期刊
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
卷 64, 期 4, 页码 675-688出版社
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.64.675
关键词
aspergillopepsin I; aspzincin; carboxypeptidase; deuterolysin; alpha-mannosidase
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 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 trypsinogen. 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 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 Zn2+ ligands of the enzyme 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 contains 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 characterized. 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 mutant capable of producing Man(5)GlcNAc(2) human-compatible sugar chains on glycoproteins 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 of human-compatible high mannose-type (Man(5)GlcNAc(2)) sugar chains in S. cerevisiae was described.
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