同工酶研究在嗜热脂肪芽孢杆菌分类中的应用

嗜热脂肪芽孢杆菌是一个尚未被严格定义的种,它几乎包括芽孢杆菌属内所有能在65℃以上生长的细菌。用聚丙烯酰胺凝胶电泳的方法对151株嗜热脂肪芽孢杆菌的9种酶(G6PDH、LDH、MDH、IDH、AlaDH、LeuDH、过氧化氢酶、过氧化物酶、酯酶)的酶谱进行测定分析。根据其酶谱的差异,可将151株菌株分成两个类型。两型之间8个酶的13或14个基因产物的相异度约为91.8％,两型间的标准遗传距离是2.55。因此这两种类型的细菌也许可以被看作为两个不同的种。     

嗜热脂肪芽孢杆菌HY—69耐热中性蛋白酶的性质研究

嗜热脂肪芽孢杆菌ＨＹ－６９的耐热中性蛋白酶已纯化。研究了纯酶的性质,该酶分子最为２４ｋｄ,由６个构成一个六聚体。酶的等电点９．１５。最适作用ｐＨ为７．５,最适作用温度为８５℃;该酶具有很好的耐热性,９０℃时酶活半寿期为２２ｍｉｎ,８０℃保温３小时,酶活仍保持６３％;酶的ｐＨ稳定性也好,该酶是金属蛋白酶,活性中心含锌离子,酶的热稳定性依赖于钙离子。测定了酶的氨基酸组成和Ｎ末端氨基酸序列。     

嗜热脂肪芽孢杆菌中转座因子IS5376的转座行为研究

ＩＳ５３７６和ＩＳ５３７７是在嗜热脂肪芽孢杆菌（Ｂａｃｉｌｕｓｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ）中发现的两个转座因子。随机取样分析的结果说明,ＩＳ５３７６由ＣＵ２１染色体向质粒ｐＦＤＣ５和ｐＦＤＣ１２的转座受温度的影响,而ＩＳ５３７７则不。温度影响的原因还不清楚,从现有证据看来,这由ＩＳ５３７６本身的性质所决定。另外,测得ＩＳ５３７６的转座作用有一定程度的专一性,还测得转座后所造成的目标序列的顺向重复为４或５ｂｐ。     

高温锻炼定向选育病毒唑高产菌的研究

利用嗜热脂肪芽孢杆菌酶促法生产病毒唑,经高温锻炼,得６５℃最适生长菌群Ｇ０,它对肌苷的病毒唑产率为原菌株的８倍。初筛后ＧＦ２０株病毒唑的产率达６２．２５％,为原菌株的２７倍。     

耐热中性蛋白酶产生条件及酶的亲和层析纯化

从云南西双版纳分离的耐热菌株中筛选到菌号为HY-69的菌株,经鉴定为嗜热脂肪芽孢杆菌(Bacillus stearothermophilus).该菌产中性蛋白酶.对该菌株的产酶条件进行了研究,酶的产量和耐热性均优于其它菌株.发酵液经超滤浓缩、硫酸铵沉淀后以亲和层析纯化.粗酶分别以Cbz-L-phe-T-sepharose 4B和Cbz-D-phe-T-sepharose 4B纯化,并对两种材料的纯化效果进行了比较,得到了PAGE和SDS-PAGE均一的酶.     

植物染色体显微切割技术的研究现状与展望

植物染色体显微切割技术的研究现状与展望马有志徐琼芳辛志勇（中国农业科学院作物育种栽培研究所,北京１０００８１）ＴｈｅＡｄｖａｎｃｅｓｏｆｔｈｅＴｅｃｈｎｉｑｕｅｏｆＰｌａｎｔＣｈｒｏｍｏｓｏｍｅＭｉｃｒｏｄｉｓｅｃｔｉｏｎＭａＹｏｕｚｈｉＸｕＱｉｏｎ...     

分离嗜热脂肪芽孢杆菌单个菌落的方法

本文研究了平板培养基的量、培养温度、培养湿度、培养基类型、培养时间对运动性较强的嗜热脂肪芽孢杆菌WF—146平板分离的影响。结果表明,控制好上述因素,嗜热脂肪芽孢杆菌WF—146在平板上培养可较好形成单菌落。     

嗜热脂肪芽孢杆菌DNA解链蛋白1的纯化及性质

以嗜热脂肪芽孢杆菌为材料,通过ＰｏｌｙｍｉｎＰ沉淀,硫酸铵分级及Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ,ＤＥＡＥ纤维素,磷酸纤维素,ＦＰＬＣ ＭｏｎｏＱ,ＦＰＬＣ Ｓｕｐｅｒｏｓｅ１２等柱层析,得到部分纯化的ＤＮＡ解链蛋白１。ＢｓｔＨ１具有依赖ＤＮＡ和Ｍｇ＾２＋的ＡＴＰ酶活力,不同类型的核酸对ＢｓｔＨ１的ＡＴＰ酶活力的促进作用不同。     

实验动物呼吸系统主要器官比较组织学研究

目的比较实验动物呼吸系统主要器官的组织学特征,为制定实验动物病理检测标准、以及毒理学、新药安全性评价提供依据。方法选取实验动物质量国家检测标准检测合格的恒河猴30只、昆明小鼠20只、SD大鼠20只、日本大耳白兔18只、比格犬16只、树鼩20只。除昆明小鼠采用颈椎脱臼致死外,其余动物麻醉后放血处死和病理解剖,对气管、肺脏进行病理大体检查和取材,常规病理制片,进行HE染色、特殊染色和免疫组化染色,显微镜下观察气管、肺脏的组织结构和细胞结构异同。结果 （1）实验动物气管上皮杯状细胞有差异：恒河猴、比格犬、日本大耳白兔杯状细胞较多,大鼠、小鼠、树鼩则较少或无。上皮分泌的黏液类型以中性黏液为主,比格犬杯状细胞分泌的黏液类型有中性黏液和酸性黏液。（2）实验动物黏膜下腺泡分布有差异：比格犬黏膜下层的腺泡最多,恒河猴、大鼠、小鼠、树鼩腺泡数量偏少,日本大耳白兔黏膜下层的混合腺泡最少。（3）实验动物的肺内支气管分支有差异：比格犬、恒河猴、日本大耳白兔由叶支气管、段支气管、小支气管、细支气管、终末细支气管和呼吸性细支气管组成,树鼩、大鼠、小鼠只由细支气管、终末细支气管和呼吸性细支气管组成。（4）实验动物细支气管组织结构有差异：恒河猴、比格犬的细支气管平滑肌为完整环形平滑肌层,没有缺失,而大鼠、小鼠、树鼩及日本大耳白兔的细支气管平滑肌薄或缺失。恒河猴、树鼩、大鼠细支气管有少量杯状细胞,其余实验动物均无杯状细胞。（5）实验动物Clara细胞形态有差异：比格犬Clara细胞呈立方形,其余动物呈柱状。结论实验动物呼吸系统组织结构的质是相同的,差异在于量的不同。研究人员在制定病理学检测标准、实验研究、药物安全性评价时应予充分考虑。     

湖北省实验动物设施环境控制中存在的问题

目的分析总结湖北省2012年度实验动物许可单位年检中环境设施的检测结果,找出问题,提出促进湖北省实验动物环境设施可靠运行的建议。方法各实验动物环境设施采用30%区域抽检方式,依据GB14925-2010《实验动物环境及设施》,进行环境指标检测。结果除气流速度、沉降菌最大平均浓度外,其他技术指标均存在不符合国家标准的情况。结论应针对实验动物设施管理加强培训,提高实验动物设施管理人员的管理水平,从而保证实验动物设施的正常运行。     

Type selective inhibition of microbial fatty acid synthases by thiolactomycin

The antibiotic, thiolactomycin, is known to selectively inhibit the Type II straight-chain fatty acid synthase (monofunctional enzyme system, e.g. Escherichia coli enzyme) but not Type I straight-chain fatty acid synthase (multifunctional enzyme system, e.g. Saccharomyces cerevisiae enzyme). We have studied the effect of thiolactomycin on the branched-chain fatty acid synthases from Bacillus subtilis, Bacillus cereus, and Bacillus insolitus. Fatty acid synthase from all three Bacilli was not inhibited or only slightly inhibited by thiolactomycin. E. coli synthase, as expected, was strongly inhibited by thiolactomycin. Branched-chain fatty acid synthase from Bacillus species is a monofunctional enzyme system but, unlike Type II E. coli synthase, it is largely insensitive to thiolactomycin.     

Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate

Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in k(cat)/K(M), by a factor of 10(3), indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 --> Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of k(cat)/K(M), supports a role of His311 as a general base in the catalysis.     

应用益生菌监测牛奶抗生素残留

目的 探索肠道益生菌在抗生素残留监测中的应用并建立牛奶中多种抗生素残留的微生物学筛检方法。方法 厌氧培养分离健康人肠道益生菌并用纸片扩散法检测其对四环素、红霉素、青霉素、庆大霉素、氯霉素和万古霉素的敏感性。根据药敏结果及产酸能力选择工作菌株。建立基于厌氧培养的益生菌抗生素残留筛检方法并优化实验条件,测定其对不同抗生素的检测限,通过测定加标模拟阳性和阴性样本计算方法的准确性。结果 短双歧杆菌A2可用于牛奶抗生素残留筛检。应用短双歧杆菌筛检牛奶中四环素、红霉素和青霉素的检测限分别为80 μg/L、40 μg/L和3 μg/L,检测加标四环素、红霉素和青霉素的牛奶样品准确性为100.0%、92.5%和90.0%,无假阴性结果。结论 应用益生菌的筛检方法敏感、简便,对环境和操作者友好,适用于监测牛奶中四环素、红霉素和青霉素等多种抗生素。     

Antibiotic inhibition of pectolytic and cellulolytic enzyme activity in twoFusarium species

Among all antibiotics tested, amoxycillin (500 ppm) completely inhibited the polygalacturonase and pectinmethylgalacturonase enzyme activity inF. oxysporum; none of the antibiotics did so inF. moniliforme. No antibiotic completely inhibited the cellulase activity in both test organisms, however, amoxycillin was better than other antibiotics in inhibiting the cellulase activity in both the organisms.     

Need for Aromatic Residue at Position 115 for Proteolytic Activity Found by Site-directed Mutagenesis of Tryptophan 115 in Thermolysin

In thermolysin, tryptophan 115 seems to be at the S2 subsite. Trp-115 was replaced with tyrosine, phenylalanine, leucine, and valine during site-directed mutagenesis in order to evaluate the role of Trp-115 in the proteolytic activity of thermolysin. The mutant enzymes with Tyr-115 or Phe-115 had as much proteolytic activity as the wild-type enzyme, but the other two mutant enzymes had no activity. We found earlier that the substitution of Trp-115 with alanine, glutamic acid, lysine, and glutamine causes the enzyme to lose all activity, so an aromatic amino acid at position 115 seems to be essential for thermolysin.     

中国有机残留物分析的研究进展及展望

有机残留物分析,是指从残留物载体中提取有机分子,利用科技检测手段进行定性、定量分析,判断有机残留物的生物来源,从而了解古代动植物的加工、利用和相关载体的功能等.中国有机残留物分析工作已开展40余年,取得了很多进展,但仍需要更多重视.本文首先回顾了中国考古遗存研究中有机残留物分析的发展历程,然后梳理了动物制品、粮食作物制...     

Occurrence and microbial degradation of fipronil residues in tropical highland rhizosphere soils of Kerala,India

The aim of the current study was to analyze the abundance and activity of soil microflora in response to fipronil residues, as well as conjointly to isolate and identify bacteria for the bioremediation of fipronil contaminated soils in the cardamom plantations of Idukki district, Kerala. Soil samples collected from rhizosphere areas of six completely different cardamom plantations were analyzed for fipronil residues, physicochemical properties, biochemical properties, and microbial abundance. Biodegradation studies using isolated bacteria were done both in liquid medium and in soil microcosm fortified with fipronil. Fipronil residues were detected in all sampling sites. Canonical correlation analysis revealed that the influence of fipronil on soil physicochemical properties was more pronounced than that on soil microbial properties. The presence of fipronil residues in the soil did not adversely affect bacterial abundance and activity. Two bacterial strains Staphylococcus arlettae and Bacillus thuringiensis could degrade fipronil in both liquid culture and soil. Paired sample T-test and degradation kinetic study recorded that the bacterial strain S. arlettae was more efficient (81.94%) in fipronil degradation than B. thuringiensis (65.98%). The results revealed the potential for in situ bioremediation of fipronil contaminated soil by bioaugmentation using efficient bacterial isolates.     

Analysis of essential leucine residue for catalytic activity of novel thermostable chitosanase by site-directed mutagenesis

Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases. The Leu64 Arg and Leu64 Gln mutations were essentially inactive and kinetic parameters such as V _max and k _cat were approximately 1/10⁷ of those of the wild-type enzyme. The Asp98 Asn mutation did not affect the K _m value significantly, but decreased k _cat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 srarr; Glu mutation affected neither K _m nor k _cat. The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity. These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2.