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The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (BdsB), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail.  相似文献   

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Li  Liu  Zheng  Zhe  Zhao  Xiao  Wu  Fengyu  Zhang  Jian  Yang  Zhennai 《Food science and biotechnology》2019,28(4):1107-1116
Food Science and Biotechnology - Bacillus methanolicus LB-1 isolated from traditional rice wine was found to produce a milk clotting enzyme (MCE), and its fermentation conditions were optimized...  相似文献   

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We have screened 39 microfungi isolates originated from soil in terms of lipolytic activity. Out of all screened, a novel strain of Mucor hiemalis f. corticola was determined to have the highest lipase activity. The extracellular lipase was produced in response to 2% glucose and 2.1% peptone. The lipase was purified 12.63-folds with a final yield of 27.7% through following purification steps; ammonium sulfate precipitation, dialysis, gel filtration column chromatography and ion exchange chromatography, respectively. MALDI-TOF MS analysis revealed 31% amino-acid identity to a known lipase from Rhizomucor miehei species. The molecular weight of the lipase was determined as 46kDa using SDS-PAGE and analytical gel filtration. Optimal pH and temperature of the lipase were determined as 7.0 and 40°C, respectively. The enzyme activity was observed to be stable at the pH range of 7.0-9.0. Thermostability assays demonstrated that the lipase was stable up to 50°C for 60min. The lipase was more stable in ethanol and methanol than other organic solvents tested. Furthermore, the activity of the lipase was slightly enhanced by SDS and PMSF. In the presence of p-NPP as substrate, K(m) and V(max) values of the lipase were calculated by Hanes-Woolf plot as 1.327mM and 91.11μmol/min, respectively.  相似文献   

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大曲细菌关系到白酒酿造的品质,其中酶的含量与活性与白酒的风味紧密关联。以芝麻香型白酒高温大曲优势细菌代表菌株Q2B1为研究对象,利用分子生物学技术对其分类学地位进行研究,并通过全基因组研究技术对产酶相关功能基因与代谢通路进行研究。结果表明,菌株Q2B1被鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。利用传统的培养技术定性定量地检测其酶活力,发现菌株Q2B1可产淀粉酶和蛋白酶,活力分别为5.852 U/mL和26.770 U/mL;其产酶相关功能基因组长度为3 475 602 bp,包括蛋白酶编码基因以及淀粉酶编码基因,在基因水平上初步揭示了Q2B1菌株合成蛋白酶和淀粉酶的分子机理,为进一步合理开发白酒大曲微生物奠定理论基础,为提高白酒品质提供科学依据。  相似文献   

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An extracellular aspartyl proteinase from Mucor mucedo DSM 809 submerged cultures was purified by a two-steps chromatographic procedure. The enzyme had a molecular weight (MW) of 32.7 kDa, and an isoelectric point (pI) value of 4.29; no evidence of N-linked glycosylation was found. As judged by mass spectrometry, the primary structure of the M. mucedo enzyme presented homology with Rhizopus spp. proteinases. The secondary structure showed 4% α-helix, 48% β-sheet and 48% random coil structure in 20 mM phosphate buffer (pH 5.8), as evidenced by circular dichroism spectroscopy. When acting on milk to provoke curd formation, the proteinase showed maximum potency at pH 5.0 and at 40 °C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55 °C for 10 min. These results indicate that the milk-clotting enzyme from M. mucedo can be considered as a potential substitute for bovine chymosin in cheese manufacturing.  相似文献   

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Three forms of pectin methylesterase (PME) were purified, from bergamot fruit (Citrus bergamia R.), to homogeneity by ion-exchange and affinity chromatography. The isoforms, named PME I, PME II and PME III, according their elution order on a heparin–sepharose column, were characterized for their relative molecular mass, activity kinetic parameters and thermostability. The molecular mass was estimated to be 42 kDa for the three forms, and the apparent Km values for citrus pectin were 0.9 mg/ml for PME I and 0.5 mg/ml for PME II and PME III. The optimum pH values lie within the range 6.5–9.0, depending on salt concentration. Thermal behaviours of the three PME isoforms were studied in a temperature range from 65 °C to 80 °C with the less abundant PME I isoform showing a higher heat resistance. Moreover, the complete exon 2 sequence of PME gene was acquired (GenBank accession no. DQ458770) using a PCR-based approach on well-known Citrus genomic DNA present in the NCBI database.  相似文献   

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6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.  相似文献   

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Bdellovibrio-and-like organisms (BALOs) are a group of highly motile delta-proteobacteria that prey on other gram-negative bacteria. However, nothing is known of the application potential of marine BALOs in safeguarding seafood safety. Here, biological characterization of two marine BALOs strains and their application in the elimination of Vibrio parahaemolyticus in oyster (Crassostrea ariakensis) at the laboratory scale were investigated.BALOs strains BDH12 and BDHSH06 were isolated from sediment of Daya bay in Shenzhen of China, with Shewanella putrefaciens strain 12 and V. parahaemolyticus strain SH06 as preys, respectively, when using double layer agar technique. They were identified as BALOs morphologically by transmission electron microscopy, while partial 16S rDNA sequencing analysis revealed that they showed no close relationships with members of the known genera Bdellovibrio, Bacteriolyticum, Bacteriovorax, or Peredibacter.Biological characterizations revealed that both strains had the optimal pH, salinity and temperature at 7.2, 3% and 30 °C, correspondingly. They could not utilize autoclaved, dead cells as hosts. Prey range analysis revealed that individually, BDH12 and BDHSH06 lysed 82.5% (47 strains) and 84.2% (48 strains) of the total 57 preys tested respectively. In combination, they lysed 98.2% (56 of 57) strains. All strains of V. parahaemolyticus, Vibrio cholerae and Vibrio alginolyticus tested could be lysed by both strains.A 7-day laboratory-scale V. parahaemolyticus elimination experiment in oyster showed that in the control, the cell counts of total vibrios and V. parahaemolyticus strain Vp plus in water and in oyster intestines were on the rise, whereas in the BALOs treated groups, their numbers were down from 8.09 ± 0.05 log CFU/ml and 8.02 ± 0.04 log CFU/ml to 2.39 ± 0.01 log CFU/ml and 2.33 ± 0.01 log CFU/ml, respectively. The same patterns could also be observed in oyster intestines. Results of this study indicate the feasibility of using BALOs to biologically control or even eliminate V. parahaemolyticus in seafood oyster.  相似文献   

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Lactobacillus plantarum UG1 isolated from dry sausage produced an antimicrobial substance that inhibited other strains of the genera Lactobacillus and Lactococcus, and some foodborne pathogens including Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Clostridium sporogenes. This antibacterial substance was inactivated by proteolytic enzymes and showed a bactericidal mode of action. Consequently, it was characterized as a bacteriocin, and was designated plantaricin UG1. This bacteriocin was stable in the pH range 4.5 to 7.0, partially inactivated by amylolytic enzymes and relatively thermostable. It was not affected by organic or lipolytic enzymes. Production of plantaricin UG1 was pH-and temperature-dependant and maximum yields were obtained in MRS broth cultures maintained at initial pH 6.5, and incubated at 25 °C to 30 °C, in the exponential to the early stationary growth phase of the producer organism. Ultrafiltration studies indicated that plantaricin UG1 has a molecular weight between 3 and 10 KDa. Curing experiments with L. plantarum UG1 resulted in the appearance of variants that lost bacteriocin production ability but were still immune to the bacteriocin. Plantaricin UG1 production appeared to be chromosomal encoded. Sensitive and insensitive Gram-positive bacteria adsorbed plantaricin UG1 irrespective of their susceptibility to it. In contrast, Gram-negative bacteria did not adsorb plantaricin UG1. The bactericidal action of plantaricin UG1 did not depend on the physiological state of the indicator culture and did not cause cell lysis. The resistance of two indicator strains to plantaricin UG1 has been studied.  相似文献   

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