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Characterization of a NaCl-tolerant β-N-acetylglucosaminidase from Sphingobacterium sp. HWLB1

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Abstract

β-N-Acetylglucosaminidases serve important biological functions and various industrial applications. A glycoside hydrolase family 3 β-N-acetylglucosaminidase gene was cloned from Sphingobacterium sp. HWLB1 and expressed in Escherichia coli BL21 (DE3). The purified recombinant enzyme (rNag3HWLB1) showed apparent optimal activity at pH 7.0 and 40 °C. In the presence of 0.5–20.0 % (w/v) NaCl, the activity and stability of rNag3HWLB1 were slightly affected or not affected. The enzyme could even retain 73.6 % activity when 30.0 % (w/v) NaCl was added to the reaction mixture. The half-life of the enzyme was approximately 10 min at 37 °C without the addition of NaCl. However, the enzyme was stable at 37 °C in the presence of 3.0 % (w/v) NaCl. A large negatively charged surface in the catalytic pocket of the enzyme was observed and might contribute to NaCl tolerance and thermostability improvement. The degree of synergy between a commercial endochitinase and rNag3HWLB1 on chitin enzymatic degradation ranged from 3.11 to 3.74. This study is the first to report the molecular and biochemical properties of a NaCl-tolerant β-N-acetylglucosaminidase.

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References

  • Bacik JP, Whitworth GE, Stubbs KA, Vocadlo DJ, Mark BL (2012) Active site plasticity within the glycoside hydrolase NagZ underlies a dynamic mechanism of substrate distortion. Chem Biol 19:1471–1482

    Article  CAS  PubMed  Google Scholar 

  • Choi H-A, Lee S-S (2012) Sphingobacterium kyonggiense sp. nov., isolated from chloroethene-contaminated soil, and emended descriptions of Sphingobacterium daejeonense and Sphingobacterium mizutaii. Int J Syst Evol Micr 62:2559–2564

    Article  CAS  Google Scholar 

  • Choi KH, Seo JY, Park KM, Park CS, Cha J (2009) Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana. J Biosci Bioeng 108:455–459

    Article  CAS  PubMed  Google Scholar 

  • Cody RM, Davis ND, Lin J, Shaw D (1990) Screening microorganisms for chitin hydrolysis and production of ethanol from amino sugars. Biomass 21:285–295

    Article  CAS  Google Scholar 

  • da Silva Junior Sobrinho I, Bataus LAM, Maitan VR, Ulhoa CJ (2005) Purification and properties of an N-acetylglucosaminidase from Streptomyces cerradoensis. Biotechnol Lett 27:1273–1276

    Article  Google Scholar 

  • De Marco JL, Valadares-Inglis MC, Felix CR (2004) Purification and characterization of an N-acetylglucosaminidase produced by a Trichoderma harzianum strain which controls Crinipellis perniciosa. Appl Microbiol Biotechnol 64:70–75

    Article  Google Scholar 

  • Fukamizo T, Kramer KJ (1985) Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem 15:1–7

    Article  CAS  Google Scholar 

  • Herlihey FA, Moynihan PJ, Clarke AJ (2014) The essential protein for bacterial flagella formation FlgJ functions as a β-N-acetylglucosaminidase. J Biol Chem 289:31029–31042

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hung KS, Liu SM, Tzou WS, Lin FP, Pan CL, Fang TY, Sun KH, Tang SJ (2011) Characterization of a novel GH10 thermostable, halophilic xylanase from the marine bacterium Thermoanaerobacterium saccharolyticum NTOU1. Process Biochem 46:1257–1263

    Article  CAS  Google Scholar 

  • Li H, Morimoto K, Katagiri N, Kimura T, Sakka K, Lun S, Ohmiya K (2002) A novel β-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose. Appl Microbiol Biotechnol 60:420–427

    Article  CAS  PubMed  Google Scholar 

  • Litzinger S, Duckworth A, Nitzsche K, Risinger C, Wittmann V, Mayer C (2010a) Muropeptide rescue in Bacillus subtilis involves sequential hydrolysis by β-N-acetylglucosaminidase and N-acetylmuramyl-l-alanine amidase. J Bacteriol 192:3132–3143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Litzinger S, Fischer S, Polzer P, Diederichs K, Welte W, Mayer C (2010b) Structural and kinetic analysis of Bacillus subtilis N-acetylglucosaminidase reveals a unique Asp-His dyad mechanism. J Biol Chem 285:35675–35684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu XS, Huang ZQ, Zhang XN, Shao ZZ, Liu ZD (2014) Cloning, expression and characterization of a novel cold-active and halophilic xylanase from Zunongwangia profunda. Extremophiles 18:441–450

    Article  CAS  PubMed  Google Scholar 

  • Lombard V, Ramulu HG, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495

    Article  CAS  PubMed  Google Scholar 

  • Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4:91–98

    Article  CAS  PubMed  Google Scholar 

  • Matsuo I, Kim S, Yamamoto Y, Ajisaka K, Maruyama J, Nakajima H, Kitamoto 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 67:646–650

    Article  CAS  PubMed  Google Scholar 

  • Mayer C, Vocadlo DJ, Mah M, Rupitz K, Stoll D, Warren RAJ, Withers SG (2006) Characterization of a β-N-acetylhexosaminidase and a β-N-acetylglucosaminidase/β-glucosidase from Cellulomonas fimi. FEBS J 273:2929–2941

    Article  CAS  PubMed  Google Scholar 

  • Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  • Minegishi H, Shimane Y, Echigo A, Ohta Y, Hatada Y, Kamekura M, Maruyama T, Usami R (2013) Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A. Extremophiles 17:931–939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miyashita Y, Ohmae E, Nakasone K, Katayanagi K (2015) Effects of salt on the structure, stability, and function of a halophilic dihydrofolate reductase from a hyperhalophilic archaeon, Haloarcula japonica strain TR-1. Extremophiles 19:479–493

    Article  CAS  PubMed  Google Scholar 

  • Molina-Guijarro JM, Perez J, Munoz-Dorado J, Guillen F, Moya R, Hernandez M, Arias ME (2009) Detoxification of azo dyes by a novel pH-versatile, salt-resistant laccase from Streptomyces ipomoea. Int Microbiol 12:13–21

    CAS  PubMed  Google Scholar 

  • Nieder V, Kutzer M, Kren V, Gallego RG, Kamerling JP, Elling L (2004) Screening and characterization of β-N-acetylhexosaminidases for the synthesis of nucleotide-activated disaccharides. Enzyme Microb Technol 34:407–414

    Article  CAS  Google Scholar 

  • Ogawa M, Kitagawa M, Tanaka H, Ueda K, Watsuji T, Beppu T, Kondo A, Kawachi R, Oku T, Nishio T (2006) A β-N-acetylhexosaminidase from Symbiobacterium thermophilum; gene cloning, overexpression, purification and characterization. Enzyme Microb Technol 38:457–464

    Article  CAS  Google Scholar 

  • Okada S, Obrien JS (1969) Tay-Sachs disease: generalized absence of a β-D-N-acetylhexosaminidase component. Science 165:698–700

    Article  CAS  PubMed  Google Scholar 

  • Park JK, Kim WJ, Park YI (2011) Purification and characterization of an exo-type β-N-acetylglucosaminidase from Pseudomonas fluorescens JK-0412. J Appl Microbiol 110:277–286

    Article  CAS  PubMed  Google Scholar 

  • Patil RS, Ghormade V, Deshpande MV (2000) Chitinolytic enzymes: an exploration. Enzyme Microb Technol 26:473–483

    Article  CAS  PubMed  Google Scholar 

  • Paul S, Bag SK, Das S, Harvill ET, Dutta C (2008) Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes. Genome Biol 9:R70

    Article  PubMed  PubMed Central  Google Scholar 

  • Premikumar L, Greenblatt HM, Bageshwar UK, Savchenko T, Gokhman I, Sussman JL, Zamir A (2005) Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog. Proc Natl Acad Sci USA 102:7493–7498

    Article  Google Scholar 

  • Qin YJ, Huang ZQ, Liu ZD (2014) A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning, heterologous expression and biochemical characterization. Extremophiles 18:271–281

    Article  CAS  PubMed  Google Scholar 

  • Rajnochova E, Dvorakova J, Hunkova Z, Kren V (1997) Reverse hydrolysis catalysed by β-N-acetylhexosaminidase from Aspergillus oryzae. Biotechnol Lett 19:869–872

    Article  CAS  Google Scholar 

  • Richard SB, Madern D, Garcin E, Zaccai G (2000) Halophilic adaptation: novel solvent protein interactions observed in the 2.9 and 2.6 Å resolution structures of the wild type and a mutant of malate dehydrogenase from Haloarcula marismortui. Biochemistry 39:992–1000

    Article  CAS  PubMed  Google Scholar 

  • Shi RR, Li ZM, Ye Q, Xu JH, Liu Y (2013) Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresour Technol 142:338–344

    Article  CAS  PubMed  Google Scholar 

  • Siroosi M, Amoozegar MA, Khajeh K, Fazeli M, Rezaei MH (2014) Purification and characterization of a novel extracellular halophilic and organic solvent-tolerant amylopullulanase from the haloarchaeon, Halorubrum sp. strain Ha25. Extremophiles 18:25–33

    Article  CAS  PubMed  Google Scholar 

  • Suzuki K, Sugawara N, Suzuki M, Uchiyama T, Katouno F, Nikaidou N, Watanabe T (2002) Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation. Biosci Biotechnol Biochem 66:1075–1083

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  PubMed  Google Scholar 

  • Tokunaga H, Arakawa T, Fukada H, Tokunaga M (2006) Opposing effects of NaCl on reversibility and thermal stability of halophilic β-lactamase from a moderate halophile, Chromohalobacter sp. 560. Biophys Chem 119:316–320

    Article  CAS  PubMed  Google Scholar 

  • Tsujibo H, Hatano N, Mikami T, Hirasawa A, Miyamoto K, Inamori Y (1998) A novel β-N-acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: gene cloning, expression, and assignment to family 3 of the glycosyl hydrolases. Appl Environ Microbiol 64:2920–2924

    CAS  PubMed  PubMed Central  Google Scholar 

  • Vyas P, Deshpande M (1991) Enzymatic hydrolysis of chitin by Myrothecium verrucaria chitinase complex and its utilization to produce SCP. J Gen Appl Microbiol 37:267–275

    Article  CAS  Google Scholar 

  • Wu GJ, Wu GB, Zhan T, Shao ZZ, Liu ZD (2013) Characterization of a cold-adapted and salt-tolerant esterase from a psychrotrophic bacterium Psychrobacter pacificensis. Extremophiles 17:809–819

    Article  CAS  PubMed  Google Scholar 

  • Yamaguchi R, Tokunaga H, Ishibashi M, Arakawa T, Tokunaga M (2011) Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians. Appl Microbiol Biotechnol 89:673–684

    Article  CAS  PubMed  Google Scholar 

  • Yang SQ, Song S, Yan QJ, Fu X, Jiang ZQ, Yang XB (2014) Biochemical characterization of the first fungal glycoside hydrolyase family 3 β-N-acetylglucosaminidase from Rhizomucor miehei. J Agric Food Chem 62:5181–5190

    Article  CAS  PubMed  Google Scholar 

  • Zhang GM, Li SY, Xue YF, Mao LW, Ma YH (2012) Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp. BG-CS10. Extremophiles 16:35–43

    Article  CAS  PubMed  Google Scholar 

  • Zhou JP, Zhang R, Gao YJ, Li JJ, Tang XH, Mu YL, Wang F, Li C, Dong YY, Huang ZX (2012) Novel low-temperature-active, salt-tolerant and proteases-resistant endo-1,4-β-mannanase from a new Sphingomonas strain. J Biosci Bioeng 113:568–574

    Article  CAS  PubMed  Google Scholar 

  • Zhou JP, Lu Q, Peng MZ, Zhang R, Mo MH, Tang XH, Li JJ, Xu B, Ding JM, Huang ZX (2015a) Cold-active and NaCl-tolerant exo-inulinase from a cold-adapted Arthrobacter sp. MN8 and its potential for use in the production of fructose at low temperatures. J Biosci Bioeng 119:267–274

    Article  CAS  PubMed  Google Scholar 

  • Zhou JP, Shen JD, Zhang R, Tang XH, Li JJ, Xu B, Ding JM, Gao YJ, Xu DY, Huang ZX (2015b) Molecular and biochemical characterization of a novel multidomain xylanase from Arthrobacter sp. GN16 isolated from the feces of Grus nigricollis. Appl Biochem Biotechnol 175:573–588

    Article  CAS  PubMed  Google Scholar 

  • Zhou JP, Lu Q, Zhang R, Wang YY, Wu Q, Li JJ, Tang XH, Xu B, Ding JM, Huang ZX (2016) Characterization of two glycoside hydrolase family 36 α-galactosidases: novel transglycosylation activity, lead–zinc tolerance, alkaline and multiple pH optima, and low-temperature activity. Food Chem 194:156–166

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 31260215), Yunling Scholars (No. 2015 56), Yunling Industry Leading Talents (No. 2014 1782), Reserve Talents Project for Young and Middle-Aged Academic and Technical Leaders of Yunnan Province (No. 2015HB033), and Applied and Basic Research Foundation of Yunnan Province (No. 201401PC00224).

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Correspondence to Zunxi Huang.

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Communicated by S. Albers.

J. Zhou and Z. Song contributed equally to this work.

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Zhou, J., Song, Z., Zhang, R. et al. Characterization of a NaCl-tolerant β-N-acetylglucosaminidase from Sphingobacterium sp. HWLB1. Extremophiles 20, 547–557 (2016). https://doi.org/10.1007/s00792-016-0848-4

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