Journal of Fermentation and Bioengineering最新文献

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 K_m and V_max 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.

The synthesis of the four possible stereoisomers of 21-methyl-8-pentatriacontene, the (R)- and (S)-enantiomers of both (Z)- and (E)-geometrical isomers, was achieved by starting from the enantiomers of 3-hydroxy-2-methylpropionate, 1-nonyne and 1,10-decandiol to evaluate the response of the male yellow-spotted longicorn beetle, Psacothea hilaris.

The interaction between homoacetogens and methanogens in lake sediments was investigated using hydrogen consumption as an indicator. Sediments samples were obtained from Lake Izunuma, Miyagi prefecture, Japan, a wintering place for migratory birds from Siberia. A batch experiment using $\text{H}{}_2\text{CO}{}_2$ as a substrate was conducted to determine the acetate generation and methane production potential of the sediments. Incubation for 4 d at 37°C gave the following stoichiometric equation: 88H₂ + 39HCO₃⁻ + 22H⁺ → 17CH₃COO⁻ + 5CH₄ + 83H₂O. The activities, ν_m, of hydrogen-utilizing homoacetogens and methanogens respectively ranged from 3.2 to 48 and from 1.8 to 3.2 mgCOD·gVSS⁻¹·h⁻¹. The population of hydrogen-utilizing homoacetogens was determined to be 2.6 × 10⁸ MPN·gVSS⁻¹, which was approximately two orders of magnitude higher than that of hydrogen-utilizing methanogens. The results suggest that homoacetogens in the sediments functioned not only as hydrogen consumers but also as major degraders of organic matter, forming acetate as the major reduction product.

A high cell density culture of Rhodococcus rhodochrous IGTS8 was investigated. Acetic acid was one of the most suitable carbon sources for cell growth and sulfate ion was more suitable than dibenzothiophene (DBT) as a sulfur source. Fed-batch culture was conducted in a 1-l jar fermentor with FB medium containing acetic acid and sulfate ion as carbon and sulfur sources. Cell growth was found to be inhibited when the concentrations of acetic acid and ammonium ion were above 3 g/l. To control the concentrations of the two components below 3 g/l, a mixture of acetic acid and ammonium acetate was supplied by means of pH-stat feeding. As a result, a cell concentration of 33 g dry cells/l was obtained after 28-h cultivation. When the cells obtained were incubated in a fresh medium containing DBT as a substrate, hydroxybiphenyl (HBP), which is the end-product of the DBT degradation pathway, was detected and its production rate gradually increased with incubation time. Incubation for 3 to 4 h was enough for the full induction of DBT-degrading enzymes, and the specific production rate of HBP was about 6.1 mmol/kg dry cells/h. A two-phase cultivation (cell growth phase and induction phase) is proposed in order to obtain a high cell density and full induction of DBT-degrading enzymes.

Extractive lactic acid fermentation has recently been paid a great deal of attention. The problem with such a process is, however, that only undissociated lactate can be extracted. Therefore, lactic acid fermentation at low pH values is desirable. In the present study, we modified the metabolism of yeast (not lactic acid producing bacteria often cultivated at pH of 6–7) by expressing the lactate dehydrogenase (LDH) gene for the production of lactate at low pH values. For this purpose, the plasmid pADNS which contains the ADH1 promoter was used as a host vector, and a heterologous gene region, cDNA-LDH-A (encoding bovine lactate dehydrogenase) digested from plasmid pLDH12 was digested and ligated into the aforementioned two host vectors. The resultant plasmids were then transformed into Saccharomyces cerevisiae DS37. Using this recombinant S. cerevisiae strain, several batch and fed-batch fermentations at aerobic, microaerobic, and anaerobic conditions were conducted at several pH values (4.5-3.5). Since the recombinant S. cerevisiae produced a considerable amount of ethanol as well as lactate (about 10 g/l), we disrupted several pyruvate decarboxylase (PDC) genes to suppress the ethanol formation. Among the PDC genes, PDC1, PDC5 and PDC6, PDC1 had the greatest effect on the cell growth and ethanol production. The plasmid which containing the LDH-A structure gene was then transformed into the mutant strain lacking the PDC1 gene. Cultivation of this strain improved the lactate yield from glucose (from 0.155 to 0.20) while suppressing ethanol formation (from 0.35 to 0.20).

The response surface method (RSM) was used to optimize the medium for the production of inulinase by Kluyveromyces sp. Y-85. The inulinase production was adequately approximated with a full quadratic equation obtained from a four-factor-five-level central composite design. Analyses of the quadratic surfaces showed that in a 24-h fermentation at 30°C, the maximum inulinase activity 59.5 U/ml appeared at extract of Jerusalem artichoke, urea, beef extract, corn steep liquor concentrations 8.0%, 2.0%, 0.2% and 4.0%, respectively. We further investigated the fermentation in 15 l fermentor and scaling-up in 1000 l tower fermentor. The inulinase activity in the scaling-up was 68.9 U/ml, which was the highest production reported at present, indicating that Kluyveromyces sp. Y-85 was an excellent strain for industrial production.

Uridine 5′-monophosphate α-d-glucose (UMPG) was evaluated as a novel and potent inhibitor of the enzymatic reaction involved in sugar nucleotide metabolism. UMPG was synthesized by chemical coupling of 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide with uridine 5′-monophosphate (UMP) to give uridine 5′-monophosphate 2″,3″,4″,6″-tetra-O-acetyl-α-d-glucose (UMPTAG), followed by deacetylation of UMPTAG with sodium methoxide. In addition to UMPG, UMPTAG showed potent inhibitory activity toward yeast UDPG pyrophosphorylase (UDPG synthetase). UMPG and UMPTAG were competitive with UDPG in the pyrophosphorolytic reaction, with inhibition constants (K_i) of 4.8 and 20.7 μM, respectively, but non-competitive with inorganic pyrophosphate. UMPG and UMPTAG also inhibited the enzyme non-competitively in the reverse reaction to synthesize UDPG from UTP and glucose 1-phosphate (G1P). The acetyl group of UMPTAG was thought to enhance its hydrophobic interaction, possibly with an active site region of the enzyme functional for binding with UDPG.

Aeromonas sobria LP004 harboring the Acinetobacter calcoaceticus lipase gene was designated as strain LP094. Lipase production of LP094 in WYGS medium [Lotrakul and Dharmsthiti, World J. Microbiol. Biotechnol., 13 : 163–166, 1997] was scaled up to a 50-l volume. LP094 lipase immobilized by ionic binding to either IR120 (Na⁺) or IRC50 (H⁺) Amberlite resins was found to retain more than 90% activity after 15 d storage at room temperature (≈ 25 to 30°C) or at 4°C. After 5 repeated lipid hydrolysis reactions, the activities of both immobilized preparations remained higher than 65%.