嗜热脂肪芽孢杆菌高温蛋白酶分解毛发角蛋白的研究

本文研究了嗜热脂肪芽孢杆菌 (Bacillusstearothermophilus)WF - 1 4 6高温蛋白酶对毛发角蛋白的水解作用。结果表明 ,在体积分数为 2 %的巯基乙醇存在的条件下 ,WF - 1 4 6高温蛋白酶对毛发角蛋白有明显的水解作用。对酶解液氨基酸分析表明 ,酶解液中含有对照液中没有的游离蛋氨酸和亮氨酸 ,且游离氨基酸总量是对照样品的 2 .4倍 ,达 1 5 8mg·L-1 。此外 ,酶解液中含有大量的肽     

嗜热脂肪芽孢杆菌高温蛋白酶的产生条件及酶学性质

对嗜热脂肪芽孢杆菌(Bacillusstearothermophilis)WF146的产蛋白酶的条件进行了研究,在58℃条件下,WF146在pH值为75的Fd培养基中振荡发酵培养48h后,发酵液中高温蛋白酶产量可达600u/mL以上。对该酶性质的研究表明,酶分子量为34kD,最适作用pH为80,最适作用温度为80℃,具有良好的pH稳定性及热稳定性。Ca2+对该酶的稳定性具有重要影响,PMSF、DFP及IAA能强烈抑制酶活力,而DTT对该蛋白酶活力无影响     

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。     

嗜热脂肪芽孢杆菌质粒的分离

从堆肥和污泥中分离到一批抗药性高温细菌,经电泳检查,发现6株高温细菌细胞中有质粒存在。其中,嗜热脂肪芽孢杆菌T653的细胞DNA提取液电泳图谱上,有三条非染色体DNA条带,用电镜直接观察,证明它们是T653细胞中的三个质粒。测得两个较小质粒的分子量分别为3.6×10~6和45×10~6道尔顿。研究了嗜热脂肪芽孢杆的T653的温度生长条件与其细胞中质粒的关系。T653细胞中三个质粒的明确功能有待进一步探讨。     

嗜热芽孢杆菌XJT—9503高温中性蛋白酶的研究

嗜热芽孢杆菌ＸＪＴ－９５０３菌的发酵液经硫酸铵和丙酮分级沉淀分离纯化得到聚丙烯酰胺凝胶电泳均一的高温中性蛋白酶制品。ＳＤＳ－ＰＡＧＥ则得酶分子量为３００００。当以酪蛋白为底物时,酶反应最适温度为６５℃,最适ＰＨ为７,在ＰＨ６．５－９范围内稳定。在６５℃、０．０２ＭＰＨ７．５的磷酸缓冲液中的半衰期为５４ｍｉｎ。金属离了铜、汞、铝强列抑制酶活,钙离子、镁离子对酶活有促进作用     

嗜热脂肪土芽孢杆菌CHB1产蛋白酶特性

目的：研究嗜热脂肪土芽孢杆菌CHBl产酶特性。方法：以蛋白酶活力为主要指标,考察温度、pH、接种量、装量等条件对CHB1产蛋白酶的影响。结果：CHB1适宜的产蛋白酶条件为：装量40mLt250mL,pH8．0,接种量5％,温度58℃,转速180r／min,时间36～44h,Tween-80对CHB1产蛋白酶具有抑制作用。结论：优化后蛋白酶产量有较大提高,最高酶活力达48U／mL,是所报道多数嗜热细菌产蛋白酶量的2—12倍。CHB1蛋白酶特性的研究及产蛋白酶量的提高,有利于揭示CHB1在堆肥化中的作用机理,提高堆肥效果。     

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;该酶分子量为２４ｋｄ,由６个单体构成一个六聚体。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稳定性也很好。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。     

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。     

The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents

Engineered extremely thermostable variants of the thermolysin-like protease from Bacillus stearothermophilus possessing an introduced disulfide bond G8C/N60C (double mutant, DM) and six additional amino acid substitutions in the exposed loop region 56-69 (Boilysin, BLN) have been probed with respect to stability toward water-miscible organic solvents and detergents. The solvent concentrations where 50% of enzyme activity were irreversibly lost (C(50)) decreased in the order methanol > 2-propanol > dimethylsulfoxide > dioxane > acetonitrile > dimethylformamide > acetone. The C(50) values were remarkably higher for the thermostable variants than for the wild-type enzymes. Therefore, the stabilization of this loop region also protects the molecule from irreversible inactivation by solvents, and inactivation seems to follow principally the same mechanism as thermal inactivation. However, in contrast to thermal inactivation where the corresponding T(50) values of DM and BLN differed by 10 K, the differences of the C(50) values of DM and BLN were not significant. Detergents had great effects on proteolytic activities which were dependent on the individual detergent and its concentration, but mostly without significant differences between the enzyme variants. These effects were inactivating (SDS, sulfobetaine) or strongly activating (CTAB, CHAPS). Triton X-100 and Tween 20 were activating or inactivating at low and high concentrations, respectively. In all detergents, stabilities of the enzymes were strongly decreased. However, the more thermostable variants were affected by the detergents to the same extent as the wild-type enzymes suggesting that the mechanism of detergent inactivation is different from that of thermal inactivation.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65℃.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 ℃ .     

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。     

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。     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

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。     

嗜热芽孢杆菌噬菌体的分离及特征研究

目的：从腾冲热海温泉中分离嗜热芽孢杆菌噬菌体,并初步分析其特征。方法：采用双层平板法分离纯化嗜热芽孢杆菌噬菌体,对分离得到的噬菌体进行电镜形态观察,按照感染复数（MOI）分别为0．01、0．1、1．0、10和100加入噬菌体纯培养液和宿主菌,55℃、160r／min培养8h后测定噬菌体滴度,并进行噬菌体的热稳定性和pH稳定性分析。结果：从腾冲热海温泉中分离得到的噬菌体为二十面体型;其感染宿主菌NHH4形成清晰的噬菌斑,最适MOI为1．0,最适感染温度为55℃,最适感染pH值为7．5。将这株噬菌体命名为TBIP1。结论：从腾冲热海温泉中分离得到的噬菌体TBIP1为典型的二十面体型,当MOI为1．0时,TBIP1感染其宿主菌产生的子代噬菌体滴度最高。