Unique Catalytic and Molecular Properties of Hydrolases from Aspergillus used in Japanese Bioindustries

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This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P_1 and P_1' like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P_1,which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp^<76> to Ser or Thr and deletion of Ser^<78>, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates contining a basic amino acid residue at P_1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K^6-I^7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His^<128>, His^<132>, and Asp^<164> provide the Zn^<2+> ligands of the enzyme according to a ^<65>Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH+D" motif and an spartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N-and 0-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser^<153>, Asp^<357>, and His^<436> residues were essential for the enzymic catalysis. The N-glycanase released high-man-nose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man_<10> GlcNAc_2 and Man_<11>GlcNAc_2,were characterized. An acidic 1,2-α-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-α-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man_5GlcNAc_2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-α-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man_5GlcNAc_2) sugar chains in S. cerevisiae was described.
社団法人日本農芸化学会の論文
2000-04-23

Unique Catalytic and Molecular Properties of Hydrolases from Aspergillus used in Japanese Bioindustries

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