Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.     

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.     

Microbial and genomic characterization of Geobacillus thermodenitrificans OS27, a marine thermophile that degrades diverse raw seaweeds

Applied Microbiology and Biotechnology - Seaweeds are a nonlignocellulosic biomass, but they are often abundant in unique polysaccharides that common microbes can hardly utilize; therefore,...     

嗜热脱氮土壤芽孢杆菌β-葡萄糖苷酶的克隆与重组表达及其酶学性质研究

【目的】克隆嗜热脱氮土壤芽孢杆菌中的β-葡萄糖苷酶基因bglB,在E.coli中异源表达,纯化并研究其酶学性质。【方法】利用PCR技术从嗜热脱氮土壤芽孢杆菌的基因组DNA中克隆得到bglB基因,将该基因克隆到表达载体pGEX-2TL上并在大肠杆菌BL21(DE3)中表达,对纯化后的β-葡萄糖苷酶的酶学性质及寡聚状态进行分析。【结果】重组表达的β-葡萄糖苷酶最适温度为65°C,最适pH为7.0,能在pH 5-10、60°C下稳定存在4 h,并能在较高的离子强度(880 mmol/L K+)下发挥其功能。Al3+离子对其有强烈的激活作用,Co2+有一定的抑制作用。最适反应条件下该酶比活力为0.043 IU/mg。该酶具有多种寡聚体形式,这些寡聚体均有β-葡萄糖苷酶活性。【结论】获得一个耐热耐盐的中性β-葡萄糖苷酶,为进一步研究β-葡萄糖苷酶的催化作用机理,提高其热稳定性提供一定的帮助。     

Purification and characterization of an extremely stable glucose isomerase from Geobacillus thermodenitrificans TH2

The D-glucose/D-xylose isomerase was purified from a thermophilic bacterium, Geobacillus thermodenitrificans TH2, by precipitating with heat shock and using Q-Sepharose ion exchange column chromatography, and then characterized. The purified enzyme had a single band having molecular weight of 49 kDa on SDS-PAGE. In the presence of D-glucose as a substrate, the optimum temperature and pH of the enzyme were found to be 80°C and 7.5, respectively. The purified xylose isomerase of G. thermodenitrificans TH2 was extremely stable at pH 7.5 after 96 h incubation at 4°C and 50°C. When the thermal stability profile was analyzed, it was determined that the purified enzyme was extremely stable during incubation periods of 4 months and 4 days at 4°C and 50°C, respectively. The K _m and V _max values of the purified xylose isomerase from G. thermodenitrificans TH2 were calculated as 32 mM and 4.68 μmol/min per mg of protein, respectively. Additionally, it was detected that some metal ions affected the enzyme activity at different ratios. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.     

Production and characterization of exopolysaccharides by Geobacillus thermodenitrificans ArzA-6 and Geobacillus toebii ArzA-8 strains isolated from an Armenian geothermal spring

The thermal ecosystems, including geothermal springs, are proving to be source of thermophiles able to produce extracellular polysaccharides (EPSs). Among the sixteen thermophilic bacilli isolated from sediment sampled from Arzakan geothermal spring, Armenia, two best EPSs producer strains were identified based on 16S rRNA gene sequence analysis and phenotypic characteristics, and designated as Geobacillus thermodenitrificans ArzA-6 and Geobacillus toebii ArzA-8 strains. EPSs production was investigated under different time, temperature and culture media’s composition. The highest specific EPSs production yield (0.27 g g⁻¹ dry cells and 0.22 g g⁻¹ dry cells for strains G. thermodenitrificans ArzA-6 and G. toebii ArzA-8, respectively) was observed after 24 h when fructose was used as sole carbon source at 65 °C and pH 7.0. Purified EPSs displayed a high molecular mass: 5 × 10⁵ Da for G. thermodenitrificans ArzA-6 and 6 × 10⁵ Da for G. toebii ArzA-8. Chemical composition and structure of the biopolymers, determined by GC–MS, HPAE-PAD and NMR, showed that both the two EPSs are heteropolymers composed by mannose as major monomer unit. Optical rotation values [α] ^25 °C_D of the two EPSs (2 mg ml⁻¹ H₂O) were − 142,135 and − 128,645 for G. thermodenitrificans ArzA-6 and G. toebii ArzA-8, respectively.

     

Isolation and identification of alpha-amylase producing Bacillus sp. from dhal industry waste

A bacterial strain was isolated from dhal industry red gram waste and identified as Bacillus. A thermostable extracellular amylase was partially purified from the strain. Optimum temperature and pH for the enzyme were found to be 60 degrees C and 6.5, respectively. The maximum amylase production was achieved with maltose as carbon source. Among the nitrogen sources, peptone and yeast extract produced maximum amylase.     

Production and characterization of thermostable alpha-amylase by thermophilic Geobacillus stearothermophilus

Studies on the alpha-amylase-producing thermophilic bacterium isolated and identified from a hot spring in Jordan and designated as Geobacillus stearothermophilus JT2 were carried out. The optimum conditions for growth and enzyme production were pH 7 and 55 degrees C. The study of the kinetics of cellular growth indicated a mu(max) of 0.22/h, a K(s) of 1.2 g/L, a tau(d) of 3.15 h and a Y(x/s) of 0.43 g cell/g starch. In addition, the activation energy for growth and death were estimated and found to be 30.5 and 210 J/mol, respectively. The effect of different carbon and nitrogen sources on the cellular growth was tested.     

Improving production of hyperthermostable and high maltose-forming alpha-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications

By cultivating Geobacillus thermoleovorans in shake flasks containing cane molasses medium at 70 degrees C, the fermentation variables were optimized by 'one variable at a time' approach followed by response surface methodology (RSM). The statistical model was obtained by central composite design (CCD) using three variables (cane-molasses, urea and inoculum density). An overall 1.6- and 2.1-fold increase in enzyme production was achieved in the optimized medium in shake flasks and fermenter, respectively. The alpha-amylase titre increased significantly in cane-molasses medium (60 U ml(-1)) as compared to that in the synthetic medium (26 U ml(-1)). Thus the cost of enzyme produced in cane molasses medium (0.823 euros per million U) was much lower than that produced in the synthetic starch-yeast extract-tryptone medium (18.52 euros per million U). The shelf life of bread was improved by supplementing dough with alpha-amylase, and thus, the enzyme was found to be useful in preventing the staling of bread. Reducing sugars liberated from 20% and 30% raw pearl millet starch were fermented to ethanol; ethanol production levels attained were 35.40 and 28.0 g l(-1), respectively.     

一株高效生物表面活性剂产生菌的筛选鉴定及其性能研究

从新疆克拉玛依油田受石油污染的土壤中筛选到一株高效生物表面活性荆产生菌,编号为XJ-T-1,经16S rDNA同源性分析和生理生化试验鉴定为产碱杆菌.该菌株具有很强的产表面活性剂能力,全培养液的排油图直径能达到13.0cm,表面张力可降至30.0mN/m.投加10%(V/V)培养7d的XJ-T-1全培养液可使原油乳状液150min脱水率达到90%以上.XJ-T-1对原油具有高效降解作用,投加量为2%～5%(V/V),pH为中性或碱性,降解时间为7d时,XJ-T-1对7500mg/L高浓度含油废水的降解率可达80%以上.     

Isolation and characterization of a moderate thermophile,Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization

An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30-52 degrees C. Similar to other biodesulfurization-competent organisms, M. phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC. The specific desulfurization activity of the 50 degrees C M. phlei GTIS10 culture was determined to be 1.1+/-0.07 micromol 2-HBP min(-1) (g dry cell)(-1). M. phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth. The dszABC operon of M. phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8. The presence of the R. erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M. phlei GTIS10. Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R. erythropolisIGTS8 reach the highest rate of DBT metabolism is near 30 degrees C whereas the temperature that shows the highest activity in resting cell cultures of M. phlei GTIS10 is near 50 degrees C, and activity is detectable at temperatures as high as 57 degrees C. In M. phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase. The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile. These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.     

Biochemical and conformational characterization of a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2

In order to search for valuable and extremely thermo-stable enzymes that could be used in the protein hydrolysis industry, the gene corresponding to a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2 (GtLAP) was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined. Meanwhile, potential applications of GtLAP in the hydrolysis of anchovy proteins were also investigated. GtLAP was overexpressed in IPTG-induced E. coli BL21 (pET28a-LAP) as a soluble protein, and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 125?±?8.75 U/mg proteins. The molecular mass of GtLAP was estimated to be 55?kDa by SDS-PAGE analysis. The optimal reaction temperature and pH of GtLAP were 70?°C and 8.0, respectively. Under optimal conditions, GtLAP showed a marked preference for Leu-p-nitroanilide, followed by Met- and Phe-derivatives. Activity of GtLAP was strongly stimulated by Ni²⁺ ions, but was strongly inhibited by Hg²⁺. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of GtLAP to various extents and further induce changes in enzymatic activity. Results of hydrolytic experiment showed that combining GtLAP with endogenous enzymes could significantly increase the degree of hydrolysis to anchovy proteins and concentrations of free amino acids in hydrolysates. In this regard, GtLAP could potentially be used in the protein hydrolysis industry.     

Statistical optimization of a high maltose-forming,hyperthermostable and Ca2+-independent alpha-amylase production by an extreme thermophile Geobacillus thermoleovorans using response surface methodology

AIM: Statistical optimization for maximum production of a hyperthermostable, Ca2+-independent and high maltose-forming alpha-amylase by Geobacillus thermoleovorans. METHODS AND RESULTS: G. thermoleovorans was cultivated in 250 ml flasks containing 50 ml of chemically defined glucose-arginine medium (g l(-1): glucose 20; arginine 1.2; riboflavin 150 microg ml(-1); MgSO4. 7H2O 0.2; NaCl 1.0; pH 7.0). The medium was inoculated with 5 h-old bacterial inoculum (1.8x10(8) CFU ml(-1)), and incubated in an incubator shaker at 70 degrees C for 12 h at 200 rev min(-1). The fermentation variables optimized by 'one variable at a time' approach were further optimized by response surface methodology (RSM). The statistical model was obtained using central composite design (CCD) with three variables: glucose, riboflavin and inoculum density. An over all 24 and 70% increase in enzyme production was attained in shake flasks and fermenter because of optimization by RSM, respectively. A good coverage of interactions could also be explained by RSM. The end products of the action of alpha-amylase on starch were maltose (62%), maltotriose (31%) and malto-oligosaccharides (7%). CONCLUSIONS: RSM allowed optimization of medium components and cultural parameters for attaining high yields of alpha-amylase, and further, a good coverage of interactions could be explained. The yield of maltose was higher than maltotriose and malto-oligosaccharides in the starch hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: By applying RSM, critical fermentation variables were optimized rapidly. The starch hydrolysate contained a high proportion of maltose, and therefore, the enzyme can find application in starch saccharification process for the manufacture of high maltose syrups. The use of this enzyme in starch saccharification eliminates the addition of Ca2+.     

Production and partial characterization of thermostable and calcium-independent alpha-amylase of an extreme thermophile Bacillus thermooleovorans NP54

AIM: An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization. METHODS AND RESULTS: The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively. The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C. Both alpha-amylase activity and production were Ca2+ independent. CONCLUSIONS: Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase. SIGNIFICANCE AND IMPACT OF THE STUDY: The calcium-independent and thermostable alpha-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.     

一株阿特拉津降解菌的分离鉴定及降解特性

从农药厂废水处理池的活性污泥中分离到一株阿特拉津降解菌X-4, 根据其生理生化特性和16S rRNA基因序列相似性分析, 将其初步鉴定为节杆菌属(Arthrobacter sp.)。该菌能以阿特拉津为唯一碳氮源生长, 42 h内对100 mg/L的阿特拉津降解效果为95.7%, 降解阿特拉津的最适温度为30 °C, pH为7.0。该菌对多种重金属离子都存在抗性, 显示了其在去除阿特拉津和重金属复合污染方面的应用潜力。对其降解基因的初步研究显示, 该菌含有trzN、atzB和atzC 3个阿特拉津降解相关基因。     

一株精氨酸脱亚胺酶产生菌的筛选及鉴定

精氨酸脱亚胺酶由于具有成为精氨酸营养缺陷型肿瘤如肝细胞瘤,黑素瘤治疗药物的潜力而被国内外多个科研机构进行研究。本文报道本研究室于无锡锡惠公园土样中筛选得到一株产精氨酸脱亚胺酶的菌株901,并对其进行了形态,生理生化特征分析及16S rRNA基因分析,该菌被鉴定为恶臭假单胞菌(Pseudomonas putida)。     

Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans

Cytochromes bd are terminal oxidases in the respiratory chains of many prokaryotic organisms. They reduce O? to 2H?O at the expense of electrons extracted from quinol. The oxidases can be divided into two subfamilies, L and S, based on the presence of either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I designated as 'Q-loop'. The L-subfamily members, e.g. the enzyme from Escherichia coli, are relatively well-studied and were shown to generate proton-motive force. The S-subfamily comprises the majority of cytochromes bd including the enzyme from Geobacillus thermodenitrificans but is very poor studied. We compared the properties of cytochromes bd from G. thermodenitrificans and E. coli at room temperature using a combination of absorption, CD and MCD spectroscopy. The G. thermodenitrificans enzyme does contain the high-spin heme b(HS) ("b(595)") despite the fact that its characteristic Q(00)-band ("α"-band) at 595nm is not seen in the absorption spectra; stoichiometry of hemes b(LS), b(HS) and d per the enzyme complex is suggested to be 1:1:1. At 1mM CO, 20-25% of ferrous heme b(HS) in the G. thermodenitrificans oxidase binds the ligand, while in case of the E. coli enzyme such a reaction is minor. In the G. thermodenitrificans oxidase, the excitonic interaction between ferrous hemes b(HS) and d decreased as compared to that in the E. coli bd. The latter may suggest that the two enzymes differ in the distance between heme d and heme b(HS) and/or in the angle between their porphyrin planes.     

产生辅酶Q10的光合细菌菌株的分离及鉴定

对水塘污泥中富集分离的13株光合细菌产生的CoQ10进行定性定量分析,筛出CoQ10含量较高的菌株2c并对其进行系统鉴定。菌株2c为革兰氏阴性菌,细胞杆状,菌体大小0.6μm～0.9μm×1.2μm～2.0μm,单极生鞭毛,片层状光合内膜,位于细胞质膜下并与之平行。光照厌氧或黑暗好氧条件下均可生长,光照下菌体产生红色素,菌体含有细菌叶绿素a和类胡萝卜素。最适生长温度30～35℃,pH7.0～8.0。多种有机化合物均可作为光合作用的电子供体和碳源,蛋白胨和硫酸铵是其生长的较好氮源,酵母膏对其生长有明显刺激作用。16S rDNA序列系统发育分析表明,菌株2c在系统进化树上与GenBank中序列号为AY751758、DQ001155、DQ001158的沼泽红假单胞菌聚为一族。菌株2c至少能稳定传代15次。初步确定菌株2c为沼泽红假单胞菌。     

一株产蛋白酶嗜碱菌株的分离、鉴定及酶学特性

【目的】筛选产蛋白酶嗜碱菌并对其进行鉴定和特性分析。【方法】利用碱性脱脂牛奶培养基分离纯化产蛋白酶嗜碱菌,通过形态特征、生理生化、16S rRNA基因序列分析以及DNA-DNA杂交实验确定菌株的分类地位,利用酪蛋白水解法分析所产蛋白酶的pH和温度作用范围、稳定性和耐氧化剂能力。【结果】从我国西藏盐碱湖样品中分离到一株产碱性蛋白酶的菌株ZL223,该菌株为革兰氏阳性菌,最适生长温度为37℃,最适生长pH9.0,16S rRNA基因序列分析显示,菌株ZL223与假强芽孢杆菌Bacillus pseudo firmus OF4亲缘关系最近,16S rRNA基因序列相似性为98.6%,DNA-DNA杂交结果显示与B.pseudofirmus OF4同源性为86%。菌株ZL223产生的蛋白酶作用的最适pH为12.0,最适温度为40℃。【结论】结合生理生化指标测定的结果,鉴定菌株为假强芽孢杆菌ZL223(B.pseudofirmus ZL223)。该菌株产生的碱性蛋白酶具有较高的pH适应性,值得进一步研究。