Environmental dynamics of Bacillus amyloliquefaciens CCMI 1051 antifungal activity under different nitrogen patterns

Aims: The aim of this study was to evaluate the influence of environmental conditions on the antifungal activity of the Bacillus sp. CCMI 1053 cultures. Methods and Results: The electrospray ionization mass spectra (ESI‐MS) analysis was used to detect the active peptides produced by Bacillus amyloliquefaciens CCMI 1051 cultures in a glucose‐containing medium to which four different nitrogen sources were added. The cultures produced different patterns of Bacillus sporulation and distinct antifungal activity of the cell‐free culture broths. Conclusions: The highest sporulation obtained corresponds to higher antifungal activity when it is formed after 3 days of microbial growth. The antifungal activity against Trichoderma harzianum CCMI 783 is more influenced by the concentration on the nitrogen source than the culture time of incubation. The association of nitrogen concentration and the time of incubation is particularly relevant in the expression of the antifungal activity. Significance and Impact of the Study: The present findings allow the reduction of the use of chemical pesticides and to limit some plant diseases. The association of the nitrogen source and the time of incubation is a novelty, which would improve the production of secondary metabolites. Both economical and environmental benefits arise from the study.     

一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究

从堆肥中分离到一株对植物病原菌尖孢镰刀菌(Fusarium oxysporum)具有强烈抗菌活性并具有较广抗菌谱的细菌Q-12菌株。通过形态观察、生理生化实验1、6S rDNA同源性序列分析以及部分特异性基因序列分析,鉴定该菌为解淀粉芽孢杆菌。该菌的最适培养基组成为:葡萄糖5g/L,NH4Cl 1g/L,牛肉膏0.8g/L,氯化镁5g/L。最适培养温度为33℃,最适培养pH为6.0,最适培养时间为40h。     

解淀粉芽胞杆菌抗真菌活性研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)具有很强的抑制植物病原真菌的能力。其菌体细胞能产生多种酶类、脂肽类抗生素、生物表面活性素、聚酮类化合物和抑菌蛋白,同时具有诱导植物产生系统抗性(ISR)的能力,因此在工农业、种植业、养殖业、食品加工业、果蔬的采后保鲜和饲料业等行业具有重要价值。本文对解淀粉芽胞杆菌抗真菌作用、抗真菌能力提高策略、抗菌化合物合成调节、抑制真菌机制及其引发的ISR等问题进行了深入探讨和综述。     

Identification and natural functions of cyclic lipopeptides from Bacillus amyloliquefaciens An6

Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their surfactant activities. However, natural functions of lipopeptides compounds have received considerably less attention. The aim of this study was to isolate and identify the lipopeptides from Bacillus amyloliquefaciens An6, and further evaluate their biological activities. An6 lipopeptides were detected by PCR using degenerated primers and MALDI‐TOF‐MS. An6 strain was found to produce surfactin, fengycin, and bacillomycin. Following their purification, the in vitro antioxidant activity of An6 lipopeptides was studied through different assays. The scavenging effect on 1,1‐diphenyl‐2‐picrylhydrazyl radicals at a dosage of 0.75 mg/mL was 81%. Its reducing power was concentration‐dependant and reached a maximum of 1.07 at 2.5 mg/mL. Moreover, they showed a strong inhibition of β‐carotene bleaching. An6 lipopeptides mixture was also found to display significant antimicrobial activity against several Gram‐positive, Gram‐negative bacteria, and fungal strains. An6 lipopeptides were insensitive to proteolytic enzymes, stable between pH 4.0 and 12.0, and resistant to high temperature. Our results provided enough evidence proving that An6 lipopeptides could be used as functional‐food components.     

水稻内生枯草芽孢杆菌G87抗菌蛋白的分离纯化及理化特性

【目的】为得到枯草芽孢杆菌(Bacillus subtilis)G87的抗菌蛋白,明确其蛋白理化特性。【方法】采用硫酸铵沉淀和柱层析法进行分离纯化。【结果】获得单一抗菌活性蛋白(峰6-2-1),此抗菌蛋白分子量为50.8 kDa,等电点为5.90。经初步分析,抗菌蛋白不含脂,而含有少量(0.62%)糖;其蛋白部分具有脯氨酸或羟脯氨酸,但不含芳香族氨基酸。抗菌蛋白在高温(≥60℃)和较碱(pH8)环境下活性明显下降,但较抗紫外线、氯仿和胰蛋白酶、蛋白酶K、胃蛋白酶。【结论】枯草芽孢杆菌G87的抗菌蛋白为不含芳烃的糖蛋白,对高温和碱性条件敏感,而对蛋白酶类和紫外线等不敏感。     

Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908

A plant pathogenic fungus, Colletotrichum lagenarium, causing watermelon anthracnose, was isolated from naturally infected leaves, stems, and fruits of watermelon. A bacterial strain, MET0908, showing a potent antifungal activity against C. lagenarium, was isolated from soil. An antifungal protein was purified by 30% ammonium sulfate saturation and concentrated using Centricon 10, DEAE-Sepharose(TM) Fast Flow column and Sephacryl S-100 gel filtration chromatography. The molecular weight of the purified protein was estimated as 40 kDa by SDS-PAGE. The purified protein was stable at 80 degrees C for 20 min and exhibited a broad spectrum of antifungal activity against various plant pathogenic fungi. Confocal microscopy image analysis and scanning electron microscopy showed that the protein acted on the cell wall of C. lagenarium. The purified antifungal protein exhibited beta-1,3-glucanase activity. The N-terminal amino acid sequence of the purified protein was determined as Ser-Lys-Ile-x-Ile-Asn-Ile-Asn-Ile-x-Gln-Ala-Pro-Ala-Pro-x-Ala. A search of the sequence with NCBI BLAST showed no significant homology with any known proteins, suggesting that the purified protein may be novel.     

Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens

Aims: To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product‐derived Bacillus amyloliquefaciens. Methods and Results: An unknown bacterial species cultured from the Yogu Farm? probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell‐free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. Conclusion: The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. Significance and Impact of the Study: This is the first report of intra‐species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin’s activity against bacterial vaginosis‐associated pathogens.     

普鲁兰酶基因在解淀粉芽孢杆菌BF7658菌株中的分泌表达

根据文献报道的核苷酸序列合成Bacillus deramificans普鲁兰酶成熟肽编码基因BdP。将BdP基因插入芽孢杆菌分泌表达载体pUC980信号肽编码区下游,获得重组质粒pUC980-BdP,重组质粒转化中温α-淀粉酶生产菌解淀粉芽孢杆菌BF7658菌株。摇瓶发酵实验表明,重组转化子发酵液有明显普鲁兰酶酶活,约48h酶活达到最高水平,为2．8ASPU／mL。酶学性质分析表明,重组酶最适作用温度约为60℃,最适反应pH为5．0,60℃保温3h仍保存50％的活性。重组酶性质适合淀粉糖化工艺的要求。     

解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究

芽孢杆菌属以多产抗菌素闻名.通过筛选几十株不同来源的芽孢杆菌,获得1株具有强抑真菌活性的芽孢杆菌.经过16S rDNA检测与Biolog分析,确定此株菌为解淀粉芽孢杆菌.本实验对摇瓶发酵的条件进行了优化,经过对发酵上清液硫酸铵盐析、透析,真空冷冻干燥获得粗提蛋白.并对粗蛋白的热稳定性、pH稳定性、抗蛋白酶水解能力、离子稳定性以及抑真菌谱进行了研究,最后使用扫描电镜对抑真菌机制进行了探索.     

解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究

芽孢杆菌属以多产抗菌素闻名。通过筛选几十株不同来源的芽孢杆菌, 获得1株具有强抑真菌活性的芽孢杆菌。经过16S rDNA检测与Biolog分析, 确定此株菌为解淀粉芽孢杆菌。本实验对摇瓶发酵的条件进行了优化, 经过对发酵上清液硫酸铵盐析、透析、真空冷冻干燥获得粗提蛋白。并对粗蛋白的热稳定性、pH稳定性、抗蛋白酶水解能力、离子稳定性以及抑真菌谱进行了研究, 最后使用扫描电镜对抑真菌机制进行了探索。     

Biosynthesis of novel daidzein derivatives using Bacillus amyloliquefaciens whole cells

Abstract

Biotransformation of daidzein was performed by using Bacillus amyloliquefaciens KCTC 13588, Lactococcus lactis subsp. lactis KCTC 3769, Leuconostoc citreum KCTC 13186, Kluyveromyces lactis var. lactis KCTC 17704, Pediococcus pentosaceus KCTC 3116, and Lactobacillus sakei KCTC 13416 cells as a biocatalyst. Four derivatives of daidzein such as daidzein-7-O-phosphate, daidzein-7-O-β-D-glucoside, daidzein-7-O-β-(6′′-O-succinyl)-D-glucoside, and 4′-Ethoxy-daidzein-7-O-β-(6′′-O-succinyl)-D-glucoside were isolated from the biotransformation reaction mixture. The structures of the molecules were elucidated by HPLC, HR-QTOF-ESI/MS and ¹H-NMR analyses. Among them 4′-Ethoxy-daidzein-7-O-β-(6′′-O-succinyl)-D-glucoside derivative is novel compound and not reported elsewhere till now.     

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Abstract A method for efficient polyethylene glycol (PEG)-mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium-succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 10⁵ transformants/μg DNA, 100–1 000-times as high as with DNA from Bacillus subtilis , suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop -6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·10⁴ transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.     

解淀粉芽孢杆菌YP6中碱性磷酸酯酶AP3的酶学性质及其溶磷作用

【背景】微生物溶磷机制多种多样,利用其解磷能力可有效促进植物生长。【目的】探究溶磷菌解淀粉芽孢杆菌YP6的溶磷机制,提高磷资源的利用率。【方法】在大肠杆菌BL21(DE3)中克隆并表达YP6中磷酸酯酶AP3基因,研究AP3的酶学性质并验证AP3的溶磷作用。【结果】AP3为碱性磷酸酯酶,最适反应pH为10.3,最适反应温度为40°C,AP3对pNPP亲和性较高,V_m为4 033.4μmol/(min·mg),K_m为12.2 mmol/L。用纯酶AP3处理24 h后,磷矿粉中的有效磷显著增加。接种菌株YP6发酵7 d后,也使土样中有效磷明显增长。【结论】揭示了碱性磷酸酯酶AP3的溶磷能力,丰富了溶磷微生物库及对微生物溶磷机制的认识。     

源于解淀粉芽胞杆菌酚酸脱羧酶的克隆与表达

【背景】酚酸脱羧酶催化分解酚酸产生的4-乙烯基酚类物质可用于食品添加剂及香精香料行业,而酚酸脱羧酶的表达水平相对较低,因此,高水平的酚酸脱羧酶是工业规模生产4-乙烯基酚类物质的先决条件。【目的】克隆解淀粉芽胞杆菌的酚酸脱羧酶基因,实现在大肠杆菌中的高效异源表达,分析酚酸脱羧酶的底物特异性,并对其表达条件进行优化。【方法】通过PCR技术获得酚酸脱羧酶的基因,构建重组基因工程菌,将测序结果与其他酚酸脱羧酶序列进行比对,利用IPTG诱导方法高效表达蛋白。将重组酚酸脱羧酶与4种不同的底物进行反应,设计响应面试验对诱导条件进行优化。【结果】酚酸脱羧酶对对香豆酸、阿魏酸、咖啡酸、芥子酸的比酶活比率为:100:23.33:15.39:10.51。结合与其他酚酸脱羧酶比对结果发现酚酸脱羧酶家族的C末端区域氨基酸序列的变异率最高,这与酚酸脱羧酶的底物特异性和催化机制有关。通过单因素和响应面试验得到酚酸脱羧酶诱导表达的最佳条件为:2×YT培养基,诱导温度30°C,接种量1.78%,诱导时机3.8 h,IPTG1.25mmol/L,诱导时间18h,此时预测酶活和实际酶活分别为47.61IU/mL和47.55...     

解淀粉芽孢杆菌在畜牧业中的应用进展

解淀粉芽孢杆菌是一种广泛应用于养殖业的益生菌。该菌可通过降低畜舍内氨气等有害气体的含量、减少畜禽粪便中钙及磷等物质的排出量并降低畜舍空气中大肠埃希菌和金黄色葡萄球菌等有害微生物的数量等方式来改善畜舍环境。同时解淀粉芽孢杆菌能促进细胞免疫因子的分泌,提高动物的免疫力,缓解畜禽的免疫应激。此外,该菌还能分解难以降解的纤维素,中和抗营养因子,促进饲料在肠道内的消化,提高饲料转化率。本文就解淀粉芽孢杆菌在上述几方面的作用进行阐述,以期为该菌在动物生产中的应用提供理论依据。