High-level expression of β-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc
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β-N-Acetylglucosaminidases (NAGase) can remove N-acetylglucosamine (GlcNAc) from the non-reducing end of chitin or chitosan. GlcNAc has many important physiological functions in organism, which can be used for the treatment of rheumatoid arthritis clinically and be used as food antioxidant, infant food additive and diabetic sweetener. Thus, it is very important to develop genetic-engineering strains with high-yield NAGase to hydrolyze chitin into GlcNAc. Here, the NAGase gene of Bacillus subtilis 168 (BsnagZ) was synthesized according to the codon bias of Pichia pastoris and expressed in P. pastoris. The expression level of BsNagZ in P. pastoris increased over the induced time and the highest activity reached 0.76 U/mL at the 7th day. The recombinant BsNagZ was purified for characterization. The optimal temperature and pH are 60 °C and 6.0, respectively. It can both keep over 80% activities after pre-incubation at 55 °C for one hour and at 4 °C for 12 h from pH 4.5 to 10.0. To further improve the expression level of BsNagZ, a recombinant strain with four copy BsnagZs was screened using a high concentration of zeocin. The highest BsNagZ activity reached 3.2 U/mL at the 12th day, which was fourfold higher than that of single-copy strain. Combined with commercial chitinase CtnSg, GlcNAc can be produced by recombinant BsNagZ when used colloidal chitin as the substrate. Our study highlights that the NAGase was first successfully expressed in P. pastoris and GlcNAc can be produced via NAGase hydrolyzing the colloidal chitin.
Keywordsβ-N-Acetylglucosaminidase Pichia pastoris GlcNAc Colloidal chitin
This work was supported by the National Natural Science Foundation of China (31240008), Technical Support Program of Hubei Province (2015BCA271), and 2016 Wuhan Yellow Crane Talents (Science) Program.
- 2.Belén GF, Abigail FS, Jacobo LS, Carmen S (2015) A novel family 19 chitinase from the marine-derived Pseudoalteromonas tunicata CCUG 44952T: heterologous expression, characterization and antifungal activity. Biochem Eng J 93(15):84–93Google Scholar
- 4.Gohel V, Singh A, Vimal M, Ashwini P, Chhatpar HS (2006) Bioprospecting and antifungal potential of chitinolytic microorganisms. Afr J Biotechnol 5(2):54–72Google Scholar
- 15.Yem DW, Wu HC (1976) Purification and properties of β-N-acetylglucosaminidase from Escherichia coli. J Bacteriol 125(1):324–331Google Scholar
- 23.Tsujibo H, Fujimoto K, Miyamoto K, Imada C, Okami Y, Inamori Y (1995) Molecular cloning of the gene which encodes β-N-acetylglucosaminidase from a marine bacterium, Alteromonas sp. strain O-7. Appl Environ Microb 61(2):804–806Google Scholar
- 24.Keyhani NO, Wang LX, Lee YC, Rosema S (1996) The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Characterization of an N,N’-diacetyl-chitobiose transport system. J Biochem Mol Biol 271(52):33409–33413Google Scholar
- 26.Matsuo I, Kim S, Yamamoto Y, Ajisaka K, Maruyama JI, Nakajima H, Kitanmoto K (2003) Cloning and overexpression of β-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide. Biosci Biotechnol Biochem 67(3):646–650CrossRefGoogle Scholar
- 28.Berkeley RC, Brewer SJ, Oritiz JM, Gillespie JB (1973) An exo-β-N-acetylglucosaminidase from Bacillus subtilis; Characterization. Biosci Biotechnol Biochem 309(1):157–168Google Scholar