Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries

This review covers the unique catalytic and molecular properties of three p roteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteina se from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic am ino 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 trypsinog ens like duodenum enteropeptidase. Substitution of Asp(76) to Ser or Thr an d deletion of Ser(78), corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing 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 try psinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1 g atom of zinc /mol of enzyme, is a single chain of 177 amino acid residues, includes thre e disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His(128), His(132), and Asp(164) provide the Zn2+ ligands of the enzym e according to a Zn-65 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 aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that c ontains both N- and O-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-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man(10) GlcNAc(2) and Man(11)GlcNAc(2), were char acterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2- alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutan t capable of producing Man(5)GlcNAc(2) human-compatible sugar chains on gly coproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production o f human-compatible high mannose-type (Man(5)GlcNAc(2)) sugar chains in S. c erevisiae was described.