Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris

The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644?U?mL^?1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2?kDa and 29.6?kDa, both larger than the predicted mass of 22.4?kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6–9 and 50°C, respectively.     

Application of Bacillus pumilus as a potential biocontrol agent of Fusarium wilt of tomato

The current study aimed at evaluating the possibility of native Bacillus pumilus species to control Fusarium wilt in tomato and examine its effect on plant growth. Biocontrol traits of B. pumilus strains, biofilm assay, root colonisation and in vivo studies under pot conditions were determined. Strain ToIrMA-KC806242 formed biofilm efficiently and could colonise and survive on tomato rhizosphere (3.1 × 10⁴ CFU/g of root). The amount of auxin production was recorded 29.7 μg/ml at the 96th hour of incubation. Siderophore production was determined positive, while ToIrMA was not able to solubilise phosphate compounds or produce cyanide hydrogen. Statistical analysis of data revealed that the increase in root and shoot length was recorded 60 and 84%, respectively, over control. In addition, about 73% reduction in disease incidence was determined in vivo experiments. In conclusion, this study suggests B. pumilus ToIrMA strain as a possible biocontrol agent in the field experiments.     

Sequence of the gene encoding an alkaline serine proteinase of Bacillus pumilus TYO-67

The complete nucleotide sequence of the gene encoding an alkaline serine proteinase (aprP) of Bacillus pumilus TYO-67 was determined. The sequence analysis showed an open reading frame of 1,149 bp (383 amino acids) that encoded a signal peptide consisting of 29 residues and a propeptide of 79 residues. The deduced 3 amino acid residues, D32, H64, and S221, were identical with 3 essential amino acids in the catalytic center of subtilases. The sequence around these residues revealed that APRP was a new member of the true subtilisin subgroup of the subtilisin family. The highest homology was found in subtilisin NAT at 64.4% in the DNA sequence. The residue S189 of APRP was different from those of other subtilases.     

Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinases

AIMS: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. METHODS AND RESULTS: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N-terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4.5 and 5.1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. CONCLUSIONS: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt.     

短小芽孢杆菌AR03对烟草赤星病菌和白粉病菌的防治

采用对峙培养法、凹玻片法、LB琼脂培养基萌发法测定短小芽孢杆菌AR03对烟草赤星病菌和白粉病菌的抑制作用.结果表明： AR03菌液对2种病菌的菌丝生长和分生孢子萌发均有明显的拮抗作用;对赤星病菌的抑制作用表现为:经AR03菌液原液(3×10⁸cfu·mL^-1)处理的菌丝隔间变短、肿胀且集结成团,内含物聚集,菌丝顶端生长膨大畸形;经该菌液处理的赤星病菌分生孢子不萌发或萌发产生畸形芽管,分生孢子变形、肿大,纵横分隔部分的组织膨胀呈泡状.AR03菌液原液、30倍液和200倍液对白粉病菌分生孢子萌发的平板抑制率分别为100%、91.4%和 69.3%,菌液对分生孢子萌发的破坏作用表现为分生孢子不萌发,细胞肿胀变形、细胞原生质解体或收缩,孢子内、外壁分离,由于原生质外泄,一些分生孢子内部呈中空状.温室防治试验结果表明： 不同浓度AR03菌悬液处理对烟草白粉病的防治效果存在显著差异,第二次药后7和15 d,AR03菌液原液的防治效果分别达到83.8%和90.3%,与对照药剂差异不显著;而100倍稀释液的防效分别为70.0%和73.3%,与对照药剂差异显著.AR03菌株防治白粉病的持效期为30 d以上.     

酸性半纤维素降解细菌的筛选与鉴定

从南方酸性水稻土和腐烂秸秆中采集样品,经初筛、复筛获得4株产生明显水解圈且D/d值较大同时传代效果稳定的酸性半纤维素降解细菌。DNS法测定4株细菌半纤维素酶活力,最终结合其产酶速度及酶活大小得到菌株NB8,液体摇瓶发酵72 h其半纤维素酶活达85.12 U/mL。经形态学观察和生理生化指标测定,初步确定该菌为芽胞杆菌属。结合16S rDNA基因序列分析结果,可以初步鉴定NB8为短小芽胞杆菌(Ba-cillus pumilus)。     

多粘芽孢杆菌(Bacillus polymyxa)β-葡萄糖苷酶基因在大肠杆菌中的表达、纯化及酶学性质分析

从多粘芽孢杆菌(Bacillus polymyxa1．794)中克隆得到β—葡萄糖苷酶基因bglA。将其构建在大肠杆菌(Escherichia coli)表达载体pET28a( )上,转化E．coli BL21,获得重组工程菌BL1979。重组表达的β—葡萄糖苷酶的酶活力达到24．7IU／mL,经镍柱纯化后的β—葡萄糖苷酶最适温度为37℃,最适pH值为7．0,该酶经纯化后纯度可达92．7％。用非变性梯度聚丙烯凝胶电泳发现该酶具有多种寡聚体形式,经荧光底物活性染色表明这些寡聚体均具有β—葡萄糖苷酶活性。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315?±?16 IU/mL acidic xylanase, 290?±?20 IU/mL alkaline xylanase, and 88?±?9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10?mM MgSO₄, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60?hr at 200?rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3?hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6?hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.