A Novel Thermostable and Alkaline Protease Produced from Bacillus stearothermophilus Isolated from Olive Oil Mill Sols Suitable to Industrial Biotechnology

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 10⁸ CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca²⁺/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca²⁺ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.     

Genome Mining,Heterologous Expression,Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost-Associated Bacillus subtilis fmb60

Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D–F (1–3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D–F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS⁺ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated.     

Biodegradation of imidacloprid in sandy loam soil by Bacillus aerophilus

A study of the biodegradation of imidacloprid in soil was carried out under laboratory conditions. Sandy soil samples were fortified with imidacloprid at 50, 100 and 150 mg kg^?1 along with 45 x 10⁷ colony forming units (cfus) of Bacillus aerophilus and the samples were compared with unamended soil. The samples were extracted with acetonitrile, cleaned up by treatment with primary secondary amine sorbent and graphitised carbon black. The residues of imidacloprid and its metabolites were analysed by high performance liquid chromatography. The parent compound, imidacloprid, was found to be more persistent in both the treatments. Among metabolites, the highest values were obtained for urea and olefin while 5-hydroxy, 6-chloronicotinic acid (6-CNA), nitrosimine and nitroguanidine (NTG) were also observed in all the treatments in amended soil. In case of unamended (control) soil, 6-CNA was found to be the most persistent metabolite followed by olefin, urea, 5-hydroxy, nitrosimine and NTG metabolites. Total imidacloprid residues for control soil samples followed first-order kinetics at 50 and 150 mg kg^?1 but in case of control imidacloprid fortified at 100 mg kg^?1, the total residues of imidacloprid and its metabolites followed pseudo-first-order kinetics. The respective half-life value for 50 mg kg^?1 was 25.08 days and 30.10 days for both 100 and 150 mg kg^?1. However, total imidacloprid residues followed pseudo-first-order kinetics for its applications at 50, 100 and 150 mg kg^?1 in sandy loam soil amended with B. aerophilus. The half-life values for 50, 100 and 150 mg kg^?1 were worked out to be 14.33, 15.05 and 18.81 days, respectively. With the use of B. aerophilus, the reduction percentage of initial applied dose imidacloprid in sandy loam soil was found to be higher in all the three doses as compared to that of the control samples.     

高效液相色谱法测定发酵液中杆菌肽A

提出了高效液相色谱法测定发酵液中杆菌肽A含量的方法。地衣芽孢杆菌发酵液经离心得到杆菌肽A的粗提液。所得粗提液再经三氯乙酸提取,离心分离。取上清液用Beckman C18色谱柱分离,用乙腈和磷酸二氢钾缓冲溶液以不同体积比混合为流动相梯度洗脱,在220 nm波长处进行测定。杆菌肽A的质量浓度在1.0~15.0 g·L-1范围内与其峰面积呈线性关系,检出限(3S/N)为0.08 g·L-1。在7.37 g·L-1和11.01 g·L-1两个添加水平做回收试验,平均回收率分别为98.1%和97.4%。     

Pressure inactivation of microorganisms

Abstract

The kinetics of inactivation by hydrostatic pressure is measured for Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, bacteriophage T4, and the spores of Bacillus subtilis. Temperature and pressure effects are discussed as well as the interrelation between germination and inactivation in the case of bacterial spores.     

Ca2+诱导的淀粉液化芽孢杆菌α-淀粉酶分子的生物活性和结构变化

在研究Ca²⁺对淀粉液化芽孢杆菌α-淀粉酶分子生物活性影响的基础上, 采用荧光光谱法和傅里叶变换红外光谱法研究了Ca²⁺诱导的酶分子结构变化. 结果表明, 当溶液中Ca²⁺浓度低于25.0 mmol/L时, Ca²⁺对酶分子具有激活作用; 而当Ca²⁺浓度高于25.0 mmol/L时, Ca²⁺对酶分子的生物活性具有抑制作用. 在Ca²⁺诱导的淀粉液化芽孢杆菌α-淀粉酶分子结构变化过程中, 酶分子仅发生二级结构的变化, 并不涉及其三级结构. 当Ca²⁺对酶分子具有激活作用时, 酶分子中的无规卷曲结构及β-折叠结构的含量下降, 而α-螺旋结构及β-转角结构的含量上升; 而当Ca²⁺对酶分子生物活性具有抑制作用时, 酶分子中的α-螺旋结构及β-转角结构的含量下降, 而无规卷曲结构及β-折叠结构的含量上升.     

弱直流电场对枯草芽孢杆菌生长和代谢活性的作用

枯草芽孢杆菌63501(Bacillus subtilis 63501)培养液在连续培养时分别通入不同强度的直流电场,研究了它们在持续直流电刺激下的生长曲线、pH曲线、ATP酶活性、胞内总蛋白含量、细胞膜通透性以及细菌微观形态变化。 结果表明,适宜的直流电能促进枯草芽孢杆菌增殖;枯草芽孢杆菌的ATP酶活性在0.045 5×10-3 A/cm2电流密度作用下,在对数生长前期、后期和稳定期分别比对照组同期提升1.9、3.5和3.8倍,表明适宜强度的电流增强了细菌细胞的代谢活性。 细菌通透性也有不同程度的提高,与透射电子显微镜观察到的细菌细胞通电后的形态变化具有一致性。     

Environmental Effects on Transglutaminase Production and Cell Sporulation in Submerged Cultivation of <Emphasis Type="Italic">Bacillus circulans</Emphasis>

In this research, the effects of pH, temperature, and oxygen on growth kinetics of a newly isolated strain of Bacillus circulans from the Amazon and their correlations with transglutaminase (TGase) production and cell sporulation were investigated. Statistical experimental methods were used to optimize these parameters, while induction of sporulation was achieved by oxygen culture control. Full factorial composite experimental design and response surface methodology were experimentally tested. The model showed that temperature has a positive and significant effect on TGase production (P < 0.05) while pH and temperature, associated with anoxic conditions, have a marked effect on cell sporulation which is consistently linked with TGase production. The contour plot of results showed that the best culture conditions for TGase production of B. circulans were 30°C, initial pH 8.5, and the highest production was obtained in late-stationary culture phase with maximal specific enzyme activity of 655 U g⁻¹ of cells (0.37 U/mL). A correlation between enzyme production and cell sporulation, as mediated by oxygen culture conditions, was also demonstrated and, although demonstrated only for B. subtilis, it corroborates the molecular mechanisms involved in this process. It can be suggested that B. circulans BL32 is a strong biological system for the industrial production of TGases.     

Recombination System Based on Cre α Complementation and Leucine Zipper Fusions

A gene encoding β-1,3-1,4-glucanase was cloned by polymerase chain reaction (PCR) from Bacillus subtilis MA139. Sequencing result showed 97% homology to the corresponding gene from Bacillus licheniformis. The open reading frame (ORF) of the gene contained 690 bp coding for a 226 amino-acid matured protein with the estimated molecular weight of 24.44 kDa. The β-1,3-1,4-glucanase gene was subcloned into an expression vector of pET28a and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a nickel–nitrilotriacetic acid (Ni–NTA) column. The purified β-1,3-1,4-glucanase demonstrated 24.05 and 12.52 U ml^-1 activities for the substrates of barley β-glucan and lichenan, respectively, and the specific activities were 728.79 and 379.1 U mg^-1 for them, respectively. The optimal temperature and pH of the purified enzyme were 40°C and 6.4, respectively. When barley β-glucan was used as the substrate, K _m was 5.34 mg ml^-1, and K _cat showed 7,206.71 S^-1, thus the ratio of K _cat and K _m was 1,349.67 ml s^-1 mg^-1. The activity of β-1,3-1,4-glucanase was affected by a range of metal ions or ethylenediaminetetraacetic acid (EDTA).     

一种甲酯化n-C16-地衣素的分离及结构鉴定

经过酸沉淀分离、有机溶剂萃取和二步柱分离,从地衣芽胞杆菌(Bacillus licheniformis)HSN 221菌株的培养液中分离纯化得到了一种地衣素类似物.经ESI-MS测定,该物质的分子量为1062Da;经对酸解后甲醇酯化的脂肪酸的GC/MS分析可知,该物质含有3-羟基正十六烷酸残基;Q-TOF MS/MS的结果显示该物质具有GlnLeuLeuVMAspMeLeuIle的肽链,因此,该物质为甲酯化的n-C16-地衣素.