Comparison of the kinetics of lipopeptide production by Bacillus amyloliquefaciens XZ-173 in solid-state fermentation under isothermal and non-isothermal conditions

This study aimed to compare the kinetics of lipopeptide production in solid-state fermentation (SSF) under isothermal and non-isothermal conditions. Models based on the logistic, modified Gompertz and Luedeking–Piret-like equations were developed to describe the time course of fermentation under different conditions. The experiments were conducted in 250 mL flasks and a 50 L fermenter. The results showed that the non-isothermal process had higher levels of product formation rate and substrate utilization rate compared to the isothermal process. The part of substrate carbon to meet microbial maintenance—energy, biomass and lipopeptides formation requirements got increased using the non-isothermal technique. In addition, fermenter conditions positively influenced the lipopeptides formation rate with significantly higher levels of substrate for the microbial growth and product formation, though the product productivity and biomass both decreased as compared to flask. This is the first report that investigates the effects of temperature changing on the kinetics of lipopeptide production by Bacillus amyloliquefaciens strain under SSF condition using soybean flour and rice straw as major substrates in flask and in fermenter.     

Production of lipopeptide antibiotic iturin A using soybean curd residue cultivated with Bacillus subtilis in solid-state fermentation

Bacillus subtilis RB14-CS, which suppresses the growth of various plant pathogens in vitro by producing the lipopeptide antibiotic iturin A, was cultured using soybean curd residue, okara, a by-product of tofu manufacture in solid-state fermentation. After 4 days incubation, iturin A production reached 3,300 mg/kg wet solid material (14 g/kg dry solid material), which is approximately tenfold higher than that in submerged fermentation. When the okara product cultured with RB14-CS was introduced into soil infested with Rhizoctonia solani, which is a causal agent of damping-off of tomato, the disease occurrence was significantly suppressed. After 14 days, the number of RB14-CS cells remained in soil at the initial level, whereas almost no iturin A was detected in soil. As the okara cultured with RB14-CS exhibited functions of both plant disease suppression and nutritional effect on tomato seedlings, this product is expected to contribute to the recycling of the soybean curd residue.     

Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciens S499

Cyclic lipopeptides (cLPs) of the surfactin, iturin and fengycin families synthesized by plant-associated Bacilli represent an important class of antibiotics as they may be tightly involved in the protective effect of selected strains against phytopathogens. However, their production by Bacillus cells developing on roots under rhizosphere conditions is still poorly understood. In this work, we combined electrospray and imaging mass spectrometry-based approaches to determine the detailed pattern of surfactins, iturins and fengycins produced in planta by Bacillus amyloliquefaciens S499. Very different production rates were observed for the three cLPs families. Whereas surfactin accumulated in significant amounts, much lower quantities of iturins and fengycins were detected in the environment of colonized roots in comparison with laboratory medium. In addition, the surfactin pattern produced by strain S499 evolving on roots is enriched in homologues with long fatty acid chains (C15) compared with the chains typically secreted under in vitro conditions. Additional experiments revealed that lipopeptide production by root-associated S499 cells is qualitatively and quantitatively dictated by the specific nutritional context of the rhizosphere (exudates enriched in organic acids, oxygen limitation) but also by the formation of biofilm-related structures around root hairs. As surfactins, iturins and fengycins retain specific functions and bioactivities, the biological relevance of their differential production observed in planta is discussed in the context of biocontrol of plant diseases.     

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...     

Coupling of fermentation and microfiltration for α-amylase production from Bacillus amyloliquefaciens

Abstract: A strain of Bacillus amyloliquefaciens secreting α-amylase was cultivated continuously in a fermentor coupled to a filtration unit. Under the tested operating conditions, the maximum flux was 91 m^-2 h^-1 during the first day and 61 m^-2 h^-1 during the 2nd day. The α-amylase retention was around 30%. Compared to a batch process, continuous cultivation with cell recycling led to lower α-amylase concentrations but to a doubling of volumetric productivities.     

Comparative analysis of PHAs production by Bacillus megaterium OUAT 016 under submerged and solid-state fermentation

In view of risk coupled with synthetic polymer waste, there is an imperative need to explore biodegradable polymer. On account of that, six PHAs producing bacteria were isolated from mangrove forest and affilated to the genera Bacillus & Pseudomonas from morpho-physiological characterizations. Among which the potent PHAs producer was identified as Bacillus megaterium OUAT 016 by 16S rDNA sequencing and in-silico analysis. This research addressed a comparative account on PHAs production by submerged and solid-state fermentation pertaining to different downstream processing. Here, we established higher PHAs production by solid-state fermentation through sonication and mono-solvent extraction. Using modified MSM media under optimized conditions, 49.5% & 57.7% of PHAs were produced in submerged and 34.1% & 62.0% in solid-state fermentation process. Extracted PHAs was identified as a valuable polymer PHB-co-PHV and its crystallinity & thermostability nature was validated by FTIR, ¹H NMR and XRD. The melting (Tm) and thermal degradation temperature (Td) of PHB-co-PHV was 166 °C and 273 °C as depicted from DTA. Moreover, FE-SEM and SPM surface imaging indicated biodegradable nature, while FACS assay confirmed cytocompatibility of PHB-co-PHV.     

Secreted expression of the pullulanase in Bacillus amyloliquefaciens BF7658

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48 h酶活达到最高水平,为2.8 ASPU/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.     

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。     

Xylanase production in solid-state fermentation: a study of its properties

Summary Xylanase production by Aspergillus niger van Tieghem was studied in solid-state cultivation. The screening of substrates was carried out in column incubators aerated with humidified air at 30°C. Results of physiological studies showed that the best yield of xylanase was 2500 U/g dry matter on a mixture of straw+bran 1:1 at 70% of moisture content.In order to compare some properties of the xylanase produced in both liquid and solid cultures, A. niger was also grown on xylan in submerged cultures. The enzymes produced in solid and liquid cultures have an optimum pH of about 3.8 and 4.5, respectively. Xylanase synthetized in solid fermentation is a little more thermostable than that from liquid culture and is maximally active at 50° C, compared to 45° C for enzyme from liquid culture.     

淀粉液化芽孢杆菌b-1,3-1,4-葡聚糖酶基因的克隆及表达的优化

为了比较不同的表达系统对β-1,3-1,4-葡聚糖酶基因(bgl)的效果,本研究将高产β-1,3-1,4-葡聚糖酶的淀粉液化芽孢杆菌Bacillus amylolique faciens BS5582的bgl基因(GenBank Accession No.EU623974)克隆到3种不同的质粒载体中,即构建pEGX-4T-1-bgl、pET20b(+)-bgl和pET28a(+)-bgl重组质粒。比较了pEGX-4T-1-bgl在不同Escherichiacoli宿主中表达效果,以及pET20b(+)-bgl和pET28a(+)-bgl在E.coliBL21(DE3)中的表达效果。结果表明,E.coliBL21(DE3)-pET28a(+)-bgl能够表达最高的重组β-1,3-1,4-葡聚糖酶酶活,其总酶活可达(322.0±8.8)U/mL,是出发菌在最适摇瓶发酵条件下产酶活的40.1%。对该重组菌的产酶条件进行了分析,结合IPTG和乳糖协同的诱导作用,在基础产酶培养基中产最高总酶活为(1883.3±45.8)U/mL,表明其具有良好的工业应用价值。     

Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens

Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of d-glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for d-glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30°C and 4°C, respectively. The K _m and V _max values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 μmole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His₆-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28°C, starting induction with 0.735% xylose when A ₆₀₀ was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.     

Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity

Bacillus amyloliquefaciens strain LP03 isolated from soil, produced an antagonistic compound that strongly inhibited the growth of plant-pathogenic fungi and a lipopeptide biosurfactant. Also, isolated strain LP03 had a marked crude oil-emulsifying activity as it developed a clear zone around the colony after incubation for 24 h at 37°C. LP03 was identified as Bacillus amyloliquefaciens by analysis of partial 16 S rRNA gene and partial gyrA gene sequence. The lipopeptide was purified by acid precipitation of cell-free culture broth, extraction of the precipitates with methanol, silica gel column chromatography, and reverse-phase, high-pressure liquid chromatography. The purified biosurfactant was analyzed biochemical structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). The masses of the two peaks were observed by HPLC chromatography. Their masses were determined to be 1,044 and 1,058 m/z with MALDI-TOF mass spectrometry. As constituents of the peptide and lipophilic part of the m/z 1,022.6, seven amino acids (Glu-Leu-Met-Leu-Pro-Leu-Leu) and β-hydroxy-C13 fatty acid were determined by ESI-MS/MS. The lipopeptide of 1,022.6 Da differed from surfactins in the substitution of leucine, valine and aspartic acid in positions 3, 4, and 5 by methionine, leucine, and proline, respectively. Novel lipopeptide was designated as bamylocin A.     

解淀粉芽胞杆菌关键酶基因过表达对鸟苷积累的影响

【目的】研究鸟苷生物合成途径中的3个关键酶编码基因(prs,purF,guaB)过表达对解淀粉芽胞杆菌(Bacillus amyloliquefaciens)发酵生产鸟苷的影响。【方法】利用穿梭表达载体PBE43,构建含有prs、purF和guaB基因的单独表达载体和prs、purF基因的串联表达载体,将它们分别转入鸟苷生产菌B.amyloliquefaciens TA208后,通过实时定量PCR测定各工程菌株内相关基因的转录水平;通过酶活检测分析关键酶基因扩增对肌苷酸脱氢酶活性的影响;通过摇瓶发酵实验考察工程菌株与对照菌株的生长、耗糖和鸟苷积累情况。【结果】转录分析结果表明prs、purF和guaB基因过表达的同时都伴随着自身转录水平的显著上调。与此同时,prs和purF基因单独表达均轻微下调了嘌呤操纵子的转录水平,但是guaB基因的过表达并不影响嘌呤操纵子和prs基因的转录。酶活分析结果表明prs和purF基因扩增并不影响肌苷酸脱氢酶的活性,guaB基因的扩增使其活性提高了126%。摇瓶发酵实验发现prs和purF基因的单独过表达均未促进宿主菌合成鸟苷,而含guaB基因过表达载体的工程菌鸟苷产量较出发菌株提高20.7%。将prs和purF基因串联表达后,鸟苷产量提高14.4%,糖苷转化率增加6.8%。【结论】过表达guaB基因能够大幅提高鸟苷产量,而prs和purF基因只有实现协同表达才能对宿主菌积累鸟苷产生积极影响,为通过代谢工程技术提高鸟苷产量奠定了研究基础。     

Comparative study of amylolytic enzymes production byAspergillus niger in liquid and solid-state cultivation

Summary Amylolytic enzymes produced by a strain ofAspergillus niger cultivated on cassava starch in liquid or solid culture were found to be mainly glucoamylases. For the same initial amount of substrate, the glucoamylase activity increased even after 60 h of culture on solid medium whereas it decreased in liquid culture. Some characteristics of the amylases produced in both culture conditions were compared. The pH optima for enzymes produced in solid and liquid cultures were 4.5 and 5.0 respectively. Glucoamylase synthetized in solid cultures was significantly more thermostable than that from liquid culture and was maximally active at 70°C compared to 50°C for the enzyme from liquid cultures. The Km values expressed as mg soluble starch/100 ml were 0.1% for crude enzyme from solid culture and 0.057% for crude enzyme from liquid culture.     

Molecular cloning of alpha-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis

The gene coding for alpha-amylase from Bacillus amyloliquefaciens was isolated by direct shotgun cloning using B. subtilis as a host. The genome of B. amyloliquefaciens was partially digested with the restriction endonuclease MboI and 2- to 5-kb fragments were isolated and joined to plasmid pUB110. Competent B. subtilis amylase-negative cells were transformed with the hybrid plasmids and kanamycin-resistant transformants were screened for the production of alpha-amylase. One of the transformants producing high amounts of alpha-amylase was characterized further. The alpha-amylase gene was shown to be present in a 2.3-kb insert. The alpha-amylase production of the transformed B. subtilis could be prevented by inserting lambda DNA fragments into unique sites of EcoRI, HindIII and KpnI in the insert. Foreign DNA inserted into a unique ClaI site failed to affect the alpha-amylase production. The amount of alpha-amylase activity produced by this transformed B. subtilis was about 2500-fold higher than that for the wild-type B. subtilis Marburg strain, and about 5 times higher than the activity produced by the donor B. amyloliquefaciens strain. Virtually all of the alpha-amylase was secreted into the culture medium. The secreted alpha-amylase was shown to be indistinguishable from that of B. amyloliquefaciens as based on immunological and biochemical criteria.