Sachio Miyairi

An increase in the transglycosylation activity of Saccharomycopsis α-amylase altered by site-directed mutagenesis

The 84th tryptophan residue in Saccharomycopsis alpha-amylase molecule was replaced by a leucine residue and the resulting site-directed mutant, W84L enzyme, showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. Analysis of the reaction products from p-nitrophenyl maltooligosaccharides indicated that these large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the tryptophan residue located at subsite 3 of the enzyme plays an important role not only to hold the substrate, but also to liberate the hydrolysis products from the substrate binding pocket.

A mutant α-amylase with enhanced activity specific for short substrates

The 210th lysine (K210) at the active site in Saccharomycopsis fibuligera α‐amylase was altered to arginine (R) or asparagine (N) by site‐directed mutagenesis. Replacement of K210 by R strengthened the 7th and weakened the 8th subsite affinities. K210 was found to contribute to both the 8th and the 7th subsites. The catalytic activity of the K210R enzyme for the hydrolysis of maltose (G2) was three‐times higher than that of the native enzyme due to an increase in the affinity of the 7th subsite adjacent to the catalytic site, whereas the activity of the K210N enzyme for G2 was decreased to 1% of that of the native enzyme by a reduction in the 7th subsite affinity.

Application of photochemical reactions of bis-azido compounds to preparation of an enzyme-polymer film

Abstract β-Glucosidase (EC 3.2.1.21) was immobilized in fibroin film by using a photo-crosslinking agent, 4,4′-diazidostilbene-2,2′-disodium sulfonate. Crosslinking and immobilization reactions proceeded by light irradiation for 20 min in air. The immobilized enzyme showed approximately 50% of its native activity with an apparent Michaelis constant of 3.1 m m . The Michaelis constant of the native enzyme was 2.3 m m . Some properties of the immobilized and native enzymes were compared.

Conversion of alpha -amylase into transglycosylation enzyme

The 210 th lysine residue in Saccharomycopsis α-amylase (Sfamy) molecule was replaced by arginine and asparagine residues. The resulting K210R and K210N enzymes cleave mainly the first glycosidic bond from the reducinng end of maltotetraose (G4), while the native enzyme hydrolyzes mainly the second bond. We changed successfully the major cleavage point in the hydrolysis reaction of G4. We estimated the 8th subsite affinities of the mutant enzymes and compared them with that of the native enzyme. These facts suggest that the K210 residue composes the 8th subsite, one of the major subsites, and that a positively charged s-amino residue is necessary for the 8th subsite affinity. The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite. The 84th tryptophan residue was replaced by leucine residues. The resulting W84L enzyme showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. These large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the W84 residue located at subsite 5 plays an important role in the addition of a water molecule to a carbonium ion intermediate and/or in the liberation of the hydrolysis product from the substrate binding pocket. The doubly mutated enzymes, W84LK210 N, are expected to form the transglycosylation products different in size from those produced by the single mutant, W84L enzyme.

Effects of Poly (amino acid) s on Photophosphorylation in a Cyanobacterial Thylakoid

National Chemical Laboratory for Industry, 1-1 Higashi, Tsukuba-shi 305, Japan Poly-L-glutamic acid accelerated by about 25% the cyclic photophosphorylation in the thylakoid a thermophilic cyanobacterium. A mixture of poly-L-glutamic acid and poly-L ornithine of a ratio 2/1 (w/w) also enhanced by 20-30% the photophosphorylation. The acceleration behaviors by poly-L-glutamic acid alone are different from those by the mixture of acidic and basic poly (amino acid) s.

Effects of basic poly(amino acid)s on a photosynthetic membrane system of a cyanobacterium.

Effects of basic poly (amino acid) s on the photosynthetic activities of the thylakoid membranes of thermophilic cyanobacterium Synechococcus sp. were examined. Poly-L-ornithine, poly-L-arginine, poly-L-lysine and poly-D-lysine uncouple the thylakoid membranes, dissipating ΔpH generated across the membranes and inhibiting cyclic photophosphorylation mediated by phenazine methosulfate. Contrary to the previous reports with some algae, poly-L-ornithine, poly-L-lysine and poly-D-lysine inhibit photosystem 1-dependent electron flow from tetramethyl-p-hydroquinone to methylviologen, presumably by affecting the function of soluble acidic protein cytochrome c-553. Poly-L-histidine gives no effect on photochemical activities of the cyanobacterial thylakoid membranes.