Extracellular production of a functional soy cystatin by Bacillus subtilis

A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.     

Colonization of wheat seedling (Triticum aestivum) roots by Pseudomonas fluorescens and Bacillus subtilis

Summary Colonization patterns of Pseudomonas fluorescens and Bacillus subtilis on roots of wheat seedlings growing on water agar were studied qualitatively by replica printing and quantitatively by the plate count method. The results indicated a stronger colonization potential for P. fluorescens (up to 10⁷ cfu/cm root) than for B. subtilis (up to 10⁵ cfu/cm root). Although the numbers of both species were lower when inoculated together, the observed colonization patterns on the roots were comparable to those found with single inoculations. For none of these bacteria was active migration along the root surface in any direction observed, indicating that distal positions are reached mainly by a passive displacement on the root tip and elongating cells. Ecological implications of the observed phenomena are discussed.     

Mechanisms of production of soil protease by proteolytic Bacillus subtilis in wetland rice soil

In sterile soil inoculated with proteolytic Bacillus subtilis, the correlation between variations in protease activity and cell numbers was investigated during incubation, and the results were compared with those obtained in different growth media. In sterile soil inoculated with B. subtilis, the extracted soil z-FLase activity (hydrolytic activity towards benzyloxycarbonyl-L-phenylalanyl-L-leucine extracted with 0.1 M phosphate buffer) was correlated with the extracted soil protein content (r=0.95; P<0.01) and the number of vegetative cells (r=0.88, P<0.05), while the extracted soil caseinase activity was well correlated with the number of spores (r=0.82, P<0.05) and not with the number of vegetative cells. Extracellular z-FLase activity in different growth media, inoculated with the proteolytic B. subtilis, was correlated with the extracellular protein concentration (r=0.73, P<0.01) and the number of cells (r=0.73, P<0.01), but extracellular caseinase activity was not correlated with either of these measurements. From these results, we concluded that soil z-FLase production might relate to vegetative growth of the proteolytic B. subtilis and soil caseinase might relate to B. subtilis sporulation.     

内生枯草芽孢杆菌B-001菌株内生定殖研究及生物学特性

用浸种、浸根、淋根、伤茎、伤叶和喷雾等接种方法测试枯草芽孢杆菌B-001菌株入侵烟草植株的途径,发现该菌可通过自然孔口和伤口入侵寄主植物;取接种植株的茎部组织切片在电镜下观察,发现该菌株在烟草的微管束和细胞内定殖。生长特性研究结果表明该菌株最适生长pH为7.5,最适生长温度为30℃,48～96h内处于稳定生长期。     

Cloning, expression, and characterization of two beta-glucosidases from isoflavone glycoside-hydrolyzing Bacillus subtilis natto

On the basis of the genomic sequence of Bacillus subtilis 168, two beta-glucosidase genes (bglH and yckE) from B. subtilis natto, which has been reported to have high isoflavone glucoside-hydrolyzing activity, were cloned and overexpressed in E. coli M15. The temperature for the optimal p-nitrophenyl-beta-D-glucoside hydrolyzing activity of both enzymes was between 37 and 45 degrees C, but BglH had a higher thermal stability than YckE. Both showed high activity at pH 6.0, but YckE was stable over a wider pH range than BglH. Recombinant BglH was inhibited 73%, 63%, and 43% by 1.0 mM Cd(2+), Fe(2+), or Cu(2+), respectively, while other divalent metal ions resulted in 0-23% inhibition, whereas YckE was inhibited by less than 20% by any of the divalent metal ions we tested. Among the substrate we used, BglH showed the highest affinity for genistin and YckE showed the highest affinity for p-nitrophenyl-beta-D-fructopyranoside. Both BglH and YckE hydrolyzed genistin and daidzin into their isoflavone aglycones, genistein and daidzein, but BglH was more efficient than YckE in isoflavone glucoside hydrolysis (20-fold higher kcat). Our results suggest that recombinant BglH may be applicable in the process of isoflavones deglycosylation.     

Improved subtilisin YaB production in Bacillus subtilis using engineered synthetic expression control sequences

Alkaline elastase YaB, a favorable meat tenderizer, is an extracellular subtilisin-type protease produced by wild strain alkalophilic Bacillus YaB. The gene ale coding for subtilisin YaB with its own expression control sequence has been cloned and expressed in Bacillus subtilis, but at levels much lower than in the parental strain Bacillus YaB. This study investigates the influence of various expression control sequences including expression control sequences of cdd and veg from B. subtilis, a synthetic expression control sequence (SECS), and engineered synthetic expression control sequences (engineered SECSs) on the expression of subtilisin YaB in B. subtilis. The engineered SECSs were generated by using the Polymerase Chain Reaction; their UP element, Shine-Dargarno (SD) sequence, or both were different from those of the native SECS. The expression efficiencies of SECS and engineered SECSs were higher than those of expression control sequences of ale, cdd, and veg. Substitution of the SD sequence of SECS resulted in higher expression of subtilisin YaB than substitution of the UP element, whereas combined substitution of both gave the highest expression. These results demonstrate that engineering of SECSs is an approach for improving subtilisin YaB production in B. subtilis. Moreover, it is suggested that these enginnered SECSs could potentially be used to express homologous and heterologous proteins in B. subtilis at high level.     

Microstructure of poly(gamma-glutamic acid) produced by Bacillus subtilis consisting of clusters of D- and L-glutamic acid repeating units

Poly(gamma-glutamic acid) (PGA) produced by a strain of Bacillus subtilis was partially hydrolyzed into various oligopeptides so that the dipeptide fraction was isolated by the preparative thin-layer chromatography. HPLC analysis was applied to the detection of each of the four stereoisomers in this fraction using chemically synthesized authentic samples. The fraction consisted of N-gamma- d-glutamyl- d-glutamic acid, N-gamma- l-glutamyl- l-glutamic acid, N-gamma- d-glutamyl- l-glutamic acid, and N-gamma- l-glutamyl- d-glutamic acid at a ratio of 5.9:6.0:1.0:1.0. On the basis of this result, a model was proposed for the microstructure of the bacterial PGA, in which d- and l-glutamic acid repeating units are alternately linked in a single chain of the molecule.     

枯草芽孢杆菌产芽孢条件的优化

为了提高枯草芽孢杆菌的芽孢数量及产芽孢率,采用分批培养法研究了营养条件、初始pH值、溶氧量对其生孢的影响。结果表明,麸皮和玉米粉分别是枯草芽孢杆菌生孢的最佳碳、氮源;无机盐和锰元素对芽孢生成有显著的影响。最佳生孢培养基组成:麸皮5 g,玉米粉10 g,NaCl 5 g,KH2PO41 g,MnSO4.H2O 0.4 g,水1 000 mL;最佳生孢条件:初始pH值为7.0,最佳装液量为250 mL锥形瓶装50 mL培养液。在150 r.min-1、30℃恒温培养72 h后,芽孢数可达到3.4×109cfu.mL-1,产芽孢率可达82.9%。     

枯草芽孢杆菌和绿色木霉协同促进芹菜生长的研究

为了芹菜高产栽培的需要,利用枯草芽孢杆菌(BLG010)和绿色木霉(H06)对芹菜进行促生长试验,试验设置了蒸馏水处理、BLG010处理、H06处理、BLG010+H06处理。结果表明,BLG010和 H06具有促进芹菜生长协同增效的现象。因此,通过测定芹菜的防御酶活性和代谢物的变化来探明 BLG010和 H06对芹菜生长呈现协同增效效果的原因。酶活性测定结果表明,相对于蒸馏水处理,BLG010和 H06均能提高芹菜的 CAT和 POD活性,并且 BLG010+H06处理的 CAT和 POD活性均比蒸馏水处理、BLG010处理、H06处理高。利用 GC-MS技术分析不同处理代谢物的结果表明,苹果酸、甘露醇和蔗糖这 3种物质的含量在不同处理间呈现规律性差异。     

Antibacterial activity of akyl gallates against Bacillus subtilis

The antibacterial activity of a series of alkyl gallates (3,4,5-trihydroxybenzoates) against Gram-positive bacteria was tested using a broth dilution method. All of the Gram-positive bacteria tested were susceptible to alkyl gallates, and this activity was found to correlate with the alkyl chain length. The antibacterial activity of alkyl gallates against Bacillus subtilis was a parabolic function of their lipophilicity and maximized with alkyl chain length between C(8) and C(11). Notably, alkyl gallates were found to be bactericidal against B. subtilis ATCC 9372, but this activity was significantly affected by the endospore formation in the culture. The antibacterial activity of alkyl gallates likely comes at least in part from their ability to inhibit the membrane respiratory chain but is not due to the prooxidant action.     

枯草杆菌β-甘露聚糖酶基因的克隆及表达

以能水解魔芋葡甘聚糖的野生筛选菌种枯草杆菌A33为材料，通过PCR技术从A33基因组中扩增出β-甘露聚糖酶基因编码序列。经过克隆、测序及BLAST比对分析，证实该基因编码β-甘露聚糖酶，属于β-甘露聚糖酶家族中的一员。该基因已注册GenBank（GenBank注册号：DQ269473）。将该基因构建到大肠杆菌表达载体pET-32a并转入大肠杆菌表达系统BL21（DE3），经过诱导获得了此酶的高效表达.     

Estimation of synergistic effect of humic fertilizer and Bacillus subtilis on lettuce plants by reflectance measurements

Previous research showed that the use of plant growth promoting rhizobacteria helped to increase nutrient use efficiency. The individual and combined effects of combined action of humic fertilizer and rhizobacteria Bacillus subtilis No. 2 on the lettuce yield, chlorophyll, total nitrogen and nitrate-(N) contents in lettuce leaves was studied. Traditional biochemical analysis and crop reflectance method were compared. Vegetation Indices and key spectrum characteristics - a median of frequency spectrum and bandwidth of frequency spectrum were used to estimate chlorophyll content in plant leaves.

The synergistic effect of bacteria and humic fertilizer was evidenced by increase in N and chlorophyll contents and in decreased nitrates content in lettuce leaves. Humic fertilizer resulted in decreased nitrates concentration in plants, whereas bacillus (B). subtilis No. 2 increased total N and chlorophyll contents. Results indicated that the application rate of humic fertilizer may be reduced when B. subtilis No. 2 is also applied.     

Synergistic effect of arbuscular mycorrhizal fungi and Bacillus subtilis on the biomass and essential oil yield of rose-scented geranium (Pelargonium graveolens)

Four different arbuscular mycorrhizal (AM) fungi, Glomus aggregatum, Glomus fasciculatum, Glomus intraradices and Glomus mosseae, were used alone and in combinations with Bacillus subtilis to evaluate their capability to increase the productivity of geranium. Mycorrhizal treatment increased the growth and total biomass invariably over non-mycorrhizal control plants. In AM alone treatment, the best result was obtained for G. mosseae treatment, where 380.9 and 335.3 g fresh herb yield per pot was recorded in 2005–2006 and 2006–2007, respectively, an increase of 75.3 and 85.9% over controls. Plants inoculated with B. subtilis alone yielded 287.8 and 252.3 g fresh herb, an increase of 32.4 and 39.9% over uninoculated controls. However, B. subtilis in combination with G. mosseae produced the highest herb yield, i.e. 410.8 and 347.8 g herbs pot^?1, an increase of 89.4 and 92.9% over untreated controls. The field experimental data validated the results of the pot experiment. Treatment with G. mosseae alone increased herb yield by 49.4%, whereas in combination with B. subtilis, it increased herb yield by 59.5%. Treatment with AM fungi and B. subtilis did not affect the essential oil content of the plant, but total oil yield was significantly increased because of the increase in biomass production.     

Antagonist Bacillus subtilis HJ5 controls Verticillium wilt of cotton by root colonization and biofilm formation

Cotton plants that are continuously subjected to monoculture suffer greatly from Verticillium wilt disease. The application of a novel bioorganic fertilizer (BOF) consisting of organic fertilizer combined with the antagonistic Bacillus subtilis strain HJ5 significantly suppressed Verticillium wilt of cotton. The disease incidence rates in soils that were treated with BOF (1 %, w/w) in the nursery stage, in the transplanted soil stage or in both stages, decreased by 42.9 %, 57.1 %, and 88.0 %, respectively, compared with controls. B. subtilis HJ5 was tagged with a plasmid-borne gfp gene encoding the green fluorescent protein to investigate its colonization behavior on cotton root surfaces. The results indicated that B. subtilis HJ5 predominantly colonizes the elongation and differentiation zones of the roots and forms micro-colonies in hydroponic and soil systems. The population of B. subtilis HJ5 in the rhizosphere and on cotton roots was also monitored. The number of B. subtilis HJ5 cells on the root surface reached a peak value of approximately 10⁷ cfu per gram of roots 3 days after exposure of the cotton seedlings to the bacteria. Probably the colonization of B. subtilis HJ5 on cotton roots is one of the mechanisms involved in protecting cotton plants from fungal infection.     

不同载体在枯草芽孢杆菌菌剂中的作用研究

载体的选择对微生物菌剂作用的发挥至关重要,考察了不同载体对枯草芽孢杆菌GY801的稳定性、土壤增殖能力、植物促生能力和抑菌抗病能力的影响。结果表明,在氨基酸微量元素型(AA-TE)液体肥料中,额外添加乳酸钠载体,可极显著提升枯草芽孢杆菌GY801的6个月留存率至87.07%,相比于只含菌对照组46.39%的留存率提升了87.7%(P<0.01);本研究还在合适的土壤增殖条件下,比较了不同载体对该菌土壤增殖能力的影响,确定海藻精载体可显著提高GY801在土中的增殖能力,对照组7、15和30 d的土壤增殖倍数分别为110、57和51倍,额外添加海藻精组可分别提升至154、94和79倍,增幅分别为40.0%、64.9%和54.9%;尖椒苗促生试验表明,海藻精载体显著增强了GY801的促生作用,与不加载体只含菌的对照组相比,显著增加了壮苗指数、根冠比、全株干重、根干重等指标,增幅分别为7.7%、8.3%、10.1%、16.7%(P<0.05);抑菌防病试验还表明,相比于仅接种GY801组13.2%的防治效果,额外添加豆粕粉载体的处理组,30 d后针对根腐病防治效果为66.8%,相比于对照组提升了4.06倍。研究表明,在枯草芽孢杆菌GY801系列菌剂中,乳酸钠是比较合适的液体菌剂稳定载体,海藻精是比较合适的土壤增殖和促生载体,而豆粕粉是相对比较合适的抑菌防病载体。     

Modification of rice starch by selective degradation of amylose using alkalophilic Bacillus cyclomaltodextrinase

A cyclomaltodextrinase (CDase) isolated from alkalophilic Bacillus sp. I-5 (CDase I-5) exists in a dodecameric form, an assembly of six dimers, each catalytic site of which is located in a narrow groove at the interface of the dimeric unit. Because of the unique geometric shape of the catalytic site, the enzyme has the ability to discriminate the molecular size of substrates. An analysis of the hydrolysis reaction of the enzyme revealed that its kcat/Km value on amylose was 14.6 s(-1) (mg/mL)(-1), whereas that for amylopectin was 0.92 s(-1) (mg/mL)(-1), showing an exceptionally high preference toward amylose. CDase I-5 was applied to modify the starch structure to produce low-amylose starch products by incubating rice starch with this enzyme. We found that the amylose content of rice starch decreased from 28.5 to 9%, while the amylopectin content remained almost constant with no significant change in the side chain length distribution. When the CDase I-5-treated rice starch was stored at 4 degrees C for 7 days, the retrogradation rate was significantly retarded as compared to that in the control sample.     

Alleviation of drought stress in maize (Zea mays L.) by using endogenous endophyte Bacillus subtilis in North West Himalayas

ABSTRACT

In the present study, we reported the isolation and molecular characterisation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing endophyte and its effect on alleviating drought stress in maize. Ten morphologically distinct indigenous plant growth-promoting rhizobacteria (PGPR) from maize roots and rhizosphere were screened for multiple plant growth-promoting (PGP) traits, and of ten, two strains (DHK and B1N1) possessing maximum PGP traits were evaluated at three water stress regimes in maize: (i) 80% field capacity (FC), (ii) 60% FC, and (iii) 40% FC for 45 days, starting 15 days after sowing to the maturity. Inoculation of DHK strain with maize at 80% FC induced a significant increase in plant biomass (root biomass, 86.67%; shoot biomass, 111.63%) together with decreased reactive oxygen species and increased activity of antioxidant enzymes (superoxide dismutase, 14.11?U/g fresh weight; peroxidase, 1.14?U/g fresh weight and catalase; 14.91?U/g fresh weight) compared to uninoculated control at same water regime (80% FC). The derivative strain also significantly accrued nutrients in maize and enhanced total chlorophyll and amino acid content in comparison with non-inoculated plants. 16S rDNA analysis of DHK strain revealed its lineage to Bacillus subtilis. Present investigations demonstrated the potential of the bacterial partner in alleviating drought stress in maize.     

酶解牦牛血发酵液制备抗氧化肽工艺优化

为进一步提高牦牛血枯草芽胞杆菌发酵液多肽含量,对牦牛血发酵液进行酶解,以酶解液的·OH清除率、多肽含量为主要指标,筛选酶解发酵液的最适蛋白酶,通过单因素和响应面试验优化酶解工艺,并对比菌酶联合制备产物与发酵液体外抗氧化活性。结果表明,碱性蛋白酶酶解发酵液最佳条件为:酶解时间3 h、p H值9.5、酶解温度60℃和酶底比190 U·g-1,此条件下制备得到酶解液多肽含量为5.52mg·mL~(-1),较发酵液提高2.39倍。菌酶联合制备产物对·O2-及脂质过氧化的IC50分别为6.22、4.87mg·mL~(-1),发酵法制备产物对·O2-及脂质过氧化的IC50分别为8.42、11.71 mg·mL~(-1),且菌酶联合制备产物的还原力优于发酵制备产物。因此,菌酶联合制备牦牛血抗氧化肽法优于传统单一发酵法。本研究结果对提高牦牛血抗氧化肽得率,增加牦牛产品附加值具有重要的意义。     

生防芽孢杆菌蛋白水解液对小白菜根系的影响

利用沿海废弃鱼蛋白为原料分别制备含各种肽和氨基酸的广谱生防枯草芽孢杆菌T2水解液、酸水解液和自然浸提液,将其分别添加到植物生长培养基中,观察并分析了3种不同处理对小白菜根系发育的影响。结果表明,T2水解液明显增加了小白菜根毛的长度和密度,有效地诱导了不定根的形成,增强了植株的根系活力,且T2水解液中植物生长素IAA的前体物质色氨酸的含量也明显高于其它两种水解液,从而为开发兼具防病和增产作用的新型生物肥料提供新思路。     

Characterization of cyclodextrin glycosyltransferase of the same gene expressed from Bacillus macerans, Bacillus subtilis, and Escherichia coli

The plasmid pHG contains a cyclodextrin glycosyltransferase (CGTase) gene (cgt) derived from Bacillus macerans. Two transformants, Bacillus subtilis (pHG) and Escherichia coli (pHG), were found to produce CGTases with the same primary structure as the enzyme from B. macerans. However, the beta-cyclodextrin coupling activity of the CGTase from E. coli (pHG) was 14-fold higher than that of the enzymes from the other strains. By contrast, no differences in alpha-cyclodextrin coupling activities were observed among these CGTases. CGTase from E. coli (pHG) was found to be less thermostable than the other CGTases. When the CGTase produced by B. subtilis was treated with increasing urea concentrations (10-1000 mM) to promote increasing degrees of protein unfolding, a bell-shaped beta-cyclodextrin coupling activity profile was obtained. Subtle differences in the conformation of the CGTase produced by E. coli are therefore proposed to be responsible for the markedly increased beta-cyclodextrin coupling activity of this enzyme.