Norimasa Onishi

Purification and properties of a galacto- and gluco-oligosaccharide-producing β-glycosidase from Rhodotorula minuta IFO879

Abstract A β-glycosidase with strong transglycosylation activity was solubilized from the cell wall fraction of Rhodotorula minuta IFO879 by a cell wall lytic enzyme, Usukizyme, and was purified to homogeneity with a yield of 53% by DEAE-Toyopearl, Butyl-Toyopearl, p -aminobenzyl 1-thio-β- d -galactopyranoside agarose and Con A agarose column chromatography. The native enzyme is a glycoprotein with a molecular weight of 144,000 and is composed of two subunits with molecular weights of about 72,000. Its isoelectric point was estimated to be 4.8 by polyacrylamide gel electrofocusing. The optimal temperature for enzyme activity is 70°C. It is stable at temperatures up to 55°C for 1 h. The optimal pH range is 4.7 to 5.2, and stability was maintained between pH 3.0 and 7.0. The purified β-glycosidase was found to be active toward β- d -fucopyranoside and α- l -arabinopyranoside as well as β- d -galactopyranoside and β- d -glucopyranoside. The K m values measured for lactose, cellobiose, o -nitrophenyl-β- d -galactopyranoside and p -nitrophenyl-β- d -glucopyranoside are 2.4, 11.1, 6.2 and 1.2 mM, respectively, and V max values for these substrates are 63.6, 270.6, 34.4 and 20.7 μmol/min per mg protein, respectively. In addition, this enzyme exhibits strong transglycosylation activities, producing 78 mg/ml galacto-oligosaccharide or 68 mg/ml gluco-oligosaccharide from 200 mg/ml lactose or cellobiose, respectively.

Gluco-oligosaccharide and galacto-oligosaccharide production by Rhodotorula minuta IFO879

Abstract Three strains of yeast, Sterigmatomyces elviae CBS8119, Rhodotorula minuta IFO879 and Sirobasidium magnum CBS6803, which are known to produce galacto-oligosaccharide (Gal-OS) from lactose, were found to process strong transglucosylation abilities and to produce gluco-oligosaccharide (Glc-OS) from cellobiose. Among them, R. minuta IFO 879 was selected as the best producer of Glc-OS. Efficient production conditions for Glc-OS from cellobiose and Gal-OS from lactose were investigated. Using toluene-treated resting cells of R. minuta IFO879, the maximal amounts of Glc-OS produced from 200 mg/ml cellobiose and of Gal-OS produced from 200 mg/ml lactose were 70 mg/ml (a yield by wieght of 35%) and 76 mg/ml (a yield by weight of 38%), respectively. Since the by-product glucose was found to inhibit oligosaccharide production, it was removed from the reaction mixture by devising a suitable culture method such that the enzymatic reaction was accompanied by cell growth to consume the glucose. Under these conditions, the productivities were markedly improved: 201 mg/ml of Glc-OS was produced from 400 mg/ml cellobiose (a yield by weight of 51%) and 230 mg/ml of Gal-OS from 360 mg/ml of lactose (a yield by wieght of 64%). The structures of the major components of Glc-OS and Gal-OS obtained by this method were identified as cellotriose and O -β- d -galactopyranosyl-(1→4)- O -β- d -galactopyranosyl-(1→4)- d -glucopyranose (4′-galactosyl-lactose), respectively.

Galacto‐oligosaccharide production from lactose by Sirobasidium magnum CBS6803

N. ONISHI, I. KIRA AND K. YOKOZEKI. 1996. Galacto‐oligosaccharide (Gal‐OS) was produced from lactose by a yeast, Sirobasidium magnum CBS6803. With toluene‐treated resting cells, 136 mg ml−1 of Gal‐OS was produced from 360 mg ml−1 of lactose at 50°C for 42 h. Then, the yield of Gal‐OS was increased by a culture method in which cell growth followed the enzymatic reaction : 224 mg ml−1 of Gal‐OS was produced at 30°C for 60 h. Finally, combination of the toluene‐treated resting cells and glucose oxidase plus catalase was applied to improve productivity by the removal of a by‐product, glucose, which inhibits the Gal‐OS production, from the reaction mixture. In this case, 242 mg ml−1 4‐galactosyl‐lactose. of Gal‐OS was produced at 50°C for 42 h without cell growth. The structure of the major product ws identified as 4‐galactosyl‐laetos.

Purification and characterization of galacto‐oligosaccharide‐producing β‐galactosidase from Sirobasidium magnum

Galacto‐oligosaccharide‐producing β‐galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p‐aminobenzyl 1‐thio‐β‐d‐galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β‐galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β‐galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The Km values for o‐nitrophenyl‐β‐d‐galactopyranoside and lactose were 14·3 and 5·5 mmol l−1, respectively, and the Vmax values for these substrates were 33·4 and 94·5 μmol min−1 mg of protein−1, respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml−1 galacto‐oligosaccharide was produced from 200 mg ml−1 lactose.

Molecular Cloning and Expression of the hyu Genes from Microbacterium liquefaciens AJ 3912, Responsible for the Conversion of 5-Substituted Hydantoins to α-Amino Acids, in Escherichia coli

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to α-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl α-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.

Purification and Characterization of Hydantoin Racemase from Microbacterium liquefaciens AJ 3912

A hydantoin racemase that catalyzed the racemization of 5-benzyl-hydantoin was detected in a cell-free extract of Microbacterium liquefaciens AJ 3912, a bacterial strain known to produce L-amino acids from their corresponding DL-5-substituted-hydantoins. This hydantoin racemase was purified 658-fold to electrophoretic homogeneity by serial chromatography. The N-terminal amino acid sequence of the enzyme showed homology with known hydantoin racemases from other microorganisms. The apparent molecular mass of the purified enzyme was 27 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and 117 kDa on gel-filtration in the purification conditions, indicating a homotetrameric structure. The purified enzyme exhibited optimal activity at pH 8.2 and 55 °C, and showed a chiral preference for L-5-benzyl- rather than D-5-benzyl-hydantoin.

Purification and Characterization of a (R)-1-Phenyl-1,3-propanediol-producing Enzyme from Trichosporon fermentans AJ-5152 and Enzymatic (R)-1-Phenyl-1,3-propanediol Production

An (R)-1-phenyl-1,3-propanediol-producing enzyme was purified from Trichosporon fermentans AJ-5152. It was NADPH-dependent and converted 3-hydroxy-1-phenylpropane-1-one (HPPO) to (R)-1-phenyl-1,3-propanediol [(R)-PPD] with anti-Prelog’s specificity. It showed maximum activity at pH 7.0 and 40 °C. Its K m and V max values toward HPPO were 20.1 mM and 3.4 μmol min−1 mg protein−1 respectively. The relative molecular weight of the enzyme was estimated to be 68,000 on gel filtration and 32,000 on SDS-polyacrylamide gel electrophoresis. An (R)-PPD-producing reaction using the (R)-PPD-producing enzyme and an NADPH recycling system was carried out by successive feeding of HPPO. A total (R)-PPD yield of 8.9 g/l was produced in 16 h. The molar yield was 76%, and the optical purity of the (R)-PPD produced was over 99% e.e.

Heat-killed cell preparation of Corynebacterium glutamicum stimulates the immune activity and improves survival of mice against enterohemorrhagic Escherichia coli

Fermentation by Corynebacterium glutamicum is used by various industries to produce L-Glutamate, and the heat-killed cell preparation of this bacterium (HCCG) is a by-product of the fermentation process. In present study, we evaluated the immunostimulating and survival effects against enterohemorrhagic Escherichia coli (STEC) infection of HCCG. HCCG significantly stimulated in vitro IgA and interleukin-12 p70 production in murine Peyer’s patch cells and peritoneal macrophages, respectively. Oral administration of 10 mg/kg body weight (BW) of HCCG for seven consecutive days stimulated IgA concentration in murine cecal digesta. Mice were orally administered HCCG for 17 consecutive days (d0–d17), and challenged with STEC on d4 to d6. Survival of mice tended to improve by 100 mg/kg BW of HCCG administration compared with those in control group. In conclusion, HCCG supplementation was found to prevent STEC infection in mice, and thus it may have the potential to stimulate the immune status of mammals. Graphical abstract Cell wall preparation of Corynebacterium glutamicum prevented of pathogenic E.coli infection through enhancement of immune function such as IgA secretion in the intestine.