Eiji Ichishima

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

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 P1 and P1′ like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, 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 P1. 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 K6-I7 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 His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn 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 Ser153, Asp357, and His436 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 Man10GlcNAc2 and Man11GlcNAc2, 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 Man5GlcNAc2 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 (Man5GlcNAc2) sugar chains in S. cerevisiae was described.

Cloning of a novel tyrosinase-encoding gene (melB) from Aspergillus oryzae and its overexpression in solid-state culture (Rice Koji)

We have cloned a novel tyrosinase-encoding gene (melB) specifically expressed in solid-state culture of Aspergillus oryzae. A tyrosinase-encoding gene (melO) from A. oryzae was already cloned and the protein structures of its catalytic and copper binding domains were investigated. However, our recent results revealed that the melO gene was highly expressed in submerged culture but not in solid-state culture. Because tyrosinase activity was also detected in solid-state culture, we assumed that another tyrosinase gene other than melO is expressed in solid-state culture. Another tyrosinase gene was screened using the expressed sequence tag (EST) library. One redundant cDNA clone homologous with the tyrosinase gene was found in the collection of wheat bran culture. Northern blot analysis revealed that the gene corresponding to the cDNA clone was specifically expressed in solid-state culture (koji making), but not in submerged culture. Molecular cloning showed that the gene carried six exons interrupted by five introns and had an open reading frame encoding 616 amino acid residues. This gene was designated as melB. The deduced amino acid sequence of the gene had weak homology (24%-33%) with MelO and other fungal tyrosinases but the sequences of the copper binding domains were highly conserved. When the melB gene was expressed under the control of the glaB promoter in solid-state culture, tyrosinase activity was markedly enhanced and the culture mass was browned with the melanization by MelB tyrosinase. These results indicated that the melB gene encodes a novel tyrosinase associated with melanization in solid-state culture.

Aorsin, a novel serine proteinase with trypsin-like specificity at acidic pH.

A proteinase that hydrolyses clupeine and salmine at acidic pH, called aorsin, was found in the fungus Aspergillus oryzae. Purified aorsin also hydrolysed benzyloxycarbonyl-Arg-Arg-4-methylcoumaryl-7-amide optimally at pH 4.0. The specificity of aorsin appeared to require a basic residue at the P(1) position and to prefer paired basic residues. Aorsin activated plasminogen and converted trypsinogen to trypsin. The trypsin-like activity was inhibited strongly by antipain or leupeptin, but was not inhibited by any other standard inhibitors of peptidases. To identify the catalytic residues of aorsin, a gene was cloned and an expression system was established. The predicted mature protein of aorsin was 35% identical with the classical late-infantile neuronal ceroid lipofuscinosis protein CLN2p and was 24% identical with Pseudomonas serine-carboxyl proteinase, both of which are pepstatin-insensitive carboxyl proteinases. Several putative catalytic residues were mutated. The k (cat)/ K(m) values of the mutant enzymes Glu(86)-->Gln, Asp(211)-->Asn and Ser(354)-->Thr were 3-4 orders of magnitude lower and Asp(90)-->Asn was 21-fold lower than that of wild-type aorsin, indicating that the positions are important for catalysis. Aorsin is another of the S53 family serine-carboxyl proteinases that are not inhibited by pepstatin.

A single free cysteine residue and disulfide bond contribute to the thermostability of Aspergillus saitoi 1,2-α-mannosidase

Aspergillus saitoi 1,2-α-mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T m values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 °C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.

Purification of serine carboxypeptidase from the hepatopancreas of Japanese common squid Todarodes pacificus and its application for elimination of bitterness from bitter peptides

An acidic serine carboxypeptidase (CPase Tpa) from the hepatopancreas of Japanese common squid Todarodes pacificus was purified. Purified CPase Tpa had a molecular mass of 36 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 6.0. The optimum pH of CPase Tpa was pH 4.0. In investigating the specificity of CPase Tpa for several peptide substances, it was found that peptides with hydrophobic or bulky amino acid residues at the P1 position reacted well. The enzymatic activity was almost completely inhibited by p-chloromercuribenzoic acid, monoiodoacetic acid, diisopropylfluorophosphate and HgCl2. This is the basis for its grouping in the serine carboxypeptidase family (EC 3. 4. 16. 5). The substrate specificity of CPase Tpa can be used to eliminate the bitterness of bitter peptides. In this study, the bitterness-reductive effect using bitter peptides prepared by hydrolyzing soy protein, casein and corn gluten with pepsin or trypsin was tested. The bitterness of soy peptide digested with pepsin was completely eliminated by treatment with CPase Tpa, whereas the bitterness of casein digested with trypsin and corn peptide digested with pepsin were somewhat less efficient. On the basis of these results, it is anticipated that CPase Tpa would be effective in eliminating the bitterness of some bitter peptides.

Isolation and characterization of cDNA encoding chicken egg yolk aminopeptidase Ey

Abstract Aminopeptidase Ey (EC 3.4.11.20) from chicken (Gallus gallus domesticus) egg yolk is a homodimeric exopeptidase with a broad specificity for N-terminal amino acid residues at P1 position of the substrate. Aminopeptidase Ey is a 300-k metalloexopeptidase, containing 1.0 g atom of zinc per mole of a subunit with a relative molecular mass of 150 k. A full-length cDNA was cloned from chicken (female) liver cDNA library. Analysis of the 3196-base pairs (bp) nucleotide sequence of the cDNA revealed a single open reading frame coding for 967 amino acid residues. The coding region of aminopeptidase Ey gene, apdE, occupies 2901 bp of the cDNA. The predicted amino acid sequence of the enzyme is 66, 65, 64 and 63% identical with those of aminopeptidases N (EC 3.4.11.2) from human, pig, rabbit and rat, respectively. Aminopeptidase Ey contains the metallo-binding sequence motif, His–Glu–Xaa–Xaa–His, found in zinc metallopeptidases. Zinc binding sites, His-386, His-390 and Glu-409, and catalytic site, Glu-387, were conserved in the homologous aminopeptidases N.

Substrate Specificities of Deuterolysin from Aspergillus oryzae and Electron Paramagnetic Resonance Measurement of Cobalt-substituted Deuterolysin

The substrate specificities of deuterolysin, a 19-kDa zinc-protease (EC 3.4.24.39) from Aspergillus oryzae, were investigated at pH 9.0 with various fluorogenic acyl-peptide-4-methylcoumaryl-7-amides (peptide- MCAs). N-Butoxycarbonyl-Arg-Val-Arg-Arg-MCA was the best substrate for deuterolysin. We therefore measured its kinetic parameters. Deuterolysin had high activity toward the peptide bonds next to pairs of basic residues in calf thymus histone H4. The specificity of cobalt-substituted deuterolysin (Co-deuterolysin) for peptide-MCAs was similar to that of native deuterolysin. The CD spectrum of Co-deuterolysin was similar to that of the native deuterolysin. The metal coordination sphere of Co-deuterolysin was analyzed by Q-band (33.9570 GHz) electron paramagnetic resonance (EPR) spectroscopy. Using computer simulation of EPR, we found the g principal values to be g xx=5.20, g yy=4.75, and g zz=2.24; the metal center was a divalent cobalt ion in a high spin state.

Acid activation of protyrosinase from Aspergillus oryzae : homo-tetrameric protyrosinase is converted to active dimers with an essential intersubunit disulfide bond at acidic pH

Aspergillus oryzae protyrosinase (pro‐TY) has a unique feature that the proenzyme is activated under conditions of acidic pH. The pro‐TY was inactive at pH 7.0. The latent enzyme was activated at pH 3.0, and was slightly activated by sodium dodecyl sulfate (SDS). The molecular masses of the pro‐TY and acid‐activated tyrosinase (acid‐TY) were 266 and 165 kDa, respectively, as estimated by gel‐filtration chromatography. The CD spectra showed that the tertiary and/or quaternary structure was changed after the acid activation. On the basis of these results, we deduce that the intersubunit polar interaction is disrupted at pH 3.0, and that the tetrameric pro‐TY dissociates to dimers. Tryptophan fluorescence spectra and binding assay of 8‐anilino‐1‐naphthalene sulfonic acid (ANS) suggested that hydrophobic amino acid residues of the active site were exposed to solvent after acid treatment. It was likely that Cys108 formed an intermolecular disulfide bond between the subunits of dimeric acid‐TY. The dimerization of acid‐TY involving the intermolecular disulfide bond is essential for the activity.

Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization

Miltpain (EC.3.4.22.-) is a cysteine proteinase that preferentially hydrolyzes basic proteins, previously found in the milt of chum salmon. Here we report a similar cysteine proteinase in the milt of the marine Pacific cod. The enzyme was isolated and purified 6900-fold and with an estimated mass of 63 kDa by gel filtration chromatography and 72 kDa by SDS/PAGE. Cod miltpain has an optimum pH of 6.0 for Z-Arg-Arg-MCA hydrolysis, and Km of 11.5 microM and kcat of 19.0 s-1 with Z-Arg-Arg-MCA. It requires a thiol-inducing reagent for activation and is inhibited by E-64, iodoacetamide, CA-074, PCMB, NEM, TLCK, TPCK, ZPCK and o-phenanthroline. This proteinase strongly hydrolyzes basic proteins such as salmine, clupeine and histone, and exhibits unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg on the substrates of P2-P1. The isoelectric point is 5.2 by isoelectric focusing. N-Terminal sequencing gave a sequence of < EVPVEVVRXYVTSAPEK. The cysteine proteinase from Pacific cod very closely matches the previously reported miltpain from chum salmon.

70 – Aminopeptidase Ey

Publisher Summary This chapter describes the structural chemistry and the biological aspects of aminopeptidase Ey. The introduction of a sensitive fluorogenic substrate, Leu-NHMec, and a chromogenic substrate, Leu-NHPhNO2, at pH 7.5 led to the discovery of aminopeptidase Ey in hens egg yolk. The enzyme has a broad specificity for N-terminal amino acid residues at the P1 position. Aminopeptidase Ey has a broad specificity for amino acid residues at the P1 position of substrates. The enzyme degrades a variety of peptides having various N-terminal amino acids: hydrophobic, basic and acidic amino acids including proline. Aminopeptidase Ey is a dimeric enzyme with homologous subunits, each having a molecular mass of 150,000 Da. It contains 1.0 mol of zinc per mol of each subunit. The enzyme molecule is seen as a dimer composed of two globular subunits by electron micrography at a magnification of 100,000×. The pI of the enzyme is about 2.8 as determined by isoelectric focusing. An asialo form of the enzyme, obtained by treatment with Arthrobacter sialidase, has a pI of 4.4.