Naoto Tonouchi

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells.

SummaryThe aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH2-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH2-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.

Effective cellulose production by a coculture of Gluconacetobacter xylinus and Lactobacillus mali.

A microbial colony that contained a marked amount of cellulose was isolated from vineyard soil. The colony was formed by the associated growth of two bacterial strains: a cellulose-producing acetic acid bacterium (st-60-12) and a lactic acid bacterium (st-20). The 16S rDNA-based taxonomy indicated that st-60-12 belonged to Gluconacetobacter xylinus and st-20 was closely related to Lactobacillus mali. Cocultivation of the two organisms in corn steep liquor/sucrose liquid medium resulted in a threefold higher cellulose yield when compared to the st-60-12 monoculture. A similar enhancement was observed in a coculture with various L. mali strains but not with other Lactobacillus spp. The enhancement of cellulose production was most remarkable when sucrose was supplied as the substrate. L. mali mutants for exocellular polysaccharide (EPS) production were defective in promoting cellulose production, but the addition of EPS to the monoculture of st-60-12 did not affect cellulose productivity. Scanning electron microscopic observation of the coculture revealed frequent association between the st-60-12 and L. mali cells. These results indicate that cell–cell interaction assisted by the EPS-producing L. mali promotes cellulose production in st-60-12.

Purification and characterization of exo-1,4-β-glucosidase from Acetobacter xylinum BPR2001

Abstract An exo-1,4-β-glucosidase (EC 3.2.1.74; G3ase) was obtained from the supernatant of cultured Acetobacter xylinum subsp. sucrofermentans BPR2001 and purified to homogeneity by ammonium sulfate precipitation, cation-exchange, gel-filtration and hydrophobic interaction chromatography. The enzyme migrated to a position corresponding to 81.2 kDa on SDS-polyacrylamide gel electrophoresis under both non-reducing and reducing conditions, suggesting that this enzyme is a monomer polypeptide. The isoelectric point was 6.0. N-Bromosuccinimide inhibited the activity of exo-1,4-β-glucosidase completely, whereas sulfhydryl reagents did not. The Km and Vmax for the hydrolysis of cellotriose as substrate were 3.7 mM and 7.4 μmol/min/mg, respectively. The enzyme specifically cleaved the non-reducing ends of β-glucosyl linkages of cellotriose or larger cello-oligosaccharides, 4-methylumberiferryl- and p-nitrophenyl-β- d -glucosides, but cellobiose was hydrolyzed only slightly and salicin not at all. The enzyme catalyzes the hydrolysis of glucosidic linkages in such a manner that the product retains the anomeric configuration of the substrate.

Increased Cellulose Production from Sucrose by Acetobacter after Introducing the Sucrose Phosphorylase Gene

A sucrose phosphorylase (SPase) gene derived from Leuconostoc mesenteroides was introduced into a cellulose-producing Acetobacter strain and expressed under the lac promoter. The activity of the SPase was detected in extracts of the transformed cells and cellulose production from sucrose by the cells was found to have increased, which strongly suggests that the increase was the result of the new metabolizing pathway. Furthermore, the level of SPase expression was increased by altering the length of the lac promoter.

Breeding of a 5-fluorouridine-resistant mutant with increased cellulose production from Acetobacter xylinum subsp. nonacetoxidans

UDP-glucose (UDP-G), the direct precursor of cellulose, is known to be produced from UTP and glucose-1-phosphate. In an attempt to increase UTP biosynthesis, 5-fluorouridine (5-FUR: a pyrimidine analog)-resistant mutants were obtained using Acetobacter xylinum subsp. nonacetoxidans 757 as the parent strain. One of the 5-FUR-resistant mutants, FUR-35, showed about 40% higher cellulose productivion compared to the parent strain. Intracellular levels of UTP and UDP-G in FUR-35 was found to be higher than those in the parent strain. The carbamyl phosphate synthetase II (CPS) activity of FUR-35 was higher than that of the parent strain and the feedback inhibition of CPS by UTP in FUR-35 had been released compared with that in the parent strain. These results suggest that the increased cellulose production of FUR-35 was attributable to its higher of intracellular UDP-G level resulting from increased UTP biosynthesis.

Cloning and Expression of thenifA Gene ofKlebsiella oxytocainK. pneumoniaeandAzospirillum lipoferum

The nif A gene in the 2.9-Kb SalI fragment from the nif gene cluster of Klebsiella oxytoca NG13, an associative nitrogen fixer in the rice rhizosphere, was cloned into the SalI site of pACYC184 to obtain pNOW7A (6.8Kb, Cmr, nif A+), in which the nif A gene was expressed from the constitutive Tcr gene promoter. A recombinant plasmid, pNT11 (15.7Kb, Sur, nif A+), was constructed by ligating pNOW7A and RSF1010 at single Eco RI sites. Constitutive expression of the nif A gene on pNOW7A in K. pneumoniae UNF714 nif A− and K. oxytoca NG13 caused derepression of the nitrogenase activity in the presence of NH4+. pNT11 induced nitrogenase activity in Azospirillum lipoferum in the presence of NH4+ to the level of 0.7% of that in the absence of NH4+.