Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K _m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - MDH methanol dehydrogenase - ADH acohol dehydrogenase - PQQ pyrroloquinoline, quinone - DTT dithiothreitol - NBT nitrobluetetrazolium - PMS phenazine methosulphate - DCPIP dichlorophenol indophenol     

Isolation and characterization of a methyl chloride utilizing,strictly anaerobic bacterium

From sludge obtained from the sewage digester plant in Stuttgart-Möhringen a strictly anaerobic bacterium was enriched and isolated with methyl chloride as the energy source. The isolate, which was tentatively called strain MC, was nonmotile, gram-positive, and occurred as elongated cocci arranged in chains. Cells of strain MC formed about 3 mol of acetate per 4 mol of CH₃Cl consumed, indicating that the organism was a homoacetogenic bacterium fermenting methyl chloride plus CO₂ according to: The organism grew with 2–3% methyl chloride in the gas phase at a doubling time of near 30 h. Dichloromethane was not utilized. The bacterium also grew on carbon monoxide, H₂ plus CO₂, and methoxylated aromatic compounds. Optimal growth with methyl chloride was observed at 25°C and pH 7.3–7.7. The G+C-content of the DNA was 47.5±1.5%. The methyl chloride conversion appeared to be inducible, since H₂ plus CO₂-grown cells lacked this ability. From the morphological and physiological characteristics, the isolate could not be affiliated to a known species.     

成人革兰阴性杆菌肺炎23例临床分析

本文以临床体征,胸片和连续二次以上细菌培养阳性为诊断标准,报告革兰阴性杆菌肺炎２３例,并就其临床表现,并发症及预后进行分析,提出８种情况应考虑革兰阴性杆菌肺炎,对临床诊断和治疗有指导意义．     

Some factors influencing the proportion of periplasmic hepatitisB virus pre-S2 antigen in the recombinant yeastHansenula polymorpha

Summary A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitisB pre-S2 antigen in the recombinant yeastHansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that is should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.     

影响甲醇酵母中外源蛋白表达的因素

甲醇酵母系统由于其在表达蛋白方面无可比拟的优越性,已越来越得到广泛的应用。但不同的个源蛋白表达量有很大差异。介绍了影响甲醇酵母中外源蛋白表达的因素,这将有助于揭示甲醇酵母中外源蛋白表达的内在机制。     

Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha

Wild-type strains of the thermotolerant methylotrophic yeast Hansenula polymorpha are able to ferment glucose, cellobiose and xylose to ethanol. H. polymorpha most actively fermented sugars to ethanol at 37 degrees C, whereas the well-known xylose-fermenting yeast Pichia stipitis could not effectively ferment carbon substrates at this temperature. H. polymorpha even could ferment both glucose and xylose up to 45 degrees C. This species appeared to be more ethanol tolerant than P. stipitis but more susceptible than Saccharomyces cerevisiae. A riboflavin-deficient mutant of H. polymorpha increased its ethanol productivity from glucose and xylose under suboptimal supply with riboflavin. Mutants of H. polymorpha defective in alcohol dehydrogenase activity produced lower amounts of ethanol from glucose, whereas levels of ethanol production from xylose were identical for the wild-type strain and the alcohol dehydrogenase-defective mutant.     

海洋分离芽胞杆菌抗白念珠菌活性物质的理化性质及类别

为寻找新型抗真菌活性物质,采用管碟法对7株分离自海洋的芽胞杆菌在不同Na Cl浓度下产生抗白念珠菌活性物质特性、活性物质的耐热性及不同p H值条件下的活性进行了比较,八大溶剂系统纸层析法对活性物质的类别进行了初步鉴定。结果表明,随着Na Cl浓度的变化产生活性物质的量也在变化,Na Cl浓度达7%时均不能产生,但在正常海洋环境盐浓度(Na Cl含量2%~3%)下都产生;活性物质有很强的耐热性和耐酸碱性,说明其较稳定;7株菌产生的抗白念珠菌活性物质均为碱性水溶性抗生素。由于目前临床上抑制人体病原真菌活性物质绝大多数为脂溶性,因而这些芽胞杆菌产生的抗白念珠活性物质有可能为新型物质,此外本研究结果为这些菌株所产生活性物质的分离纯化提供了依据。     

血培养标本中病原菌的分布特征、耐药性变迁和耐药基因分型

目的 分析嘉兴市中医医院血培养标本中病原菌的分布特征、耐药性变迁和耐药基因分型。方法 将本院2013年1月至2016年12月送检的血培养标本进行培养、转种、分离鉴定和药敏试验,采用聚合酶链反应（PCR）技术进行产超广谱β-内酰胺酶（ESBLs）革兰阴性菌和革兰阳性菌的耐药基因检测,并对阳性菌株、科室分布、耐药性等相关数据进行分析。结果 2013年1月至2016年12月共送检血培养标本27 003份,共分离出病原菌（剔除重复菌株）978株,阳性率为3.62%,2015‒2016年阳性率显著低于2013‒2014年,差异有统计学意义（P<0.01）。血培养阳性株数前三位为ICU、儿科和肾内科,2015‒2016年与2013‒2014年相比,ICU的阳性株数构成比显著下降,儿科、呼吸内科和其他科室显著上升,差异有统计学意义（P<0.01）。血培养致病菌检出率最高的为大肠埃希菌,占18.51%,其次为表皮葡萄球菌,占10.53%,不可忽视的是真菌,占4.91%。2015‒2016年与2013‒2014年相比,金黄色葡萄球菌检出率显著上升,粪肠球菌和近平滑假丝酵母菌的检出率显著下降,差异有统计学意义（P<0.05）。血培养分离的大肠埃希菌对氨苄西林的耐药率最高,肺炎克雷伯菌对呋喃妥因的耐药率最高。2015‒2016年与2013‒2014年相比,大肠埃希菌对氨苄西林/舒巴坦和庆大霉素的耐药率显著下降（P<0.01）,肺炎克雷伯菌对庆大霉素的耐药率显著上升（P<0.01）。分离的表皮葡萄球菌和金黄色葡萄球菌对青霉素的耐药率最高,2015‒2016年与2013‒2014年相比,表皮葡萄球菌对红霉素和氯洁霉素的耐药率显著下降（P<0.01）,金黄色葡萄球菌对环丙沙星和左氧氟沙星的耐药率显著下降（P<0.01）。大肠埃希菌ESBLs（+）菌株对氨苄西林、氨苄西林/舒巴坦、头孢唑啉等的耐药率以及所有基因型表达率均显著高于ESBLs（‒）菌株（P<0.05或P<0.01）;肺炎克雷伯菌ESBLs（+）菌株对头孢唑啉、头孢曲松、庆大霉素等的耐药率以及TEM、SHV、CTX-MⅡ基因型表达率均显著高于ESBLs（‒）菌株（P<0.05或P<0.01）。表皮葡萄球菌检出mecA基因（+）72株,金黄色葡萄球菌检出mecA基因（+）53株。屎肠球菌和粪肠球菌分离株中,未发现有vanA（+）、vanB（+）和vanM（+）万古霉素耐药株。结论 了解本院血培养致病菌整体变化趋势、病区分布特点及耐药性变迁和耐药基因分型,对临床合理使用抗生素和院内感染的控制有重要意义。     

非水溶性甲胺磷降解酶的检测

甲胺磷农药是一种水溶性广谱、剧毒杀虫剂,化学名称：Ｏ,Ｓ－二甲基胺基硫代磷酸酯．目前,它在我国的生产和使用量已相当可观,并由此造成了严重的生态破坏和环境污染［１］．近几年,作者针对上述问题,结合国内外对甲胺磷代谢和有关酶知识缺乏了解的实际［２］,开展...