Improving the specific activity of β-mannanase from Aspergillus niger BK01 by structure-based rational design.
β-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic β-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57Å crystal structure of ManBK. The protein adopts a typical (β/α)8 fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18±2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher kcat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications.
Institute of Biotechnology, Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan.
Jian-Wen
JW
Huang
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Chun-Chi
CC
Chen
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Chun-Hsiang
CH
Huang
Genozyme biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co., Ltd., Dongguan, 523808, China.
Ting-Yung
TY
Huang
Institute of Biotechnology, Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan.
Tzu-Hui
TH
Wu
Genozyme biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co., Ltd., Dongguan, 523808, China.
Ya-Shan
YS
Cheng
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
Tzu-Ping
TP
Ko
Genozyme biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co., Ltd., Dongguan, 523808, China.
Cheng-Yen
CY
Lin
Institute of Biotechnology, Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan; Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan. Electronic address: jrliu@ntu.edu.tw.
Je-Ruei
JR
Liu
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: guo_rt@tib.cas.cn.
Rey-Ting
RT
Guo
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D044902:beta-Mannosidase / Q000737:chemistry / Q000378:metabolism*
D044902:beta-Mannosidase
Aspergillus niger; Crystal structure; Rational design; Thermophilic; β-Mannanase