Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

实验大鼠泰泽菌检测方法的比较研究

目的　比较实验大鼠泰泽菌检测方法─nested PCR、IFA、免疫抑制诱发试验 触片染色镜检和组织病理学诊断。方法　根据泰泽菌 16SrDNA合成引物 ,对 16个菌株作nested PCR扩增并进行特异性、敏感性试验、验证。将此PCR应用于 2 0只免疫抑制Wistar大鼠和 5只非免疫抑制SD大鼠泰泽菌检测 ,并作IFA、常规细菌学检测和组织病理学诊断。结果　nested PCR中仅有泰泽菌出现 196bp特异性扩增条带 ;而 15株非泰泽菌均未出现此扩增条带。该PCR能检出 10pg泰泽菌DNA。将此PCR应用于大鼠泰泽菌检测 ,结果未检出阳性样品。nested PCR与常规细菌学检测、组织病理学诊断结果相一致。采用IFA方法 ,以购得的大鼠泰泽菌抗原片对上述 2 5份大鼠血清进行检测 ,结果有 6份血清产生非特异性反应。结论　采用IFA对动物群进行筛查出现阳性结果 ,须采用免疫抑制诱发试验、PCR和组织病理学诊断技术组合进一步验证。本研究建立的nested PCR方法 ,特异、敏感、快速 ,结合组织病理学诊断技术对实验动物泰泽菌感染可做出精确诊断。     

酪酸梭菌活菌胶囊对胆道结石患者术后肠功能紊乱的治疗作用

目的观察酪酸梭菌活菌胶囊(商品名:阿泰宁)对胆道结石患者术后肠功能紊乱的疗效及对术后消化吸收功能的改善作用。方法选取40例行胆总管切开探查术且术后出现腹泻、腹胀、消化不良等肠功能紊乱的胆总管结石患者。随机分为观察组和对照组,每组20例。两组患者均予以生理盐水补液、止泻药止泻和低脂饮食等常规治疗,观察组患者加服酪酸梭菌活菌胶囊,3粒/次,2次/d,连用28d。观察两组患者用药后的临床疗效及不良反应。结果观察组患者临床总有效率(100.0%)显著高于对照组(80.0%)(P0.05);腹泻和腹胀的时间显著短于对照组(P0.01);同时治疗后观察组患者总胆固醇、甘油三酯、球蛋白水平显著高于对照组(P0.05),电解质紊乱发生率低于对照组(P0.05)。治疗期间所有患者未见不良反应。结论酪酸梭菌活菌胶囊治疗胆道结石患者术后肠功能紊乱疗效显著,能够显著改善患者对脂质的消化吸收能力,对患者术后消化吸收功能的改善具有积极的作用,值得临床推广应用。     

Hydrogen oxidation by membranes from autotrophically grown Alcaligenes eutrophus H16: role of the cyanide-resistant pathway in energy transduction

Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, aK _m for acetate of 160 mM, and ak _cat of 16,800 min^-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.Abbreviations Acetyl-P Acetyl-phosphate - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide     

Electrotransformation studies in Clostridium cellulolyticum

Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays. Electrotransformation was then performed under optimized conditions (6 to 7.5 kV cm⁻¹ field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer). Transformants were only obtained when 7 or 7.5 kV cm⁻¹ pulses were applied. Transformation efficiencies evaluated from the growth curves of transformed cells were between 10⁵ and 10⁷ transformants per microgram of plasmid DNA for five different replicon-based plasmids. Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain. Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively. Journal of Industrial Microbiology & Biotechnology (2001) 27, 271–274. Received 12 September 2000/ Accepted in revised form 25 November 2000