title: Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance.
abstract: Enzymatic degradation of chitin has attracted substantial attention because chitin is an abundant renewable natural resource, second only to lignocellulose, and because of the promising applications of N-acetylglucosamine in the bioethanol, food and pharmaceutical industries. However, the low activity and poor tolerance to salts and N-acetylglucosamine of most reported β-N-acetylglucosaminidases limit their applications. Mining for novel enzymes from new microorganisms is one way to address this problem.
  
  
  
  A glycoside hydrolase family 20 (GH 20) β-N-acetylglucosaminidase (GlcNAcase) was identified from Microbacterium sp. HJ5 harboured in the saline soil of an abandoned salt mine and was expressed in Escherichia coli. The purified recombinant enzyme showed specific activities of 1773.1 ± 1.1 and 481.4 ± 2.3 μmol min-1mg-1towards p-nitrophenyl β-N-acetylglucosaminide and N,N'-diacetyl chitobiose, respectively, a Vmaxof 3097 ± 124 μmol min-1mg-1towards p-nitrophenyl β-N-acetylglucosaminide and a Kiof 14.59 mM for N-acetylglucosamine inhibition. Most metal ions and chemical reagents at final concentrations of 1.0 and 10.0 mM or 0.5 and 1.0% (v/v) had little or no effect (retaining 84.5 - 131.5% activity) on the enzyme activity. The enzyme can retain more than 53.6% activity and good stability in 3.0-20.0% (w/v) NaCl. Compared with most GlcNAcases, the activity of the enzyme is considerably higher and the tolerance to salts and N-acetylglucosamine is much better. Furthermore, the enzyme had higher proportions of aspartic acid, glutamic acid, alanine, glycine, random coils and negatively charged surfaces but lower proportions of cysteine, lysine, α-helices and positively charged surfaces than its homologs. These molecular characteristics were hypothesised as potential factors in the adaptation for salt tolerance and high activity of the GH 20 GlcNAcase.
  
  
  
  Biochemical characterization revealed that the GlcNAcase had novel salt-GlcNAc tolerance and high activity. These characteristics suggest that the enzyme has versatile potential in biotechnological applications, such as bioconversion of chitin waste and the processing of marine materials and saline foods. Molecular characterization provided an understanding of the molecular-function relationships for the salt tolerance and high activity of the GH 20 GlcNAcase.
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Junpei
authors.initials: J
authors.lastname: Zhou
authors.affiliation: College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Song
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Junjun
authors.initials: J
authors.lastname: Li
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Xianghua
authors.initials: X
authors.lastname: Tang
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Junmei
authors.initials: J
authors.lastname: Ding
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Nanyu
authors.initials: N
authors.lastname: Han
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China. huangzunxi@163.com.|College of Life Sciences, Yunnan Normal University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People's Republic of China. huangzunxi@163.com.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China. huangzunxi@163.com.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China. huangzunxi@163.com.
authors.forename: Zunxi
authors.initials: Z
authors.lastname: Huang
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry*; D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000648:ultrastructure*; D039903:Actinobacteria / Q000201:enzymology*; D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D055049:Salt Tolerance; D012492:Salts / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D012492:Salts; D006026:Glycoside Hydrolases; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine
keywords: Activity; Microbacterium; N-Acetylglucosaminide tolerance; Salt tolerance; β-N-Acetylglucosaminidase