Journal of Fermentation and Bioengineering最新文献

A cellulose triacetate (CTA) membrane containing trioctylmethyl ammonium chloride (TOMAC) as a carrier and o-nitrophenyloctyl ether (NPOE) as a plasticizer was developed for the in situ separation of fermented lactic acid. The separation characteristics of the CTA-TOMAC-NPOE membrane were investigated. The permeation rate of lactate through the CTA-TOMAC-NPOE membrane was comparable to that through a supported liquid membrane. TOMAC played the role of a mobile carrier in lactate transport. It was found that the CTA-NPOE membrane containing TOMAC has the advantages of allowing a high flux of lactate and having a low solubility of TOMAC in aqueous solution.

A novel arylesterase from Agrobacterium radiobacter IFO 12607 catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA) to form deacetyl 7-ACA, but is inactive with cephalosporin C. A DNA fragment carrying the gene encoding the 7-ACA-deacetylating enzyme was cloned from the chromosomal DNA of this bacterium. The open reading frame encoding the enzyme was 642 bp long, corresponding to a protein of 214 amino acid residues (molecular mass=23,085). The deduced amino acid sequence did not contain the sequence GXSXG, typical of the many serine esterases including Bacillus cephalosporin C deacetylase, but has the pentapeptide motif sequence GDSLT (amino acid position 9–13) which is also a consensus sequence of some serine esterases. The newly cloned gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product purified from E. coli exhibited the same catalytic properties as the enzyme purified from A. radiobacter. Site-directed mutagenesis of S11A or S11C within the pentapeptide motif sequence led to complete loss of the enzyme activity. Thus, the Ser-11 residue within the GDSLT motif sequence was determined to construct the catalytic center. These results together with those of our previous studies indicated that the 7-ACA-deacetylating enzyme from A. radiobacter IFO 12607 is a new member of the family of lipolytic serine esterases containing the GDSLT sequence as their catalytic center.

To determine the role of the C-terminal region of Cellvibrio gilvus β-glucosidase, a deletion mutant was constructed lacking five amino acid residues (RGRAR), three of which were arginine, from the C-terminal end. The mutant, designated ΔRGRAR, could be folded into an active form when expressed with the molecular chaperons $\text{GroEL}\text{ES}$. In comparison with the native enzyme, the optimum pH of the mutant ΔRGRAR shifted to the acidic region and the pH stability to the neutral region, while its heat stability decreased. No significant difference in the kinetic parameter K_m was observed. It was concluded that the RGRAR residues located at the C-terminal end are quite important for the stability of the enzyme and protein folding.

A non-self-transmissible multiple-copy plasmid, pEC3, isolated from the phytopathogenic bacterium, Erwinia carotovora subsp. carotovora, can be mobilized with the help of a self-transmissible plasmid. When donors carrying a cya or a crp mutation were used, the mobilization efficiencies of pEC3 were markedly decreased. The covalently closed circular conformation of plasmid DNA containing both oriT and mob genes of pEC3 was found to be relaxed after the addition of cyclic AMP (cAMP). The relaxed DNA contained a specific single-strand DNA nick within the oriT region. RNA transcription of the mob operon was also increased by the addition of cAMP. These results indicate that the expression of the mob operon of pEC3 is positively regulated by cAMP and that the mob gene products are involved in nicking within the oriT region.

An endo-1,4-β-d-galactanase (EC 3.2.1.89) was purified to homogeneity from a solid-state culture of Aspergillus sojae. The molecular weight of the galactanase was estimated to be 39,700 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration chromatography indicated the native enzyme to be a monomer. The isoelectric point of the galactanase was 3.60. The optimum pH and temperature of the enzyme activity were 4.5 and 50°C, respectively. The galactanase was stable from pH 6.0 to 10.0, and up to 35°C. The K_m value for arabinogalactan from soybean was 0.82 mg/ml. The activity of the enzyme was significantly inhibited by Mn²⁺, Hg²⁺, Ag⁺, and Fe³⁺, and no stimulation by metal ions was apparent. After the hydrolysis of arabinogalactan from soybean, the major products were galactobiose and galactose, and no liberation of arabinose was observed in the reaction mixture.

The nucleotide sequence of a β-glucanase gene from an extremely thermophilic anaerobe NA10 was determined. The open reading frame extended over 3000 bp and encoded a polypeptide with a molecular mass of 113 kDa. The deduced amino acid sequence of this protein exhibited high homology to a bifunctional cellulase CelB of Caldocellum saccharolyticum. Based on the homology to CelB, the NA10 β-glucanase appears to comprise three domains: N-terminal, central, and C-terminal domains. Among these, N- and C-terminal domains apper to be catalytic domains, and the central domain to be a cellulose binding domain. These domains were joined with each other by proline and threonine rich segments (PT box). The GST-fused C-terminal domain showed CMCase and MUCase activities, but the activities of the GST-fused N-terminal domain were very weak. Zymogram analysis revealed that recombinant Escherichia coli containing the β-glucanase gene produced a protein with a molecular mass of approximately 113 kDa, which was in good agreement with that deduced from the DNA sequence. However, Western blot analysis indicated that the amount of this full length protein was very small. Several smaller abundant proteins which exhibited CMCase activity were also detected. Northern blot analysis indicated that there appear to be putative internal transcriptional initiation sites. Generation of small molecular mass species appear to be due to alternative transcription and translation from the initiation sites within the gene, or partial proteolysis.

Class II fumarase (fumC) and Mn-containing superoxide dismutase (sodA) genes were cloned and sequenced from Thermus thermophilus HB27. The fumC and sodA genes existed in tandem. Comparison of the DNA and amino acid sequences of the funC-sodA regions between T. thermophilus HB27 and Thermus aquaticus YT1 revealed that coding regions showed high homology but noncoding regions were less homologous between the two strains. The deduced amino acid sequences of T. thermophilus fumC and sodA genes showed 58.2% and 53.2% identities to those of the corresponding Escherichia coli genes, respectively. The T. thermophilus fumC gene was expressed in E. coli and the heat-resistant fumarase activity was detected at 70°C.

A 6955-bp sequence of a PstI-HindIII DNA fragment containing the manganese-superoxide dismutase (MnSOD) gene of Thermus aquaticus YT-1 was determined. The gene (sod) encoded a polypeptide consisting of 204 amino acid residues (the mature enzyme without the initiation methionine) with a calculated Mr of 22,773. The deduced amino acid sequence of the sod gene showed 92% identity to that of Thermus thermophilus HB8 determined chemically. The sod gene was well expressed in Escherichia coli and a heat-stable active enzyme was produced. An open reading frame encoding fumarase was found 91 bp upstream of the MnSOD gene in tandem form. The fum gene encoded a polypeptide consisting of 466 amino acid residues with a calculated M_r of 50,950. The deduced amino acid sequence of the fum gene product has similarity to that of the fumC of E. coli (57% identity), suggesting that this gene encodes a class II type fumarase.