Generation and identification of variants with improved purification yield of Bowman-Birk protease inhibitors carrying protein binding loops

Replacing the chymotrypsin inhibitory loop of soybean Bowman-Birk inhibitor (sBBI) with a VEGF binding peptide (BBI-AV) significantly reduces the overall purification yield when BBI-AV is produced as a fusion protein in a Bacillus subtilis expression system. The low purification yield is primarily due to a higher fraction of molecules with incorrect disulfide bond configurations after production and also after disulfide bond shuffling induced by 2-mercaptoethanol. To improve production yields, site-saturation libraries were generated at 39 out of the 66 amino acid residues of BBI-AV. Initial screens were designed to select for variants with higher trypsin inhibitory activities than the parent after treatment with a reducing agent. Secondary screens were developed to select for variants with the highest purification yields, and to also eliminate any false positives. From the screens, it was found that positively charged substitutions in the exposed hydrophobic patch region (sites 27, 29, 40, 50 & 52) are especially productive. In fact, one substitution, F50R, improves the purification yield to nearly the same level as wild-type sBBI. Productive amino acid substitutions were combined to select for the variant with the best overall yield after purification. Several variants were obtained with higher purification yields than even sBBI. The octuple variants, A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T and A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q, are particularly productive having greater than a five fold increase in final purification yield over the parent.     

Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and potential mode of action

The bacterial strain E1R-j, isolated as an endophyte from wheat roots, exhibited high antifungal activity to Gaeumannomyces graminis var. tritici (Ggt). Strain E1R-j was identified as Bacillus subtilis based on morphological, physiological and biochemical methods as well as on 16S rDNA analysis. This strain inhibited mycelium growth in vitro of numerous plant pathogenic fungi, especially of Ggt, Coniothyrium diplodiella, Phomopsis sp. and Sclerotinia sclerotiorum. In greenhouse experiments, soil drenches with cell densities of 10⁶, 10⁹ and 10¹² CFU ml⁻¹ E1R-j reduced significantly take-all disease, caused by Ggt, in wheat seedling by 62.6%, 68.6% and 70.7%, respectively, compared to the inoculated control, 4 weeks after sowing. Growth parameters such as lengths and fresh weights of roots and shoots of Ggt-inoculated control plants were significantly lower compared to Ggt-inoculated and E1R-j treated plants. Field experiments in the season 2006/2007, heights of wheat plants in the Ggt inoculated plots were significantly reduced compared to the non inoculated treatments. Yield parameters such as kernels per head and thousand kernel weight (TKW) in inoculated control plants were lower compared to the other treatments. In the experimental year 2007/2008, independent treatments with the bacterial strain E1R-j and the fungicide Triadimefon reduced take-all disease in wheat roots by 55.3% and 61.9%, compared to the inoculated control plants. In this season plant height in inoculated control was significantly lower and also the yield parameters seeds per head and especially TKW were drastically reduced compared to the other treatments. E1R-j treatment alleviated the detrimental effects of take-all on grain yield parameters to a similar extent as Triadimefon application. SEM studies revealed that in the presence of E1R-j, hyphae of Ggt showed leakage, appeared ruptured, swollen and shriveled. Following root drench, strain E1R-j was able to colonize endophytically roots and leaves of wheat seedlings. While the population of the bacterial strain in wheat roots steadily increased from the second to the fourth leaf stage, in the leaf tissue the population of the strain rapidly declined. TEM studies also showed that cells of E1R-j were present in roots of wheat seedlings and effectively retarded infection and colonization of Ggt in root tissue; suppression of Ggt by E1R-j was accompanied by disintegration of hyphal cytoplasm. In addition, in the presence of E1R-j cells in Ggt-infected root tissue morphological defense reactions were triggered such as formation of wall appositions and papillae. The results presented indicate that the endophytic strain E1R-j of B. subtilis meets demands required for biocontrol of take-all.     

微流控芯片监测绿色荧光蛋白在枯草芽孢杆菌中的表达

目前主要使用激光共聚焦扫描显微镜观察绿色荧光蛋白的表达,但需要昂贵的仪器并耗费大量时间。本研究开发了一种新型激光诱导的微流芯片检测系统来监测绿色荧光蛋白在枯草芽孢杆菌中的表达。该系统主要由激光装置、光路系统、微流控芯片、光电倍增管和计算机处理系统等5部分组成。对该系统的测试结果显示,随着诱导强度的增强监测信号峰也随之增强,并且与激光共聚焦显微镜观察的结果一致。利用该芯片系统能够快速准确地筛选和鉴定用绿色荧光蛋白作为标记的细胞克隆,可以替代PCR鉴定方法。但该系统仅仅能够监测表达强度,不能够满足蛋白定位等高水平研究,因此,该系统适合应用于环境的微生物监测、药物筛选和其他无需观察蛋白定位等研究。     

短小芽孢杆菌2080碱性蛋白酶的纯化与性质

短小芽孢杆菌（Bacillus pumilus）2080碱性蛋白酶的发酵液经超滤、硫酸铵沉淀、CM Sepharose Fast Flow和DEAE Sepharose Fast Flow离子交换层析得到了纯化的组分。SDS-PAGE电泳分析显示其分子量约为61kDa。酶学性质研究表明,该纯化酶的最适pH为10．5,最适温度为50℃。     

A modeled structure for amidase-03 from Bacillus anthracis

Homology models of amidase-03 from Bacillus anthracis were constructed using Modeller (9v2). Modeller constructs protein models using an automated approach for comparative protein structure modeling by the satisfaction of spatial restraints. A template structure of Listeria monocytogenes bacteriophage PSA endolysin PlyPSA (PDB ID: 1XOV) was selected from protein databank (PDB) using BLASTp with BLOSUM62 sequence alignment scoring matrix. We generated five models using the Modeller default routine in which initial coordinates are randomized and evaluated by pseudo-energy parameters. The protein models were validated using PROCHECK and energy minimized using the steepest descent method in GROMACS 3.2 (flexible SPC water model in cubic box of size 1 Å instead of rigid SPC model). We used G43a1 force field in GROMACS for energy calculations and the generated structure was subsequently analyzed using the VMD software for stereo-chemistry, atomic clash and misfolding. A detailed analysis of the amidase-03 model structure from Bacillus anthracis will provide insight to the molecular design of suitable inhibitors as drug candidates.     

枯草芽孢杆菌CAS15嗜铁素基因dhbC的克隆、表达及功能鉴定

通过PCR技术扩增得到dhbC基因,对其进行序列分析发现,dhbC基因片段长为1197bp,预期编码398个氨基酸,蛋白分子量大小为43.8kD。将目的片段连接到表达载体pET-30a(+),转化大肠杆菌Escherichia coli BL21(DE3)获得重组菌株BL21(DE3)/pET-30a-dhbC,以IPTG在30oC诱导4h实现高效表达,获得一个分子量为48.8kD的融合蛋白。重组蛋白可溶性分析结果表明:融合蛋白主要为可溶性蛋白。Western blotting分析结果表明:重组蛋白可与兔抗His-tag多克隆抗体发生特异性反应,在48.8kD处有特异条带,与预期结果一致,证明重组质粒中含有dhbC基因。通过同源重组的策略将dhbC基因敲除后重新导入,验证了dhbC基因与嗜铁素的生物合成密切相关。     

Peroxidase from Bacillus sp. VUS and its role in the decolorization of textile dyes

Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li²⁺, Co²⁺, K²⁺, Zn²⁺, and Cu²⁺ where as it showed less activity in the presence of Ca²⁺ and Mn²⁺. Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while sodium azide (NaN₃) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage of the dye.     

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, and anion SO₄⁻ increased enzyme activity while strong inhibition was observed in the presence of Hg²⁺, Cl⁻, HCO₃⁻, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V_max and K_m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K_i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.     

Analysis of dye binding by and membrane potential in spores of Bacillus species

Aims:  To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential.
Methods and Results:  Staining by four dyes and autofluorescence of B. subtilis spores that lack some ( cotE , gerE ) or most ( cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC₆(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC₆(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC₆(3) bound well to all germinated spores.
Conclusions:  The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate.
Significance and Impact of the Study:  The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.