Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain  

作　　者：Fan Li Xuejun Zhu Yanfei Li Hao Cao Yingjiu Zhang 

机构地区：[1]Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun130012, China [2]College of Life Science, Jilin Agricultural University, Changchun 130118, China

出　　处：《Acta Biochimica et Biophysica Sinica》2011年第4期324-334,共11页生物化学与生物物理学报（英文版）

基　　金：This work was supported by grants from the National Natural Science Foundation of Chain （No. 30870518）, the National High Technology Research and Development Program of China （No. 2007AA100601-2） and the Platform base construction projects of Jinlin University.

摘　　要：A gene encoding a special thermophilic multifunctional amylase OPMA-N was cloned from Bacillus sp. ZW2531- 1. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a β-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain （1-124 aa） on the function and properties of OPMA-N. Both OPMA- N （12.82U/mg） and its N-terminal domain-truncated AOPMA-N （12.55 U/mg） only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaltotriose, and isomaltotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal 13-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaltotriose and less maltose, and it seemed to contribute to OPMA-N＇s thermostability since OPMA-N showed higher activity than AOPMA-N in a temperature range from 40 to 80℃ and the halflife （t^12） was 5 h for OPMA-N and 2 h for AOPMA-N at 60℃. Both OPMA-N and AOPMA-N were Ca2＋- independent, but their activities could be influenced by Cu2＋, Ni2＋, Zn2＋, EDTA, SDS （1 mM）, or Triton-X100 （1%）. Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and sub- sequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and AOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.A gene encoding a special thermophilic multifunctional amylase OPMA-N was cloned from Bacillus sp. ZW2531- 1. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a β-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain （1-124 aa） on the function and properties of OPMA-N. Both OPMA- N （12.82U/mg） and its N-terminal domain-truncated AOPMA-N （12.55 U/mg） only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaltotriose, and isomaltotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal 13-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaltotriose and less maltose, and it seemed to contribute to OPMA-N＇s thermostability since OPMA-N showed higher activity than AOPMA-N in a temperature range from 40 to 80℃ and the halflife （t^12） was 5 h for OPMA-N and 2 h for AOPMA-N at 60℃. Both OPMA-N and AOPMA-N were Ca2＋- independent, but their activities could be influenced by Cu2＋, Ni2＋, Zn2＋, EDTA, SDS （1 mM）, or Triton-X100 （1%）. Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and sub- sequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and AOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.

关 键 词：OLIGOSACCHARIDES substrate/productspecificity STARCH 

分 类 号：Q550.4[生物学—生物化学] TQ925.1[轻工技术与工程—发酵工程]