Shinsaku Hayashida's research while affiliated with Kyushu University and other places

Two saccharifying α-amylases with different molecular masses were purified from Bacillus subtilis IFO 3108. The higher molecular mass α-amylase 1 (RBSA-1, MM 67 kDa) was able to adsorb to α-cyclodextrin (α-CD) sepharose CL-6B and hydrolyze raw starch. RBSA-1 showed weak adsorption to raw corn starch over the wide pH range of 5.0–9.0. At low pH (5.0–6.0), RBSA-1 exhibited high adsorption to raw potato starch. The lower molecular mass α-amylase 2 (BSA-2, MM 45 kDa) exhibited enzymatic properties similar to RBSA-1, however, was unable to adsorb to α-CD sepharose CL-6B and failed to hydrolyze raw starch. On the other hand, a liquefying type α-amylase (RBLA), purified from Bacillus sp., exhibited remarkable adsorption to raw starches tested over a wide pH range (5.0–9.0). This pH range corresponded to that suitable for the digestion of raw starches. The adsorption of RBSA-1 and RBLA to α-CD sepharose CL-6B was pH-independent, however, the extent of adsorption of RBSA-1 (70–85%) was much greater than that of RBLA (30–45%). The hydrolysis of raw corn starch was specifically inhibited by α-CD for both enzymes. The Ki value (0.44 mM) for RBSA-1 was much lower than that for RBLA (3.44 mM).

Glucoamylase I (GAI) from Aspergillus awamori var. kawachi hydrolyzes raw starch efficiently and is composed of three functional domains: the amino-terminal catalytic GAI' domain (A-1 to V-469), the threonine- and serine-rich O-glycosylated Gp-I domain (A-470 to V-514), and the carboxy-terminal raw starch-binding Cp domain (A-515 to R-615). In order to investigate the role of the Gp-I domain, an additional repeat of Gp-I and internal deletions of the entire Gp-I sequence or parts of the Gp-I sequence were introduced within Gp-I. All mutant genes as well as the wild-type gene were inserted into a yeast-secretion vector, YEUp3H alpha, and expressed in Saccharomyces cerevisiae. Wild-type GAI expressed in yeast cells (GAY), GAGpI, having an extra Gp-I, and GA delta 470-493, lacking the A-470-to-T-493 sequences of Gp-I, were successfully secreted into the culture medium. On the other hand, GA delta 470-507, lacking A-470 to S-507, and GA delta GpI, lacking the entire Gp-I (A-470-to-V-514) sequence, failed to be secreted and remained in the yeast cells. The carbohydrate content of GAGpI was 1.2 times higher than that of GAY and 2.4 times higher than that of the original GAI. The raw starch digestibility of GAGpI was almost the same as that of GAY but was 1.5 times faster than that of GAI.(ABSTRACT TRUNCATED AT 250 WORDS)

The digestion of raw starch by a glucoamylase (GA MU-H) from a mutant strain of Aspergillus awamori var. kawachi was closely correlated with mannoside chains O-linked to the Gp-I region (A470-V514), but not sugar chains N-linked to catalytic GAI' domain of GA MU-H. The partial replacement of mannose residues by glucose residues led to a significant decrease raw starch digestion. By the substitution of D2O for H2O in the reaction mixture, the raw starch digestion of GA MU-H decreased to 80% of that at 30 degrees C, although the rate of hydrolysis of soluble starch by and the ability to bind beta-cyclodextrin of GA MU-H were unchanged. Glycerol, known as an antichaotropic reagent, decreased the raw starch digestion of GA MU-H significantly. However, it did not have any effect on the enzymatic activity for soluble starch when soluble starch was the substrate. The efficient digestion of raw starch with raw starch-digesting glucoamylase needed the mannoside chains O-linked to the Gp-I region, which were suggested to contribute to digestion of raw starch through the interaction with water.

Sixteen biphenyl and polychlorinated biphenyl (PCB)-degrading strains were isolated from soil at various locations and characterized. All the isolates were gram negative bacteria that include eleven species of Pseudomonas, one Achromobacter, one belonging to the CDC Group VE, Biotype I, while three strains were unidentified. Most of these strains grew on basal salts media supplemented with a wide variety of aromatic compounds, which include substituted biphenyls and biphenyl related compounds. Southern blot analysis using two previously cloned bph genes as probes revealed that four (three Pseudomonas, one unidentified) out of the sixteen strains showed significant hybridization with the well studied bph genes of Pseudomonas pseudoalcaligenes KF707. The immunological cross reactivity of the 2,3dihydroxybiphenyl dioxygenases and hydrolases from the four strains corresponded well to the DNA homology. The existence of almost identical bph genes in different soil bacteria indicates that certain chromosomal bph operons have a mechanism of transfer within bacterial populations in the environment. However, the twelve other biphenyl utilizing isolates did not show any significant hybridization with the KF707 bph probe, while all of the sixteen strains did not hybridize with the Moraxella sp. KF704 bph probe. These results suggest the diversity of PCB-degrading genotypes.

Saccharomyces cerevisiae wy2 segregated to 2 mater and 2 non-mater in relation to mating ability. The non-mater segregants behaved as the normal type of homothallic life cycle. On the other hand, the mater segregants gradually formed spores during successive subcultures, indicating that slow interconversion of mating-type happened to occur during subcultures. We termed this novel type of life cycle "delayed homothallism". The results of complementation tests with standard ho strains and introduction of a wild type HO gene showed that delayed homothallism was caused by a defective HO gene. The amino acid sequence deduced from the nucleotide sequence of the wy2 HO gene differed from the wild type HO gene in three amino acid residues. In the carboxy terminus of HO protein, there are three repeats of cysteine and histidine that are postulated to play a role in binding of HO protein to DNA. However, wy2 HO protein lacked one such repeat at residues Cys470-His475, where His was replaced by Leu.

Carboxy-terminal deletions were introduced into the raw starch-binding domain (A-515 to R-615) encoded by the gene for glucoamylase I (GAI) from Aspergillus awamori var. kawachi. Genes coding for proteins designated GA596 (A-1 to E-596), GA570 (A-1 to A-570), and GA559 (A-1 to N-559) were constructed and resulted in truncated proteins. All of the mutant genes were expressed heterologously in Saccharomyces cerevisiae. GA596 adsorbed to raw starch and digested it. GA570 and GA559 did not adsorb to raw starch or to an alpha-cyclodextrin-Sepharose CL-4B gel under our experimental conditions. However, GA570 was able to digest raw starch, and the digestion of raw starch by GA570 was inhibited by beta-cyclodextrin. Residue Trp-562 of GAI, which was suggested previously to contribute to formation of an inclusion complex with raw starch, was replaced by Leu (GAW562L), Phe (GAW562F), and Gly (GAW562G). GAW562L and GAW562F adsorbed to raw starch and an alpha-cyclodextrin gel, but GAW562G did not. Although GAW562L digested raw starch to the same extent as wild-type GAI (designated GAY), GAW562F and GAW562G exhibited less ability to digest raw starch. On the basis of our results, it appears that the sequence around Trp-562, PL(W-562)YVTVTLPA, is the minimal sequence necessary for digestion of raw starch and that hydrophobic residue Trp-562 contributes to formation of an inclusion complex. The sequence near Trp-589, which has abundant hydrogen bond-forming residues and the charged amino acid residues needed for stable adsorption to raw starch, probably assists in the formation of the inclusion complex.

The extradiol ring-cleavage dioxygenases derived from seven different Pseudomonas strains were expressed in Escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. The substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing Pseudomonas putida F1, 1,2-dihydroxynaphthalene dioxygenase from a NAH7 plasmid, and catechol 2,3-dioxygenase from a TOL plasmid pWW0 were compared. Among the dioxygenases, that from Pseudomonas pseudoalcaligenes KF707 showed a very narrow substrate range. Contrary to this, the dioxygenase from pWW0 showed a relaxed substrate range. The seven extradiol dioxygenases from the various Pseudomonas strains are highly diversified in terms of substrate specificity.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).

Multicomponent enzyme complexes of biphenyl (BP) dioxygenase (Dox) encoded by the gene cluster, bphA1A2A3A4 in Pseudomonas pseudoalcaligenes strain KF707 [Taira et al., J. Biol. Chem. 267 (1992) 4844-4858] and toluene Dox encoded by the gene cluster, todC1C2BA in P. putida strain F1 [Zylstra et al., J. Biol. Chem. 264 (1989) 14940-14946], show high homologies (approx. 60%) for the corresponding subunit component in spite of the fact that they have discrete substrate specificities. We constructed hybrid gene clusters by replacing the gene component(s) between the large and small subunits of terminal Dox in the bph and tod gene clusters, and analyzed the function of a novel hybrid aromatic ring Dox. Escherichia coli cells expressing the hybrid gene clusters, todC1::bphA2A3A4, todC1C2::bphA3A4 and bphA1::todC2::bphA3A4, gained the ability to convert benzene-toluene and their derivatives to the dihydrodiols, indicating that the hybrid terminal Dox composed of TodC1::BphA2 and BphA1::TodC2 forms a functionally active multicomponent Dox associated with ferredoxin (Fer) (BphA3) and Fer reductase (BphA4). Moreover, hybrid Dox (composed of TodC1::BphA2A3A4 and TodC1C2::BphA3A4) showed a wide substrate specificity rather similar to that of the wild-type toluene Dox (TodC1C2BA). On the other hand, the hybrid Dox (BphA1::TodC2::BphA3A4) showed oxidative activities for the same compounds, but the rate of oxidation was dependent upon the substrate. These results suggest that (i) the two subunits of terminal Dox are critically involved in the substrate specificity for BP, benzene and their derivatives, and (ii) the electron transport proteins, Fer and Fer reductase, are exchangeable with one another between the BP Dox and toluene Dox complexes.

Glucoamylase I (GAI) bound to β-cyclodextrin (β-CD) with the binding constants of Kd = 17.7 μm and n = 1.69. Binding resulted in formation of an inclusion complex, as indicated by the fact that organic guest compounds for β-CD inhibited the binding of GAI to β-CD. Titration of GAI (A(1)-R(615)) with β-CD or Amylose A showed critical spectral perturbations at 286 nm that reflected the aromatic side chains of Tyr and Trp, but no spectral changes were observed in GAI' (A(1)-V(469)) and Gp-I (A(470)-V(514)). Titration of GAI with derivatives of β-CD, namely, 6-hydroxypropyl-β-CD (H-CD), 2,6-O-dimethyl-β-CD, and 2,3,6-O-trimethyl-β-CD, yielded spectral changes only in the case of H-CD. Therefore, the Cp region (A(515)-R(615)) of GAI seems stereospecifically able to recognize the structure of the secondary OH-side but not the primary OH-side of β-CD. Chemical modifications specific for aromatic amino acids of the GAI-CD inclusion complexes were done with N-bromosuccinimide and tetranitromethane. In the presence of β-CD, one Trp residue was protected from oxidation but Tyr residues were not. The analysis of a Cp region/β-galactosidase fusion protein indicated that Trp(562) contributed to formation of inclusion complexes.