Optimization of cultural conditions for thermostable β-1,3-1,4-glucanase production by Bacillus subtilis ZJF-1A5

The optimization of cultural conditions for β-glucanase production by Bacillus subtilis ZJF-1A5 was investigated in flask trials. Temperature had gre at effect on β-glucanase production which maximized at optimal temperature of 37 ℃ and decreased significantly when temperature was over 37℃.Charge quantity af fected β-glucanase production significantly. Adding oxygen vector N-dodec ane or acetic ether benefited β-glucanase production, but it depended on the conc entrat ion and charge quantity. The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting β -gluc anase production and the cultivation time span to reach the highest β-gluc anase activity. The optimal cultural conditions for β-glucanase production obtai ned wi th CCD were as follows: inoculum age and size (16 h, 3.82%(v/v)), shaking speed 2 10 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37℃ for 50 h. Repeated experimental results accorded with those predicted b y a second-order polynomial model. The amount of β-glucanase, α-amylase and neut ral protease produced by B subtilis ZJF-1A5 was associated partially with c ell g rowth. Those three enzymes' activities increased following the cell growth and i ncreased significantly when cells entered the stationary phase.     

植物内生菌Bacillus amyloliquefaciens ES-2菌株对苹果青霉病的抑制效果

本文主要研究了不同贮藏温度下植物内生菌Bacillus amylolique faciens ES-2菌株对苹果青霉病的抑制效果。ES-2菌株的各处理液在苹果果实和PDA培养基上对苹果青霉病菌均有抑制作用。较低的贮藏温度有利于拮抗菌对病菌的抑制效果;24h后接种病菌孢子的果实其病斑直径一般都高于48h后接种的果实。实验证明,ES-2菌株防治苹果青霉病的机理是由于ES-2产生的抗菌物质的作用。     

Purification and characterization of keratinase from a new Bacillus subtilis strain

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50℃ was 8.5 and the optimum temperature at pH 8.5 was 55℃. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT),mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO)stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.     

苏云金杆菌SD—5菌株的生物毒力研究

本文比较了 SD—5孢晶混合物、纯化芽孢及纯化晶体对菜青虫的毒力,研究了芽抱与晶体的比例对毒力的影响,表明 SD—5对菜青虫的毒力以晶体为主;测定了五批中试产品的生物毒力,感染3龄菜青虫72小时的 LC50为稀释2740—3692倍;并研究了 SD—5对霜天蛾、大袋蛾及烟青虫的毒力,结果表明SD—5对这些害虫具有较高毒力,具有美好的应用前景。     

Antitumor effects of human interferon-alpha 2b secreted by recombinant bacillus Calmette-Guérin vaccine on bladder cancer cells

Objective:Our objective was to construct a recombinant bacillus Calmette-Guérin vaccine(rBCG) that secretes human interferon-alpha 2b(IFNα-2b) and to study its immunogenicity and in vitro antitumor activity against human bladder cancer cell lines T24 and T5637.Methods:The signal sequence BCG Ag85B and the gene IFNα-2b were amplified from the genome of BCG and human peripheral blood,respectively,by polymerase chain reaction(PCR).The two genes were cloned in Escherichia coli-BCG shuttle-vector pMV261 to obtain a new recombinant plasmid pMV261-Ag85B-IFNα-2b.BCG was transformed with the recombinant plasmid by electroporation and designated rBCG-IFNα-2b.Mononuclear cells were isolated from human peripheral blood(PBMCs) and stimulated with rBCG-IFNα-2b or wild type BCG for 3 d,and then cultured with human bladder cancer cell lines T24 and T5637.Their cytotoxicities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.Results:BCG was successfully transformed with the recombinant plasmid pMV261-Ag85B-IFNα-2b by electroporation and the recombinant BCG(rBCG-IFNα-2b) was capable of synthesizing and secreting cytokine IFNα-2b.PBMC proliferation was enhanced significantly by rBCG-IFNα-2b,and the cytotoxicity of PBMCs stimulated by rBCG-IFNα-2b to T24 and T5627 was significantly stronger in comparison to wild type BCG.Conclusions:A recombinant BCG,secreting human IFNα-2b(rBCG-IFNα-2b),was constructed successfully and was superior to control wild type BCG in inducing immune responses and enhancing cytotoxicity to human bladder cancer cell lines T24 and T5637.This suggests that rBCG-IFNα-2b could be a promising agent for bladder cancer patients in terms of possible reductions in both clinical dosage and side effects of BCG immunotherapy.     

脱毛蛋白酶生产菌株Bacillus cereus SZ-4的筛选及遗传稳定性检测

采用平皿菌落分离法、透明圈法及摇瓶发酵法从盐腌皮上分离、筛选得到脱毛蛋白酶生产菌株Bacillus cereus SZ-4,该菌株经发酵后得到的粗酶液酶活力为72/mL;对其遗传稳定性检测结果表明,Bacillus cereus SZ-4遗传稳定性较差,在1～4代菌种之间差异不是很显著,可用于发酵生产接种。     

芽孢杆菌葡萄糖脱氢酶结构与功能研究进展

芽孢杆菌葡萄糖脱氢酶在辅酶再生方面具有良好的应用前景,因而受到了广泛关注.本文结合葡萄糖脱氢酶的三维结构和生理生化特性,尝试建立起其结构与功能方面的相关关系,为葡萄脱氢酶的体外改造提供有价值的信息,并指出了目前应用上存在的问题,对发展趋势做了展望.     

地衣芽孢杆菌微生物菌肥在水稻上的肥效研究

为验证地衣芽孢杆菌微生物菌肥对农作物的肥效,对水稻喷施地衣芽孢杆菌微生物菌肥进行田间效应试验,水稻喷施地衣芽孢杆菌微生物菌肥与基质比：增产率为7.29%;单因子差异生物统计检验达极显著水平;平均为亩纯增收56.6元。     

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29"

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel P-100.The protein was absorbed on DEAE-cellulose and Bio-Gel P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pl value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited in-hibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia scle-rotiorum. The 50% inhibitory concentrations (IC50) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B291 also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germi-nated spores.     

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel® P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel® P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. The 50% inhibitory concentrations (IC₅₀) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B29I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores.