Characterization of a new family 75 chitosanase from Aspergillus sp. W-2.
A new chitosanase gene of glycoside hydrolase (GH) family 75, csnw2, was cloned from an isolated strain Aspergillus sp. W-2 (CGMCC7018). The mature CsnW2 protein, fused to His-tag at C-terminus, was expressed in Pichia pastoris X-33 and purified with the affinity chromatography of Ni(2+)-NTA. The novel recombinant CsnW2 showed maximal activity with chitosan at pH 6.0 and 55°C. Moreover, it had good pH stability and thermostability at a broad pH range of 3.0-10.0 and a temperature range of 30-70°C, respectively. The enzymatic activity of the CsnW2 could be significantly enhanced by Ca(2+), Mn(2+) and Mg(2+) at a concentration of 1mM, but strongly inhibited by Fe(2+), Zn(2+), Ge(2+), Ni(2+) and Cu(2+) above 1mM. The CsnW2 showed specific hydrolytic activity against chitosan and preferred to hydrolyze chitosan with high degree of deacetylation. The main products of chtiosan (92% deacetylation) were chitosan oligosaccharides (COS) with degree of polymerization (DP) arranging from 2 to 6. Combined with the hydrolysis of COS from DP2 to DP6, CsnW2 was considered to be an endo-acting chitosanase.
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian 116023, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Jianping
J
Zhang
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian 116023, PR China. Electronic address: caohl@dicp.ac.cn.
Hailong
H
Cao
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian 116023, PR China.
Shuguang
S
Li
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian 116023, PR China.
Yong
Y
Zhao
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian 116023, PR China.
Wenxia
W
Wang
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian 116023, PR China.
Qingsong
Q
Xu
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian 116023, PR China; Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, PR China.
Yuguang
Y
Du
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian 116023, PR China. Electronic address: yinheng@dicp.ac.cn.
Heng
H
Yin
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D021901:DNA, Intergenic; D004789:Enzyme Activation; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D010802:Phylogeny; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D011994:Recombinant Proteins; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D021901:DNA, Intergenic; D007477:Ions; D008670:Metals; D012337:RNA, Ribosomal, 18S; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase
Aspergillus sp.; Chitosanase; Glycoside hydrolyase family 75