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。     

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。     

嗜热脂肪芽孢杆菌耐热木糖异构酶的特性

嗜热脂肪芽抱杆菌在木糖或木聚糖诱导下产生木糖异构酶。从破碎细胞中分离到该酶。经硫酸铵沉淀,热处理及SephadexG-200柱层析等步骤获得纯化了19倍的酶制备物。该酶反应的最适pH值为7.5,在pH6．2～8．0范围内稳定,最适反应温度为80℃,低于此温度时酶有很好稳定性。该酶对底物木糖的Km值为6．67mmol／L,Mg₂₊、Co₂₊和Mn₂₊对该酶有激活作用,而Zn₂₊、Cu₂₊和Fe相似文献   

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。     

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。     

嗜热脂肪芽孢杆菌与北京棒状杆菌细胞融合初探

对两种不同属间的细菌,嘈热脂肪芽孢杆菌与北京棒状杆菌原生质体的形成,再生及融合进行了研究。初步确定了适应此两种出发菌株的破壁、再生及融合的最佳条件。在此条件下,其破壁率均可达98%以上;LT1菌与LT2菌的再生率分别可达52%与51%;融合率可达2.55×10(?)。试验结果表明,此两种不同属间的细菌细胞融合是完全可能的。10(?)以上的融合率应用于工业遗传育种是完全可行的。     

固定化嗜热脂肪芽孢杆菌合成低聚半乳糖

利用海藻酸钙、明胶和壳聚糖为固定化载体包埋嗜热脂肪芽孢杆菌细胞合成低聚半乳糖 (GOS)。通过比较三种方法的酶活力回收、最适反应条件、GOS的得率和和载体机械强度 ,选择明胶作为固定化细胞的载体。反应体系的温度、pH、乳糖浓度、乳糖的转化率和载体的传质阻力对GOS合成有明显影响。在CSTR反应器中水解 60 %乳糖 ,GOS最大得率为31 2 % ,经过 96h( 8批反应 ) ,产物得率为原来的 88%。在空速 0 0 9h- 1条件下 ,利用填充床反应器连续水解乳糖 ,GOS的得率和反应器生产能力分别为 31 5%和 1 7 4g (L·h) ,连续反应1 40h,GOS得率下降 2 0 %。产物经过活性炭柱层柱分离纯化 ,通过13C NMR鉴定四糖的化学结构为 β D Gal ( 1→ 3) D Gal ( 1→ 6) D G ( 1→ 4) D Glu。     

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。     

Partial purification and characterization of lipase from Geobacillus stearothermophilus AH22

The lipase was partially purified by ion exchange chromatography and gel filtration column chromatography, and was characterized from Geobacillus stearothermophilus AH22 strain. The lipase was purified 18.3-folds with 19.7% recovery. The lipase activity was determined by using p-nitrophenyl esters (C₂–C₁₂) as substrates. The K_m values of the enzyme for these substrates were found as 0.16, 0.02, 0.19 and 0.55?mM, respectively, while V_max values were 0.52, 1.03, 0.72 and 0.15?U?mg^?1. The enzyme showed maximum activity at 50?°C and between pH 8.0 and 9.0. The enzyme was found to be quite stable at pH range of 4.0–10.0, and thermal stability between 50 and 60?°C. It was found that the best inhibitory effect of the enzyme activity was of Hg²⁺. The inhibitory effect as orlistat, catechin, propyl paraben, p-coumaric acid, 3,4-dihydroxy hydro-cinnamic acid was examined. These results suggest that G. stearothermophilus AH22 lipase presents very suitable properties for industrial applications.     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Engineering of the alpha-amylase from Geobacillus stearothermophilus US100 for detergent incorporation

AmyUS100DeltaIG is a variant of the most thermoactive and thermostable maltohexaose forming alpha-amylase produced by Geobacillus stearothermophilus sp.US100. This enzyme which was designed to improve the thermostability of the wild-type enzyme has acquired a very high resistance to chelator agents. According to modeling structural studies and with the aim of enhancing its resistance towards chemical oxidation, a mutant (AmyUS100DeltaIG/M197A) was created by substituting methionine 197 to alanine. The catalytic proprieties of the resulting mutant show alterations in the specific activity and the profile of starch hydrolysis. Interestingly, AmyUS100DeltaIG/M197A displayed the highest resistance to oxidation compared to the AmyUS100DeltaIG and to Termamyl300, the well-known commercial amylase used in detergent. Further, performance of the engineered alpha-amylase was estimated in the presence of commonly used detergent compounds and a wide range of commercial detergent (liquid and solid). These studies indicated a high compatibility and performance of AmyUS100DeltaIG/M197A, suggesting its potential application in detergent industry.     

热稳定碱性脂肪酶产生菌的筛选及发酵条件的优化

从渤海湾盐碱地被油污染的土样中分离筛选出6株产热稳定碱性脂肪酶菌株。其中菌株1-7产脂肪酶能力较强,其最高酶活为8.67U/mL。根据其16S rDNA序列分析和Biolog生理生化分析,初步鉴定为醋酸钙不动杆菌(Acinetobacter calcoaceticus)。初步酶学性质研究表明该菌所产脂肪酶具有较好的热稳定性,最适作用pH为9.0。摇瓶实验表明,该菌株最适产酶培养基为(g/L):玉米粉10,黄豆饼粉20,K_2HPO_4 1,NaNO_3 5,橄榄油10。最适产酶条件为:初始pH 8.0,培养温度37℃,培养时间29 h。接种量为2%,250 mL摇瓶装液量为30 mL。     

应用响应面法优化嗜热脂肪土芽孢杆菌培养基

目的：筛选并优化嗜热脂肪芽孢杆菌CHB1液体发酵培养基。方法：通过Plackett-Burman试验和响应面分析方法,确定培养基的主要影响因素和最佳浓度。结果：利用SAS软件进行分析,确定对响应值影响最大的3个因素为豆粕、酵母粉、K2HPO4。最佳培养基组成为0.51%、豆粕浓度为0.425%、K2HPO4浓度为0.994%。根据模型预测得到的理论最大菌数为2.94×10^8cfu。在初始条件下实验,菌数为2.40×10^8cfu,在优化的最佳培养基条件下,实际的菌数为3.06×10^8cfu。菌数比优化前提高了27.3%。试验值与预测值的误差为4.08%。结论：实验值与模型预测值拟合良好。     

嗜热脂肪土芽孢杆菌CHB1生长特性与培养条件研究

目的:研究嗜热脂肪土芽孢杆菌CHB1的生长特性与培养条件。方法:以菌体生长量为主要评价指标,利用单因子试验与正交试验相结合的方法对影响CHB1生长的主要因素进行分析。结果:CHB1最低和最高生长温度分别为45℃和74℃,最佳培养温度为60℃;最低和最高起始生长pH值分别为6.5和9.0,最适起始pH为8.0;菌体生长到达对数期的时间为15~18h;接种量2%,装液量40mL,转速180r/min。结论:CHB1为高温菌,生长pH范围偏碱性,条件优化后总菌体浓度可达1.1×109CFU/mL。     

Effects of superheated steam on Geobacillus stearothermophilus spore viability

Aims: To examine the effect of processing with superheated steam (SS) on Geobacillus stearothermophilus ATCC 10149 spores. Methods and Results: Two inoculum levels of spores of G. stearothermophilus were mixed with sterile sand and exposed to SS at 105–175°C. The decimal reduction time (D‐value) and the thermal resistance constant (z‐value) were calculated. The effect of cooling of spores between periods of exposure to SS was also examined. A mean z‐value of 25·4°C was calculated for both inoculum levels for SS processing temperatures between 130°C and 175°C. Conclusions: Spore response to SS treatment depends on inoculum size. SS treatment may be effective for reduction in viability of thermally resistant bacterial spores provided treatments are separated by intermittent cooling periods. Significance and Impact of the Study: There is a need for technologies that require short thermal processing times to eliminate bacterial spores in foods. The SS processing technique has the potential to reduce microbial load and to modify food texture with less energy in comparison to commonly used hot air treatment. This work provides information on the effect of SS processing parameters on the viability of G. stearothermophilus spores.     

Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1

We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.     

Properties of Geobacillus stearothermophilus levansucrase as potential biocatalyst for the synthesis of levan and fructooligosaccharides

The production of levansucrase (LS) by thermophilic Geobacillus stearothermophilus was investigated. LS production was more effective in the presence of sucrose (1%, w/v) than fructose, glucose, glycerol or raffinose. The results (T_op 57°C; stable for 6 h at 47°C) indicate the high stability of the transfructosylation activity of G. stearothermophilus LS as compared with LSs from other microbial sources. Contrary to temperature, the pH had a significant effect on the selectivity of G. stearothermophilus LS‐catalyzed reaction, favoring the transfructosylation reaction in the pH range of 6.0–6.5. The kinetic parameter study revealed that the catalytic efficiency of transfructosylation activity was higher as compared with the hydrolytic one. In addition to levan, G. stearothermophilus LS synthesized fructooligosaccharides in the presence of sucrose as the sole substrate. The results also demonstrated the wide acceptor specificity of G. stearothermophilus LS with maltose being the best fructosyl acceptor. This study is the first on the catalytic properties and the acceptor specificity of LS from G. stearothermophilus. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1405–1415, 2013     

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic Bacterium Geobacillus stearothermophilus

A group I intron that can be spliced in vivo and in vitro was identified in the flagellin gene of the thermophilic bacterium Geobacillus stearothermophilus. We also found one or two intervening sequences (IVS) of flagellin genes in five additional bacterial species. Furthermore, we report the presence of these sequences in two sites of a highly conserved region in the flagellin gene.