环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

环状芽孢杆菌C-2几丁酶基因的克隆

环状芽孢杆菌（Ｂａｃｉｌｕｓｃｉｒｃｕｌａｎｓ）Ｃ２总ＤＮＡ经ＰｓｔＩ部分酶切后分离２～１０ｋｂ的片段,插入质粒ｐＵＣ１９的ＰｓｔＩ位点,转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）,利用几丁质平板从约８０００个重组子中筛选到一个几丁酶基因阳性克隆（命名为ｐＣＨＴ１）。用１２种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长３０ｋｂ,其中各有一个ＫｐｎＩ,ＳａｃＩ和ＳｓｐＩ位点。把该克隆片段反向插入ｐＵＣ１９的ＰｓｔＩ位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Ｓｏｕｔｈｅｒｎ杂交证实了该片段来自于Ｂ．ｃｉｒｃｕｌａｎｓＣ２基因组,且以单拷贝形式存在,它不能与来自于其它７株几丁酶产生菌的总ＤＮＡ杂交。     

Catabolic pathways of glucose in Bacillus circulans var. alkalophilus

Enzymes and the metabolic pathways of glucose catabolism of Bacillus circulans var. alkalophilus were studied. The metabolism of the microbe was mixed acid fermentative yielding mainly acetic and formic acids as end products from glucose. It was estimated that B. circulans var. alkalophilus partitions 90%–93% of the carbon from glucose into the Embden-Meyerhof-Parnas (EMP) pathway and 7%–10% into the hexose monophosphate (HMP) and Entner-Doudoroff (ED) pathways. Rather low activities of glucose dehydrogenase and gluconokinase appeared in the early logarithmic and late stationary phases, whereas NADH oxidase was markedly high. This result can be explained by a demand to reduce NADH to NAD⁺ for the EMP pathway; when acetic and formic acids are produced, no NADH is regenerated to NAD⁺, which is required in the early steps of EMP and HMP pathways. A small percentage (1.6%–2.4%) of the total CO₂ was formed from (6-C) of glucose, which means that the tricarboxylic acid cycle was functional but its contribution was insignificant. Large differences do not seem to exist between alkaliphilic and neutrophilic bacilli in the use of glucose pathways. Received: January 29, 1999 / Accepted: July 30, 1999     

Effect of culture conditions on growth and esterase production by the moderate thermophile Bacillus circulans MAS2

Growth and esterase production (activity on p-nitrophenyl caprylate) by the newly isolated Bacillus circulans MAS2 bacterial strain were studied. The growth rate at 50°C was high (0.9 h^-1) on LB medium with glucose added. Esterase production followed growth with the majority of activity being intracellular during exponential growth phase. During stationary phase, the esterase activity was released in the culture medium. The strain was able to grow at 35– 55°C with maximum growth rate at 50°C, showing a pattern typical of a moderate thermophile. Growth occurred at pH 6–9 with a maximum at 8, with a similar pattern for the esterase production. Addition of glucose, fructose, sucrose or sodium acetate greatly promoted both growth and esterase production while starch, inulin, tributyrin or glycerol showed no effect. Complex nitrogen sources such as tryptone or yeast extract increased growth and esterase production while mineral sources (ammonium chloride or sulfate), glycine or glutamate showed no effect. An increase of tryptone plus yeast extract and glucose concentrations stimulated growth and esterase production which reached 160 U L⁻¹. Received 17 March 1999/ Accepted in revised form 25 June 1999     

Magnetic porous corn starch for the affinity purification of cyclodextrin glucanotransferase produced by Bacillus circulans

Magnetic porous corn starch was prepared as an affinity adsorbent for the efficient and simple scale-up procedure for one-step purification of cyclodextrin glucanotransferase (CGTase) from Bacillus circulans. Magnetic affinity separation enabled isolation of CGTase from cultivation media (volumes between 10 and 400 mL) with ca 60–70% recovery after elution with alkaline buffers containing soluble starch; the enzyme purification factor was 19–25 in different batches. The majority of ballast proteins were removed during the purification process, which shows high selectivity of the affinity material used.     

Detergent-compatible,organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: Purification and characterization

Proteases are now recognized as the most indispensable industrial biocatalyst owing to their diverse microbial sources and innovative applications. In the present investigation, a thermostable, organic solvent-tolerant, alkaline serine protease from Bacillus circulans MTCC 7942, was purified and characterized. The protease was purified to 37-fold by a three-step purification scheme with 39% recovery. The optimum pH and temperature for protease was 10 and 60°C, respectively. The apparent molecular mass of the purified enzyme was 43 kD as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The K_m and V_max values using casein-substrate were 3.1 mg/mL and 1.8 µmol/min, respectively. The protease remained stable in the presence of organic solvents with higher (>3.2) log P value (cyclohexane, n-octane, n-hexadecane, n-decane, and n-dodecane), as compared to organic solvents with lower (<3.2) log P value (acetone, butanol, benzene, chloroform, toluene). Remarkably, the protease showed profound stability even in the presence of organic solvents with less log P values (glycerol, dimethyl sulfate [DMSO], p-xylene), indicating the possibility of nonaqueous enzymatic applications. Also, protease activity was improved in the presence of metal ions (Ca²⁺, Mg²⁺, Mn²⁺); enhanced by biosurfactants; hardly affected by bleaching agents, oxidizing agents, and chemical surfactants; and stable in commercial detergents. In addition, a protease–detergent formulation effectively washed out egg and blood stains as compared to detergent alone. The protease was suitable for various commercial applications like processing of gelatinous film and as a compatible additive to detergent formulation with its operative utility in hard water.     

Increased Production of Antioxidative Sesaminol Glucosides from Sesame Oil Cake through Fermentation by Bacillus circulans Strain YUS-2

Bacillus circulans strain YUS-2 was isolated as the strongest antioxidant-producer in fermentation of sesame oil cake (SOC, defatted residue yielded from sesame seed oil production). Two major strong antioxidants from fermented SOC were purified and identified as known sesaminol triglucoside and sesaminol diglucoside, however, our results demonstrated that the fermentation process with B. circulans YUS-2 was highly effective to gain the extraction efficiency of the sesaminol glucosides.     

Associated roles of hemolysin and p60 protein for the intracellular growth of Bacillus subtilis

Hemolysin expressing Bacillus subtilis strain (B. subtilis ble/hlA) was used as a carrier for listerial protein p60 to study the impact of this protein on bacterial virulence independent of other gene products of Listeria monocytogenes. Bacillus subtilis ble/hlyA exhibited longer cell chains than B. subtilis ble/hlyA/iap. Recombinant Bacillus strains are able to adhere to the mouse macrophage-like J774 and human epithelial-like Int407 cell lines. The bacterial number of B. subtilis ble/hlyA/iap strain that adhered to the Int407 cell lines was 2.52-fold higher, and its invasion level strain was 2.66-fold higher than that observed for the hemolytic strain. Microscopy analysis of infected monolayers showed that recombinant B. subtilis cells were localized inside the cytoplasm of epithelial cells, near to the nuclei, in cellular compartments with low internal pH. Furthermore, in cells infected with bacteria, the actin structures rapidly changed and accumulation of a fat, wide actin layer around the nucleus zone was observed.     

Continuous production of cyclodextrins in an ultrafiltration membrane reactor,catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrins (CDs) are cyclic oligosaccharides of wide industrial application, whose synthesis is catalyzed by Cyclodextrin glycosyltransferase (CGTase) from starch. Here, CDs were produced using CGTase from Bacillus circulans DF 9R in continuous process and an ultrafiltration membrane reactor. The batch process was conducted as a control. This method allowed increasing the yield from 40 to 55.6% and the productivity from 26.1 to 99.5 mg of CD per unit of enzyme. The method also allowed obtaining a high‐purity product. The flow rate remained at 50% of its initial value after 24 h of process, improving the results described in the literature for starch hydrolysis processes. CGTase remained active throughout the process, which could be explained by the protective effect of the substrate and reaction products on CGTase stability. In addition, batch processes were developed using starches from different sources. We concluded that any of the starches studied could be used as substrate for CD production with similar yields and product specificity. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:695–699, 2015     

Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313

A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology (RSM) and were found to be 48°C and 4.9, respectively. The enzyme was stable up to 60°C and its pH stability was in the range of 5.0–8.0. The K _m and V _max of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, E _a, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a K _i of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a K _i of 9.5 mg/ml. The enzyme was inhibited by Hg²⁺, Mn²⁺, Fe³⁺ and Cu²⁺ and enhanced by Co²⁺ and Mg²⁺. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose in the starch hydrolysate. Journal of Industrial Microbiology & Biotechnology (2002) 28, 193–200 DOI: 10.1038/sj/jim/7000220 Received 11 December 2000/ Accepted in revised form 22 October 2001     

Bacteriocin-like substance production by Bacillus licheniformis strain P40

AIMS: To investigate the production of bacteriocin-like compounds by Bacillus spp. isolated from the Amazon basin. METHODS AND RESULTS: An antimicrobial substance produced by Bacillus licheniformis strain P40 was inhibitory to a broad range of indicator strains, such as Listeria monocytogenes, Bacillus cereus and clinical isolates of Streptococcus spp. The compound was stable at 100 degrees C, but lost its activity when treated at 121 degrees C/103.5 kPa for 15 min. It was resistant to the proteolytic action of trypsin and papain but sensitive to pronase E and was stable within a wide range of pH (3-11). The substance was bactericidal and bacteriolytic to L. monocytogenes. CONCLUSIONS: An antibacterial peptide produced by Bacillus licheniformis was characterized, presenting a broad spectrum of activity against pathogenic and spoilage organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of a substance active against important pathogens addresses an important aspect of food safety.     

A statistical approach for the enhanced production of thermostable alkaline protease showing detergent compatibility activity from Bacillus circulans

Abstract

Response surface methodology (RSM) was employed to enhance the production of a thermostable alkaline protease from Bacillus circulans. Significant influences of peptone, yeast extract, and glucose on protease production were noted with a one-variable-at-a-time optimization strategy. Then, a full factorial central composite design (CCD) was applied to study the effects of glucose, peptone, and yeast extract to determine the optimal concentrations of these compounds for protease production by B. circulans under shake flask fermentation conditions. The statistical reliability and significance of the model was validated by an F-test for analysis of variance (ANOVA); enzyme production was improved significantly under optimized conditions. The enzyme was purified by ammonium sulphate fractionation, and gel filtration chromatography. Maximum enzyme activity was observed at 60°C temperature, and at pH 10. Alkaline protease from B. circulans showed excellent compatibility and stability in the presence of commercial detergents like Ariel, Surf Excel, Tide, Rin, Nirma, Wheel, and Doctor and showed excellent blood destaining effectiveness with commercial detergents.     

Modelling and optimization of fermentation factors for enhancement of alkaline protease production by isolated Bacillus circulans using feed-forward neural network and genetic algorithm

Aim: Modelling and optimization of fermentation factors and evaluation for enhanced alkaline protease production by Bacillus circulans. Methods and Results: A hybrid system of feed‐forward neural network (FFNN) and genetic algorithm (GA) was used to optimize the fermentation conditions to enhance the alkaline protease production by B. circulans. Different microbial metabolism regulating fermentation factors (incubation temperature, medium pH, inoculum level, medium volume, carbon and nitrogen sources) were used to construct a ‘6‐13‐1’ topology of the FFNN for identifying the nonlinear relationship between fermentation factors and enzyme yield. FFNN predicted values were further optimized for alkaline protease production using GA. The overall mean absolute predictive error and the mean square errors were observed to be 0·0048, 27·9, 0·001128 and 22·45 U ml^?1 for training and testing, respectively. The goodness of the neural network prediction (coefficient of R²) was found to be 0·9993. Conclusions: Four different optimum fermentation conditions revealed maximum enzyme production out of 500 simulated data. Concentration‐dependent carbon and nitrogen sources, showed major impact on bacterial metabolism mediated alkaline protease production. Improved enzyme yield could be achieved by this microbial strain in wide nutrient concentration range and each selected factor concentration depends on rest of the factors concentration. The usage of FFNN–GA hybrid methodology has resulted in a significant improvement (>2·5‐fold) in the alkaline protease yield. Significance and Impact of the Study: The present study helps to optimize enzyme production and its regulation pattern by combinatorial influence of different fermentation factors. Further, the information obtained in this study signifies its importance during scale‐up studies.     

Evaluation of immunomagnetic separation in combination with ALOA Listeria chromogenic agar for the isolation and identification of Listeria monocytogenes in ready-to-eat foods

A study was conducted to evaluate the performance of the ALOA (chromogenic media) in combination with immunomagnetic separation (IMS) for the detection of Listeria monocytogenes in ready-to-eat food products. IMS-ALOA method was found to be equivalent to Health Canada's reference culture method as well as comparable to BAX-PCR method in terms of the sensitivity of the methods for the detection of L. monocytogenes in ready-to-eat foods such as turkey roast, beef roast, mixed vegetable salads, potato and egg salad, soft cheese and smoked salmon. The IMS-ALOA method gave 100% sensitivity in the inclusivity tests with 42 pure L. monocytogenes strains. Exclusivity testing with five other species of Listeria genus and 29 pure non-L. monocytogenes strains from 21 different genera showed 97% specificity. The method was able to detect L. monocytogenes at levels near or below 1 cfu/25 g regulatory limit in ready-to-eat food matrices after 24 h enrichment, with a turnaround time of 3 days compared to 7-8 days for culture method. IMS-ALOA method is a valuable alternate test method for the screening of L. monocytogenes in a variety of foods especially ready-to-eat foods.     

Growth dynamics of Bacillus circulans colony

We have investigated the growth dynamics of Bacillus circulans colony exhibiting the knotted-branching pattern by swarming on a hard agar medium. The knotted-branching pattern consists of many circular clusters, so-called subcolonies, and their trajectories. We analysed the processes of a subcolony because they are presumably the key elements for the formation of knotted-branching pattern. It was found that a subcolony has three processes, i.e. "generation", "growth", and "migration" by microscopic and time-resolved observations. An embryonic small subcolony (child subcolony) formed around an existing subcolony (parent subcolony) grows larger and migrates away from the parent subcolony. We proposed a simple model to explain the migration and the growth processes. It is assumed that the internal part of the subcolony is unfavorable for the bacteria and that the motion of the child subcolony on the agar medium can be modeled using a frictional force. The experimental data were quantitatively analysed in order to compare with models. Our models are consistent with the experimental results on following three points: (1) the radius of a subcolony increases linearly with the incubation time, (2) a subcolony stops just after formation and then starts to migrate suddenly, and (3) the trajectory of a subcolony predicted by the model agrees with the experimental one.     

Isolation, characterization and evolution of a new thermophilic Bacillus licheniformis for lactic acid production in mineral salts medium

The high fermentation cost of lactic acid is a barrier for polylactic acid (PLA) to compete with the petrochemical derived plastics. In order to lower the cost of lactic acid, the industry needs a microorganism that can ferment various sugars at high temperature (50 °C) and at the same time using low cost mineral salts (MS) medium. One such bacterium, BL1, was isolated at 50 °C and identified as Bacillus licheniformis. BL1 can ferment glucose to optically pure l-lactate with a maximum specific productivity of 7.8 g/h l in LB medium and 0.7 g/h l in MS medium at 50 °C. BL1 can also consume 10% and 15% glucose in 20 and 48 h, respectively. After serial transfer of BL1 and BL2 in different concentrations of xylose and MS medium respectively, the final mutant BL3 could efficiently ferment glucose and xylose with specific productivity of 1.9 g/h l and 1.2 g/h l in strict MS medium.     

Refolding the unfoldable: A systematic approach for renaturation of Bacillus circulans xylanase

Xylanases are important polysaccharide‐cleaving catalysts for the pulp and paper, animal feeds and biofuels industries. They have also proved to be valuable model systems for understanding enzymatic catalysis, with one of the best studied being the GH11 xylanase from Bacillus circulans (Bcx). However, proteins from this class are very recalcitrant to refolding in vitro. This both limits their high level expression in heterologous hosts, and prevents experimental approaches, such as peptide ligation or chemical modifications, to probe and engineer their stability and function. To solve this problem, a systematic screening approach was employed to identify suitable buffer conditions for renaturing Bcx in vitro. The fractional factorial screen employed identified starting conditions for refolding, which were then refined and developed into a generic protocol for renaturing preparative amounts of active Bcx in a 50–60% yield from inclusion bodies. The method is robust and proved equally proficient at refolding circularly permuted versions that carry cysteine mutations. This general approach should be applicable to related GH11 xylanases, as well as proteins adopting a similar β‐jellyroll fold, that are otherwise recalcitrant to refolding in vitro.     

Evaluation of ALOA plating medium for its suitability to recover high pressure-injured Listeria monocytogenes from ground chicken meat

AIMS: To evaluate a chromogenic plating medium for the isolation of sublethally injured cells of Listeria monocytogenes from processed foods. METHODS AND RESULTS: The inactivation of L. monocytogenes at pressures up to 400 MPa and 12 degrees C in ground chicken meat was employed to examine the recovery of high-pressure injured cells. Before and after different repair incubation periods at 30 degrees C in a nonselective broth, samples were plated onto a selective and differential agar [Agar Listeria according to Ottaviani and Agosti (ALOA)] and in the same medium supplemented with 4% sodium chloride (ALOA-S), and incubated at 37 degrees C. Sublethally injured cells were able to grow when directly plated onto the ALOA medium, without a previous repair incubation period. However, only uninjured cells grew on the ALOA-S medium. CONCLUSIONS: Sublethally injured cells of L. monocytogenes can be quantified by subtracting counts on ALOA-S medium from counts on ALOA medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Possible applications include direct enumeration on ALOA of stressed cells of L. monocytogenes in foods with more than 100 colony forming units per gram.     

健康家兔肝脏内李司忒菌的分离鉴定

我们在健康家兔中发现有些动物的肝脏有灶性坏死,疑为李司忒菌感染所致,从而进行了细菌分离。结果培养出2株革兰氏阳性杆菌,经鉴定结果为单核细胞增多性李司忒菌1/2血清型。     

Performance of a new chromogenic plating medium for the isolation of Listeria monocytogenes from marine environments

AIMS: This study investigated the performance of a new chromogenic plating medium for the detection of Listeria monocytogenes from naturally contaminated samples obtained from marine environments in Morocco in comparison with the conventional plating media PALCAM and Oxford. METHODS: A total of 479 marine samples (sea water, sediment and mussels) were collected from 16 littoral sites in the region of Agadir (western centre of Morocco). They were examined for the presence of L. monocytogenes using a slight modification of the standardized French method (AFNOR V 08-055) for the detection of L. monocytogenes from food and three different isolation media: PALCAM, Oxford and a new chromogenic plating medium. RESULTS AND SIGNIFICANCE OF THE STUDY: The Oxford and the new chromogenic plating media were found relatively more efficient than the PALCAM medium for the isolation of L. monocytogenes (chi-square test, P < 0.05) from marine samples. However, the new chromogenic plating medium was significantly more selective for L. monocytogenes (P < 0.005) than the two other isolation media as 87.5% of the suspect colonies on this medium were indeed confirmed through identification of the isolates vs 12.7% for Oxford and only 3.8% for the PALCAM medium.