Journal of Fermentation and Bioengineering最新文献

The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was successfully expressed in cytoplasm of Saccharomyces cerevisiae, using two cytoplasmic linear plasmids, pGKL1 and pGKL2. In order to integrate the glaA gene, two pGKL1-derived plasmids, designated pKTF951 and pKTF952, were constructed. The glaA gene was placed downstream of the ORF2 promoter (UCS2) between ORF1 and ORF3 of pGKL1 to obtain pKTF951. pKTF952 contained an additional ORF5 promoter (UCS5) from pGKL2, connected downstream of the UCS2 in pKTF951. pKTF951 and pKTF952 were respectively transformed to S. cerevisiae carrying the original pGKL1 and pGKL2. Southern analysis revealed that in vivo homologous recombination took between the indigenous pGKL1 and the recombinant pKTF951 or pKTF952 within the common ORF1 and ORF3 regions, which resulted in the replacement of ORF2 by the glaA gene in pGKL1. Transformants carrying the resultant linear recombinant plasmids, pNS951 or pNS952, produced GAI. S. cerevisiae (pNS952) secreted 2.6-fold more GAI than S. cerevisiae (pNS951).

Modulation of the enantioselectivity of two lipases exhibiting different specificities in the esterification of 2-octanol with lauric acid was investigated by utilizing surfactant-coating and molecular imprinting techniques. The enantioselectivity of the surfactant-coated lipase from Pseudomonas cepacia (PS) was enhanced in the presence of (R)-2-octanol as a print molecule, whereas the enantioselectivity of the lipase from Candida cylindracea (AY) was hardly changed by the imprinting technique. In the case of lipase AY, however, the selectivity of the native enzyme toward the (R)-isomer was changed to a preference for the (S)-isomer as a result of being coating with surfactant molecules. The molecular imprinting effect was prominent in the case of lipase PS, its enantioselectivity in the formation of 2-octanoate in isooctane being enhanced around two-fold compared with that of the native enzyme.

Alkaline pulping liquors are problematic for anaerobic treatment due to their toxicity to methanogens and their relatively large fraction of inert lignin. In previous research, toxicity due to wood extractives was shown to be highly eliminated during aerobic wastewater treatment, but not during anaerobic treatment. These observations have led to the proposal of a detoxification strategy denominated upfront dilution, based on the sequenced anaerobic-aerobic treatment of the pulping liquor, recirculating the aerobic effluent to dilute the incoming influent to sub-toxic concentrations. In this study, the treatment of highly toxic hemp stem wood black liquor (HSWBL) in lab-scale UASB reactors with upfront dilution was compared with direct anaerobic treatment and with direct aerobic treatment. Direct anaerobic treatment of 12 g COD/l HSWBL led to almost complete inhibition of the methanogenic activity within 14 d. However, recirculation of 75% of the aerobic post-treatment effluent for upfront dilution of the toxic HSWBL, enabled anaerobic treatment at loading rates up to 21.5 g COD/l_UASB·d without significant inhibition of the methanogenic activity. Extensive detoxification was confirmed during anaerobic-aerobic treatment of 20 g COD/l HSWBL recirculating 86% of the aerobic effluent. COD and BOD removal was 72% and 97%, respectively, after anaerobic-aerobic treatment at an overall loading rate of 3.6 g COD/l·d, while 30–35% of the incoming COD was recovered as methane. Batch-assays demonstrated significant detoxification after anaerobic-aerobic treatment of HSWBL. Treatment efficiencies and detoxification during anaerobic-aerobic and aerobic treatment were similar. However, the anaerobic-aerobic treatment system provided 50% lower surplus sludge production, production of 0.16 m³ methane/kg COD_removed as an energy source, less nutrient dosage and substantial reductions in aeration costs. During anaerobic-aerobic treatment as well as aerobic treatment significant lignin removal was obtained, ranging from 28–58%. Lignin removal could be attributed to biodegradation of low molecular weight lignin (MW < 2.2 kD). The lignin which survived biological treatment was extensively polymerized to MW of > 34 kD.

Carboxypeptidase Y (CPY; EC 3. 4. 16. 1) encoded by the PRC1 gene is a yeast vacuolar protease. It enters the vacuole as a zymogen, proCPY, which is activated by vacuolar enzymes, proteinase A (PrA) and proteinase B (PrB). We previously showed that active CPY was efficiently secreted from the Saccharomyces cerevisiae ssl1 (supersecretion of lysozyme) mutant carrying a multicopy plasmid which contains the PRC1 gene fused to an inducible GAL10 promoter. In this study, we detected PrA and PrB activities in the culture supernatant of the ssl1 mutant harboring the CPY expression plasmid. The N-terminal amino acid sequence of extracellular CPY coincided with that of the original vacuolar one. Furthermore, studies using protease inhibitors suggested that CPY was secreted into the medium in the form of a precursor and was mainly activated by extracellular PrB. The ssl1 mutant secreted CPY, PrA and PrB into the medium even with a single copy of the PRC1 gene. On the other hand, a cytoplasmic marker enzyme, glucose-6-phosphate dehydrogenase, and a vacuolar membraneassociated enzyme, α-mannosidase, were not detected in the medium, whether the PRC1 gene was overexpressed or not. It is suggested that secretion of vacuolar proteases is caused by characteristics of the ssl1 mutation or overexpression of the PRC1 gene.

An increase in extracellular nucleotides appeared to conform to simple diffusion, when Saccharomyces cerevisiae cells were suspended in 20% ethanol at 15°C. However, in the case of cells grown in the presence of 8% ethanol, the leakage of nucleotide appeared to be repressed significantly in the initial phase. The addition of glucose led to a continuation of the repression of the leakage. Under this condition, the addition of iodoacetamide as an inhibitor in glycolytic pathway, or stilbestrol as an inhibitor of plasma membrane ATPase, resulted in a rapid increase in the leakage of nucleotides, indicating that the membrane permeability barrier is dependent on membrane ATPase. Ouabain, an inhibitor of the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}\text{-ATPase}$, had no effect. The addition of 10 mM CaCl₂ for inducing lateral phase separation in the inner membrane, which caused a faster release of nucleotides in general, had a little effect on nucleotide leakage, when cells grown in the presence of 8% ethanol were suspended in 20% ethanol containing glucose. Moreover, the addition of 10 mM KCl, together with CaCl₂, had almost no effect on leakage. Ca²⁺ influx was found to be smaller in cells grown in the presence of ethanol when compared to cells grown in the absence of ethanol, when fura-2 loaded cells were suspended in K-MOPS buffer containing 50 mM CaCl₂. Divalent cations such as Ba²⁺, Mg²⁺ and Mn²⁺ had effects similar to Ca²⁺ on membrane permeability. The decrease in cell viability corresponded to the amount of leakage of nucleotides over the experimental time course. These results suggest that the high activity of membrane ATPase which is induced by growth in the presence of 8% ethanol ensures homeostasis of ions in the cytoplasm at high concentrations of ethanol, and reduces the effects of membrane surface-acting substances such as divalent cations on the membrane integrity. Thus the maintenance of the cell membrane as a permeability barrier appears to lead to a high ethanol-endurability.

Bacterial cellulose (BC) was produced from fructose by Acetobacter xylinum subsp. sucrofermentans BPR3001A was performed in continuous culture and the effect of lactate on the production was investigated. In continuous culture with feeding of CSL-Fru medium containing 30 g·l⁻¹ fructose at a dilution rate of 0.07 h⁻¹, a higher production rate (0.62 g·l⁻¹·h⁻¹) was obtained than that (0.40 g·l⁻¹·h⁻¹) in a batch culture using CSL-Fru medium with 70 g·l⁻¹ initial fructose. However, when the dilution rate or fructose concentration in the feed medium were increased, the total yield of BC declined because the residual fructose concentration in the drawn broth increased. Supplementing 12.5 g·l⁻¹ lactate to the feed medium increased the cell concentration and fructose consumption at a steady state, resulting in a production rate of 0.90 g·l⁻¹·h⁻¹ and a BC yield of 36% at a dilution rate of 0.1 h⁻¹. The ATP content of viable cells was maintained at a higher level by feeding a lactate-supplemented medium rather than the unsupplemented CSL-Fru medium. In a batch culture using lactate as the main carbon source, 77% of the lactate consumed was oxidized to CO₂ and only 6.9% was converted to BC. These findings indicated that lactate functioned as an energy source, not as a substrate for BC biosynthesis. Increased intracellular ATP resulting from lactate oxidation may have improved the fructose consumption and BC production in the continuous culture.

The EST2 gene, encoding an isoamyl acetate-hydrolyzing esterase, was disrupted in a diploid strain of Saccharomyces cerevisiae UT-1 (MATa/MATα ura3/ura3 trp1/trp1 EST2/EST2), which is derived from the industrial sake yeast Kyokai no. 701 (strain K-701), by using two disruption plasmids (pDest2U, est2::URA3; and pDest2T, est2::TRP1) sequentially. Genomic Southern blot analysis revealed that both loci of the EST2 gene on the chromosome of strain UT-1 were disrupted. The resultant mutants were named UTUT-1 and UTUT-2 (a/MATα ura3/ura3 trp1/trp1 est2::URA3/est2::TRP1). Deficiency in Est2p esterase was also confirmed by activity staining of the gel after native-polyacrylamide gel electrophoresis of cell extracts of the two mutant strains. Small scale sake brewing was carried out using these sake yeasts and the strains they were derived from, and their brewing properties were compared. The fermentation profiles of the four strains (strains K-701, UT-1, UTUT-1, and UTUT-2) were largely similar. The components of the resulting sake were also similar except for the acetate ester concentration, although strains UTUT-1 and UTUT-2 produced approximately 2-times more isoamyl acetate than the wild type K-701. These resuts strongly suggest that the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in the sake mash. Strains UTUT-1 and UTUT-2, deficient in Est2p esterase, are suitable for sake brewing.

To produce levansucrase, a fructosyltransferase enzyme, in a recombinant Escherichia coli harboring the levU gene of Zymomonas mobilis, fermentation strategies were examined in terms of induction methods and plasmid stability. Although the recombinant levansucrase was induced rapidly by IPTG, high instability of the plasmid and formation of inclusion bodies were observed. Segregational instability was aggravated by the excretion of β-lactamase into the culture broth during subculturing, which caused an overgrowth of plasmidfree cells. A combination of methicillin (2, 6-dimethoxyphenyl-penicillin) and ampicillin to provide selective pressure was effective in preventing the growth of plasmid-free cells. The population of plasmid-harboring cells was maintained above 95% of the total cells for more than 100 generations under this condition. In order to replace IPTG, which is toxic and too expensive for use in a large scale, d-lactose was tested and found to be favorable as an alternative inducer.

In order to study the inhibitory effect of the partial pressure of carbon dioxide (pCO₂) on bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR3001A, the BC concentration, oxygen consumption rate, viable cell concentration, and ATP content of the cells were investigated during cultivation in a 50-l jar fermentor sparged with air containing 10% (v/v) CO₂. A high pCO₂ (0.15–0.20 atm) reduced the BC yield, BC production rate per unit volume of broth (the volumetric BCPR), and viable cell concentration. However, it enhanced the oxygen consumption rate and the ATP content per viable cell, and consequently the BC production rate per viable cell (the specific BCPR). Thus, a high pCO₂ reduced BC production due to a reduction of cell growth and not by inhibiting BC biosynthesis. A possible reason for the reduction of the BC yield arising from a high pCO₂ is the dissipation of substrate for excess ATP generation.

This paper studies the temperature effect on the specific growth rate of the microorganisms (μ_c) as particular aspect of the mathematical relationships between μ_c of Thiobacillus ferrooxidans and the concentrations of substrate (ferrous iron) and product (ferric iron). Until now, there have been few studies of this relationship; also, there has been little work studying the influence of temperature on growth kinetics of this bacteria. In this paper, an extensive experimental phase has been developed, with the aim of determining with precision the influence of temperature on the maximum specific growth rate (μ_max). The influence on the growth rate of T. ferrooxidans exerted by the initial pH of the medium and by agitation rate has also been studied.