A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52 % amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55 % of the maximum activity when assayed at 40–75 °C, 23 % at 20 °C, 16 % at 85 °C, and even 8 % at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62 % xylanase activity and stability at the concentration of 3–30 % (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.     

High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan

To produce xylobiose from xylan, high-level expression of an endoxylanase gene from Bacillus sp. was carried out in Bacillus subtilis DB104. A 1.62-kb SmaI DNA fragment, coding for an endoxylanase of Bacillus sp., was ligated into the Escherichia coli/B. subtilis shuttle vector pJH27Δ88, producing pJHKJ4, which was subsequently transformed into B. subtilis DB104. A maximum endoxylanase activity of 105 U/ml was obtained from the supernatant of B. subtilis DB104 harboring pJHKJ4. The endoxylanase was purified to homogeneity by ion-exchange chromatography and the production profile of xylooligosaccharides from xylan by the endoxylanase was examined by HPLC with a carbohydrate analysis column. Xylobiose was the major product from xylan at 40 °C and its proportion in the xylan hydrolyzates increased with the reaction time; at 12 h, over 60% of the reaction products was xylobiose. These results suggest that xylobiose, which has a stimulatory effect on the selective growth of the intestinal bacterium Bifidobacterium, can be mass-produced effectively by the endoxylanase of Bacillus sp. cloned in B. subtilis. Received: 2 January 1998 / Received revision: 4 March 1998 / Accepted: 4 March 1998     

Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity

Psychrobacter, a micro-organism originally isolated from Antarctic sea water, expresses an extremely active hormone-sensitive lipase (HSL) which catalyzes the hydrolysis of fatty acid esters at very low temperature and is therefore of great potential industrial and pharmaceutical interest. An insoluble form of the entire enzyme has previously been cloned and expressed in Escherichia coli, subsequently refolded and shown to be active, whilst a shorter but completely inactive version, lacking the N-terminal 98 amino acids has been expressed in soluble form. In this study the entire enzyme has been expressed as a fully soluble protein in E. coli in the presence of either the osmolyte trehalose, plus high salt concentration, or the membrane fluidizer benzyl alcohol. Trehalose promotes protein mono-dispersion by increasing the viscosity of the growth medium for bacterial cells, thereby helping circumvent protein aggregation, whilst the heat-shock inducer benzyl alcohol stimulates the production of a network of endogenous chaperones which actively prevent protein misfolding, whilst also converting recombinant aggregates to native, correctly folded proteins. The resultant recombinant protein proved to be more stable than its previously expressed counterpart, as shown by CD and enzymatic activity data which proved the enzyme to be more active at a higher temperature than its refolded counterpart. By light scattering analysis it was shown that the newly expressed protein was monomeric. The stability of the full length native protein will help in understanding the structure of PsyHSL and the role of its regulatory N-terminal for eventual application in a myriad of biotechnological processes.     

Antifungal activity of Bacillus sp. isolated from compost

Four strains ofBacillus isolated from lupine compost exhibited an antifungal activity against six plant fungal pathogens (Rhizoctonia solani, Bipolaris sorokiniana, Sclerotinia sclerotiorum, Trichothecium roseum, Fusarium solani, Fusarium oxysporum). It was significantly influenced by the composition of the cultivation media.     

Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability

Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca²⁺ on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca²⁺ with Ba²⁺, Mn²⁺, Mg²⁺ and Sr²⁺ in the phytase resulted in the expression of > 90% of the maximal activity with calcium-phytate as the substrate, while Fe²⁺ and Zn²⁺ rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg²⁺, Sn²⁺ and Sr²⁺, respectively. On replacing Ca²⁺ on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg²⁺ and Sr²⁺, respectively. Only Ca²⁺ resulted in a marked increase in the melting temperature (T_m) of the enzyme by 12-21 °C, while Ba²⁺, Mn²⁺, Sr²⁺ or Cu²⁺ resulted in a modest (2-3.5 °C) increase in T_m. In the presence of 1-5 mM Ca²⁺, the optimum temperature of the phytase activity was increased from 40 °C to 70 °C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region.     

Purification and characterization of an endoxylanase from the culture broth of Bacillus cereus BSA1

An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE-cellulose chromatography and followed by gel filtration through Sephadex G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55 degrees C and at pH 7.0 and remained reasonably stable in a wide range ofpH (5.0-8.0) and temperature (40-65 degrees C). The Km and Vmax values were found to be 8.2 mg/ml and 181.8 micromol/(min mg), respectively. The enzyme had no apparent requirement ofcofactors, and its activity was strongly inhibited by Cu++, Hg++. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhihited xylanase activity. Since the enzyme was active over wide range ofpH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.     

Cysteine enhances activity and stability of immobilized papain

Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. After immobilization, the optimum temperature of papain activity increased by 20°C (from 60 to 80°C) and its optimum pH activity shifted from 6.5 to 8.0. Catalytic efficiency (k_cat/K_m) and specific activity of the immobilized enzyme do not significantly change after immobilization. The temperature profile of this form of immobilized papain showed a broad range of activity compared with both free and immobilized form of papain in the absence of cysteine. This significant behavior in terms of activation energy is also discussed.     

Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. Strain NG-27

Alkalophilic Bacillus sp. strain NG-27 produces a 42-kDa endoxylanase active at 70 degrees C and at a pH of 8.4. The gene for this endoxylanase was cloned and sequenced. The gene contained one open reading frame of 1,215 bases. An active site characteristic of the family 10 beta-glycanases was recognized between amino acids 303 and 313, with the active glutamate at position 310. Though highly thermostable, the enzyme contains no cysteine residue.     

Antifungal activity of Bacillus coagulans against Fusarium sp

The antifungal activity of Bacillus coagulans against three pathogenic species of Fusarium was examined. Fungal growth was determined by colony forming units, dry matter and ergosterol level. Biosynthesis of Fusarium mycotoxins was also investigated. The strongest inhibition of fungal growth was noticed when Bacillus coagulans was co-inoculated at the beginning of culture. Estimation of ergosterol level as a determinant of fungal growth showed the greatest degree of Fusarium sp. inhibition. Addition of Bacillus coagulans to Fusarium culmorum culture inhibits the DON (deoxynivalenol) production.     

固定化的栅藻深度脱氮和除磷能力

将栅藻包埋固定在筛网上,经饥饿处理,在平行板式生物反应器中对人工污水进行深度净化后,测定藻细胞生长期和饥饿时间对NH4^＋-N和PO4^3-P去除效率影响以及净化前后藻细胞生理变化的结果表明,生长静止初期藻细胞的氮和磷去除率高于对数期的,细胞饥饿48h的氮和磷去除率大于饥饿24和12h,第2个循环中处理4h的氨氮和磷的去除率可分别达到90％和70％以上。     

Characterization of endo-alpha-N-acetylgalactosaminidase from Bacillus sp. and syntheses of neo-oligosaccharides using its transglycosylation activity

Endo-alpha-N-acetylgalactosaminidase was purified to homogeneity from the culture fluid of Bacillus sp. isolated from soil and characterized. The molecular mass of the enzyme was estimated as 110 kDa. The enzyme was stable at pH 4.0-10.0, up to 55 degrees C, and was most active at pH 5.0. The substrate specificity of the enzyme was strict for the disaccharide, galactosyl beta1, 3 N-acetyl-d-galactosamine, bound to aglycone in alpha configuration. On the other hand, the specificity of the enzyme for the aglycone structure was fairly relaxed. The enzyme could transfer the disaccharide from para-nitrophenyl substrate to various acceptors, such as monosaccharides, disaccharides, and sugar alcohols. Using this transglycosylation activity of the endoglycosidase, it may be possible to synthesize neo-oligosaccharides.     

Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence

New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins in E. coli by high cell density cultivation. Received: 8 September 1999 / Received revision: 3 January 2000 / Accepted 4 January 2000     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Functions of the COOH-terminal region of cyclodextrin glucanotransferase of alkalophilic Bacillus sp. #1011: relation to catalyzing activity and pH stability

Cyclodextrin glucanotransferase (beta-CGTase) of alkalophilic Bacillus sp. #1011 degrades starch to mainly beta-cyclodextrin (beta-CD). This enzyme is considered to contain an extra-polypeptide in its COOH-terminal region in addition to its NH2-terminal domain which exhibits the starch-degrading activity. To analyze the functions of this extra-polypeptide in the beta-CGTase, two mutated enzymes, in which DNA regions encoding 10 or 13 amino acids from the COOH-terminus were deleted, were obtained. The mutated enzymes degraded starch to glucose, maltooligosaccharides and alpha-CD, in addition to beta-CD. Furthermore, the pH stability of the mutated enzymes in the alkaline pH range (pH 9-11) was reduced.     

Heterologous expression,purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.     

Improvement of acetoin reductase activity enhances bacitracin production by Bacillus licheniformis

Bacitracin fermentation by Bacillus licheniformis in this work showed three characteristics: (1) the extracellular propionate, butyrate, acetoin and 2,3-butanediol accumulates under conditions of low dissolved oxygen (zero after 4 h cultivation), reaching a total content of approximately 11.1 g/L; (2) cell growth occurs quickly subsequent to cell autolysis and the second growth; and (3) there is a low content of 2,3-butanediol, a reduced product of acetoin catalyzed by acetoin reductase, in the culture process. In this study, addition of MnCl₂ (0.3 mg/L) to the production medium increased the acetoin reductase activity, redirected the NADH oxidation partly from the propionate- and butyrate-production pathways to the 2,3-butanediol synthesis pathway, reduced the intracellular NADH/NAD⁺ ratio, and facilitated cell growth, ultimately achieving a 11.6% increase in bacitracin production (1076 U/mL) versus the control. The results provide useful information regarding large-scale bacitracin production by B. licheniformis.     

芽孢杆菌SK007的分类鉴定及其拮抗植物病原菌的功能分析

【背景】农业生产中,发掘和利用具有生防功能的微生物资源是保障粮食安全和提高作物产量的重要举措。【目的】明确土壤中芽孢杆菌SK007的分类地位,验证其对多种植物病原菌的拮抗作用,挖掘潜在的生防功能。【方法】通过16SrRNA基因和基因组分析方法确定分离菌株SK007的分类地位;采用平板对峙法研究该菌株对番茄灰霉病菌、白菜黑斑病菌、烟草赤星病菌、小麦赤霉病菌、马铃薯干腐病菌等植物病原菌的拮抗作用;采用AntiSMASH分析和预测菌株SK007的抗生素相关基因。【结果】基于16SrRNA基因、全基因组序列、平均核苷酸一致性和DNA同源性分析,结果表明菌株SK007属于Bacillus velezensis,并且具有产生脂肽类抗生素和聚酮类抗生素的基因,对多种植物病原真菌有较强的抗性。此外,菌株SK007基因组中抗生素基因簇数目较多,丰富度高。【结论】芽孢杆菌SK007在拮抗植物病原菌方面有许多优良性状,具有促进作物抗病和增产的潜力。     

一株盐湖芽孢杆菌LG的鉴定及其抗金黄色葡萄球菌活性研究

【目的】从运城盐湖中分离获得一株耐盐细菌LG,对其进行分类鉴定及抗菌特性研究。【方法】利用16S rRNA基因序列分析对菌株进行分类鉴定。以金黄色葡萄球菌为指示菌,采用杯碟法对菌株LG发酵上清液进行抗菌活性检测,利用扫描电镜和透射电镜观察其抗菌效果。研究不同因素对上清液抗菌活性的影响,并采用PCR技术对菌株基因组进行功能基因筛查。【结果】系统发育分析表明该菌为Bacillus属成员,在0–25%的NaCl浓度范围内生长良好,为耐盐细菌。电镜观察发现,菌株LG发酵上清液处理金黄色葡萄球菌可导致细胞结构明显出现异常,细胞质泄漏。抗菌稳定性研究表明,菌株LG发酵上清液活性稳定,表现出了良好的对紫外光、温度、pH和NaCl的耐受性。采用特异性引物,通过PCR筛查发现菌株LG基因组中含有聚酮合酶(PKS I)基因和非核糖体肽合成酶(NRPS)基因,表明该菌具有产多种代谢产物的潜力。【结论】极端环境中的微生物资源可作为抗菌活性物质的潜在新来源。