Hisaharu Taguchi

Purification of two alcohol dehydrogenases from Zymomonas mobilis and their properties

SummaryTwo alcohol dehydrogenases (ADHI and ADHII, EC 1.1.1.1) were purified to homogeneity from the cell extract of Zymomonas mobilis. The subunit molecular weights of ADHI and ADHII were 40,000 and 38,000, respectively, and both enzymes were homologous dimers. The optimal pHs of ADHI in ethanol oxidation and acetaldehyde reduction reactions were 9.5 and 4.5, and those of ADHII were 9.5 and 6.5, respectively. The optimal temperatures of ADHI and ADHII were 55° C and 45° C, respectively. ADHI was heat-inactivated at 65° C at a 10-fold higher rate than ADHII. ADHI and ADHII were inhibited by 4 μM and 1 mM p-chloromercuribenzoate, respectively, and the inhibitions were reversed by the addition of 70 mM 2-mercaptoethanol. ADHII activity was enhanced by 0.02 to 2 mM CoCl2 and inhibited by 0.4 mM o-phenanthroline; and the activity of inactivated ADHII was restored by addition of 1 mM CoCl2 or ZnCl2.ADHI was active on most primary alcohols but not secondary alcohols. ADHII was active on only ethanol, n-propanol, allylalcohol, and furfuryl alcohol.In the anaerobic culture of Z. mobilis, ADHII activity accounted for more than 80% of total alcohol dehydrogenase activity. In aerobic culture, ADHII was the main enzyme but was produced only in the early growth phase.

Genetic construction of yeast strains for high ethanol production

SummaryTo improve ethanol production capability, a flocculent yeast,Saccharomyces cerevisiae TJ1, was hybridized with other high-alcohol producing strains by protoplast fusion. A fusant strain of strain TJ1 hybridized with an Awamori-brewing strain, N1, produced 12.4 (w/v)% of ethanol even at 40°C. The fusant showed good flocculence and good sedimentation. In a repeated batch culture with cell reuse at 37°C, an average productivity of 13 g/ℓ·h was attained.

Hyperproduction of phenylalanine by Escherichia coli: application of a temperature-controllable expression vector carrying the repressor-promoter system of bacteriophage lambda

For the high production of phenylalanine by Escherichia coli, we cloned the pheAFR and aroFFR genes (FR = feedback resistant), which encoded chorismate mutase P-prephenate dehydratase and 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase that are feedback inhibition-free as to the endproducts, into a temperature-controllable expression vector composed of the PR and PL promoter and a temperature sensitive repressor, cI857, of bacteriophage lambda. The plasmid obtained was designated as pSY130-14, and the temperature dependency of expression of the cloned genes and of phenylalanine production was investigated at different temperatures between 30 and 42°C using the strain AT2471 harbouring the plasmid. Above 35°C, the pheAFR gene and aroFFR gene expressions, and activities of both enzymes continued to increase up to 42°C. The cell concentration remained constant up to 38.5°C, but started to decrease sharply above 40°C, while the cell concentration of the host strain, AT2471, remained constant at all temperatures tested. The concentration of phenylalanine also depended on the temperature, and the highest production of phenylalanine, 18.6 g l−1, was obtained from glucose at 38.5°C in a 2.5 1 reactor.

Dewatering characteristics of activated sludges and effect of extracellular polymer

Shiratos expression theory, which is based on Ruths filtration theory and Terzaghis consolidation theory, was applied to an analysis of the dewatering characteristics in activated sludges. It was ascertained that the process could be divided into periods of filtration and consolidation. It was further confirmed that Ruths average specific resistance (αav) was increased and Shiratos modified consolidation coefficient (Ce) decreased by adding the extracellular polymer substance extracted from the sludges. That is to say, the extracellular polymer was found to have a bad effect on the dewatering process through the two periods. From the results of compression-permeability testing, the addition of extracellular polymer was found not to affect the local porosity (ϵ), but it did have a great influence on the local specific filtration resistance (α).

Effect of glucose concentrations on xanthan gum production by xanthomonas campestris

Abstract The effect of the glucose concentration on xantham gum production by Xanthomonas campestris ATCC 13951 was studied resulting that the glucose concentration between 30 and 40 g/kg broth was best for xanthan gum production. Controlling the glucose concentration at between 30 and 40 g/kg broth by intermittent addition of glucose prevented the inhibition of cell growth and the cessation of xanthan gum production, which were observation with a higher glucose concentration. By means of a glucose feeding strategy, the xanthan gum concentration reached 43 g/kg broth after 96-h cultivation.

Hydrolysis of cellulose by cellulases of Sporotrichum cellulophilum in an ultrafilter membrane reactor

Cellulose has been continuously hydrolysed by the crude enzymes of Sporotrichum cellulophilum in an ultrafilter membrane reactor with the intermittent addition of cellulose. The product concentration decreased gradually from 30 to 16 g l−1 during 5 days reaction. Glucose accounted for 55–60% of the product. This reactor was five times more efficient per unit weight of enzyme used than the batch reaction. The slower flow rate gave higher product concentration at a flow rate of 3–30 ml h−1 but the production of cumulative total sugar was almost same at a flow rate > 15 ml h−1. In the long term hydrolysis at 30 to 60°C, the production of cumulative total sugar was highest at 50°C. The inactivation in the reactor was due to a protease present in the cellulase preparation, but not to heat denaturation.

Mechanistic analysis of xanthan gum production in a stirred tank

Abstract The overall effect of agitation on xanthan gum production by Xanthomonas campestris ATCC13951 in a stirred vessel was mechanistically analyzed considering local variation of the specific production rate due to variation of shear stress in the vessel. The whole liquid volume in a fermentor was roughly divided into three regions; the micromixing region around the impeller with high shear stress, the macromixing region dominated by a circulating flow and the stagnant region. The value of the shear rate was first ascertained by experiments in order to obtain a picture of shear rate variation in a radial direction from the impeller, and the equivalence between the volumes of the high shear stress region and micromixing region was confirmed. The shear stress obtained using a correlation between the shear rate at the impeller tip and Reynolds number of Wichterle et al. was used as a representative of the shear stress in the micromixing region, and the shear stress estimated by use of an empirical correlation between the average shear rate in a fermentor and agitation speed derived by Metzner et al. was adopted as a representative of the shear stress in the macromixing region. The information about the circulation time distribution was also used to take into account oxygen deficiency during circulation of liquid elements in the macromixing region, considering that oxygen from the gas phase was supplied mainly in the high shear region. The calculated values of xanthan gum concentrations which were obtained by the proposed simulation method agreed well with the experimental data in the time course of xanthan gum production at various agitation speeds. Experimental results of the relationship between the overall specific production rate and ND (N, agitation speed, and D, impeller diameter) was also verified by the proposed method.

Synthesis of cephalosporin C by a methionine analogue resistant mutant of Cephalosporium acremonium

SummaryDL-seleno-methionine resistant mutants of Cephalosporium acremonium were isolated which have an enhanced capacity to utilized sulfate for the synthesis of cephalosporin C. Of these mutants, one designated as SMR-I3 produced three-fold more cephalosporin C from sulfate than its parent CW19. Mutant SMR-I3 required less dl-methionine for maximal synthesis of cephalosporin C, but an excess of dl-methionine inhibited the synthesis of the antibiotic. Furthermore, the mutant accumulated excessive methionine in the amino acid pool and possessed superior activity for sulfate uptake. These observations indicate that in the mutant SMR-I3, the biosynthesis of methionine from sulfate is very active and excess methionine becomes available for the synthesis of cephalosporin C.

15β-Hydroxylation of Lithocholic Acid by cunninghamella sp.

Abstract The microbiological transformation of lithocholic acid (3α-hydroxy-5β-cholanic acid) into 3α, 15β-dihydroxy-5β-cholanic acid by Cunninghamella sp. is Investigated. The structure has been determined on the basis of two-dimensional 1 H-NMR.

Transformation of lithocholic acid to a new bile acid, 3α, 15β-dihydroxy-5β-cholanic acid by Cunninghamella blakesleeana ST-22

SummaryA fungus identified as Cunninghamella blakesleeana (Lendner) can carry out 15β-hydroxylation of lithocholic acid to a new bile acid (3α,15β-dihydroxy-5β-cholanic acid). By optimizing the fermentation conditions, the amount of the product increased from 0.17 g/l to 1.2 g/l. Hydrophilicity measurements and in vitro cholesterol solubilization tests showed that 3α, 15β-dihydroxy-5β-cholanic acid was as effective as ursodeoxycholic acid in cholesterol solubilization.