a 779 base pair bacteriorhodopsin gene. J Am Chem Soc. 1994; 11:8799-800.
- [21]Carr PA, Park JS, Lee YJ, Yu T, Zhang S, Jacobson JM. Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res. 2004; 32:162.
- [22]Cheng PN, Lam TL, Lam WM, Tsui SM, Cheng AW, Lo WH et al.. Pegylated recombinant human arginase (rhArg1-peg5, 000 mw) inhibits the in vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion. Cancer Res. 2007; 67:309-17.
- [23]Tsui SM, Lam WM, Lam TL, Chong HC, So PK, Kwok SY et al.. Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellularcarcinoma cell (HCC). Cancer Cell Int. 2009; 9:9. BioMed Central Full Text
- [24]Ikemoto M, Tabata M, Miyake T, Kono T, Mori M, Totani M et al.. Expression of human liver arginase in Escherichia coli. Purification and properties of the product. Biochem J. 1990; 270:697-703.
- [25]Stone EM, Glazer ES, Chantranupong L, Cherukuri P, Breece RM, Tierney DL et al.. Replacing Mn2+ with Co2+ in human arginase I enhances cytotoxicity toward L-arginine auxotrophic cancer cell lines. ACS Chem Bio. 2010; 5:333-42.
- [26]Zakalskiy AE, Zakalska OM, Rzhepetskyy YA, Potocka N, Stasyk OV, Horak D et al.. Overexpression of (His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography. Protein Expr Purif. 2012; 81:63-8.
- [27]Costanzo LD, Ilies M, Thorn KJ, Christianson DW. Inhibition of human arginase I by substrate and product analogues. Arch Biochem Biophys. 2010; 496:101-8.
- [28]Hwang JH, Kim HE, Cho DY. Isolation and properties of arginase from a shade plant, ginseng (Panax ginseng C.A.Meyer) roots. Phytochem. 2001; 58:1015-24.
- [29]Subrahmanyam TS, Reddy SR. L-Ornithine and L-lysine need their α-carboxyl groups for effective inhibition of bovine liver arginase. Indian J Biochem Biophys. 1986; 23:359-61.
- [30]Adriano WS, Filho EHC, Silva JA, Giordano RLC, Gonçalves LRB. Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan. Braz J Chem Eng. 2005; 22:529-38.
- [31]Zhan YP, Liu JZ, Mao PT, Zhang HJ, Liu Q, Jiao QC. Biotransformation of L-ornithine from L-arginine using whole-cell recombinant arginase. World J Microbiol Biotechnol. 2013; 29:2167-72.
- [32]Li M, Yang J, Qu H, Zhang Q, Bai F, Bai G. Novel immobilization of arginase I via cellulose–binding domain and its application in producing of L-Ornitine. Appl Biochem Microbiol. 2014; 50:43-8.
- [33]Zhang GM, Hu Y, Zhuang YH. Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris. Biocatal Biotransform. 2006; 24:371-9.
- [34]Shevchenko AJ, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem. 1996; 68:850-8.
- [35]Chinard FP. Photometric estimation of proline and ornithine. J Biol Chem. 1952; 199:91-5.
- [36]Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54.
- [37]Chiu SH, Chung TW, Giridhar R, Wu WT. Immobilization of β-cyclodextrin in chitosan beads for separation of cholesterol from egg yolk. Food Res Int. 2004; 37:217-23.
- [38]Gaffar R, Kermasha S, Bisakowski B. Biocatalysis of immobilized chlorophyllase in a ternary micellar system. J Biotechnol. 1999; 75:45-55.