Culture Conditions for Production of Thermostable Amylase by Bacillus stearothermophilus

Bacillus stearothermophilus grew better on complex and semisynthetic medium than on synthetic medium supplemented with amino acids. Amylase production on the complex medium containing beef extract or corn steep liquor was higher than on semisynthetic medium containing peptone (0.4%). The synthetic medium, however, did not provide a good yield of extracellular amylase. Among the carbohydrates which favored the production of amylase are, in order starch > dextrin > glycogen > cellobiose > maltohexaose-maltopeptaose > maltotetraose and maltotriose. The monosaccharides repressed the enzyme production, whereas inositol and d-sorbitol favored amylase production. Organic and inorganic salts increased amylase production in the order of KCI > sodium malate > potassium succinate, while the yield was comparatively lower with other organic salts of Na and K. Amino acids, in particular isoleucine, cysteine, phenylalanine, and aspartic acids, were found to be vital for amylase synthesis. Medium containing CaCl(2) 2H(2)O enhanced amylase production over that on Ca -deficient medium. The detergents Tween-80 and Triton X-100 increased biomass but significantly suppressed amylase synthesis. The amylase powder obtained from the culture filtrate by prechilled acetone treatment was stable over a wide pH range and liquefied thick starch slurries at 80 degrees C. The crude amylase, after (NH(4))(2)SO(4) fractionation, had an activity of 210.6 U mg. The optimum temperature and pH of the enzyme were found to be 82 degrees C and 6.9, respectively. Ca was required for the thermostability of the enzyme preparation.     

高产耐热脂肪酶嗜热脂肪地芽孢杆菌的选育

采用氮离子注入技术对耐热脂肪酶产生菌嗜热脂肪地芽孢杆菌（Geobacillus stearothermophilus）L4进行诱变,筛选获得酶活力有较大提高且传代稳定的正突变菌株L4-3;再对L4-3进行紫外线诱变,得到脂肪酶活力提高的正突变菌株L4-3-2,其脂肪酶活力达25．71U／mL,较原始菌株M提高511．9％。高产突变株L4-3-2所产脂肪酶的最适作用温度为50℃,70℃保温60min的剩余酶活为82％,最适作用pH为7．0～8．0,为一种耐热碱性脂肪酶。     

嗜热脂肪地芽孢杆菌NAD激酶克隆表达及酶学性质研究

NAD激酶能催化NAD生成NADP。本研究采用PCR技术从嗜热脂肪地芽孢杆菌基因组中获得NAD激酶基因,以pET30a（＋）为表达载体、E．coliBL21（DE3）为宿主菌,实现其在大肠杆菌中异源表达,并进行酶学性质研究。结果显示,嗜热脂肪地芽孢杆菌中NAD激酶编码基因大小为816bp,酶分子量大约为35kD。酶学性质分析表明,来源于嗜热脂肪地芽孢杆菌的NAD激酶最适反应温度和pH分别为35℃、pH7．5,在35qC中保温2h后仍能保持80％左右的活性。Mn2＋、Ca2＋对该酶有较强的激活作用,在最适反应条件下该酶的比活力为4．43U／mg。动力学性质分析结果显示NAD激酶对底物NAD催化的k和圪。,分别为1．46mmol／L和0．25tzmol／（L·min）。NAD激酶在大肠杆菌的异源表达为以NAD为底物生物合成NADP提供了更多生物资源。     

嗜热菌来源的生淀粉酶分离纯化及其酶学性质

从嗜热菌库中分离到两株能水解生淀粉的菌株173和174,通过扩增和测定两株菌的16S rDNA序列并进行比对结果表明,所分离两株菌属于Geobacillus属的细菌.液体摇瓶发酵菌株173、174,其产生的生淀粉酶(简称RSDE173、RSDE174)活力分别达14.5 U/mL和12.9 U/mL.通过生淀粉吸附-熟淀粉洗脱系统和TOYOPEARL HW-55F系统进行分离纯化,得到纯化的RSDE173和RSDE174,纯化倍数分别为50和29,活力回收率分别为34%和41%.有关RSDE173和RSDE174酶学性质研究显示.对熟淀粉水解的最适作用温度均为70℃,而对生淀粉水解则分别在50℃～60℃和40℃～60℃下表现出高水解活力;对不同底物的最适作用pH值均为5.0～5.5;它们对大多数试验离子的敏感性较低,但个别离子如Co2 、Cu'2 对RSDE173或u'2 对RSDE174的酶活力有一定的抑制作用.纯化的这两种生淀粉酶对不同来源生淀粉的底物专一性并不相同.RSDE173底物专一性顺序为红薯淀粉>小麦淀粉>玉米淀粉>木薯淀粉>糯米淀粉;而RSDE174的糯米淀粉>小麦淀粉>红薯淀粉>玉米淀粉>木薯淀粉.RSDE173对生红薯淀粉有很好的降解,其水解糊化淀粉与生红薯淀粉的比值为1.48;而RSDE174优先降解生糯米淀粉,其相应比值为1.69.     

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.     

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.     

Production of amylase by newly isolated moderate halophile,Halobacillus sp. strain MA-2

Production of extracellular amylase was demonstrated under stress conditions of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately halophilic bacterium of spore-forming Halobacillus sp. strain MA-2 in medium containing starch, peptone, beef extract, and NaCl. The maximum amylase production was secreted in the presence of 15% (w/v) Na(2)SO(4) (3.2 U ml(-1)). The isolate was capable of producing amylase in the presence of NaCl, NaCH(3)COOH, or KCl, with the results NaCl>NaCH(3)COOH>KCl. Maximum amylase activity was exhibited in the medium containing 5% (w/v) NaCl (2.4 U ml(-1)). Various carbon sources induced enzyme production. The potential of different carbohydrates in the amylase production was in the order: dextrin>starch>maltose>lactose>glucose>sucrose. In the presence of sodium arsenate (100 mM), maximum production of the enzyme was observed at 3.0 U ml(-1). Copper sulfate (0.1 mM) decreased the amylase production considerately, while lead nitrate had no significant enhancement on amylase production (p<0.05). The pH, temperature, and aeration optima for enzyme production were 7.8, 30 degrees C, and 200 rpm, respectively, while the optimum pH and temperature for enzyme activity was 7.5-8.5 and 50 degrees C, respectively.     

Significant improvement of Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase production using response surface methodology

The medium optimization for the production of the Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase was carried out in shake flask cultures using safflower high oleic oil. In the first step of optimization, a two level fractional factorial design allowed the identification of the concentration of nutrient broth and temperature as the main variables significantly affecting lipase production (P<0.05). In a second step, a D-optimal design was applied to determine the variables optimal values, defined as those yielding maximal lipase production in shaken flasks, thus demonstrating that the optimal concentration of nutrient broth was 3.8 g/l and the optimal culture temperature was 39.5°C. The model was experimentally validated, yielding a lipase production of 2283.70 ± 118.36 U/mL which represents a 6.7-fold increase in comparison to the non-optimized medium.     

通过信号肽筛选优化耐高温α-淀粉酶在枯草芽孢杆菌中的分泌

以大肠-枯草穿梭载体p MA5质粒为基本骨架,以来源于嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus NUB3621的耐高温α-淀粉酶基因为目标基因,利用POE-PCR法,成功构建针对淀粉酶的信号肽筛选载体。从枯草芽孢杆菌168基因组中扩增得到46个信号肽,利用POE-PCR法,使46个信号肽分别与线性化的筛选载体形成对应的multimer产物,直接转化枯草芽孢杆菌1A751,得到含不同信号肽的重组菌株。发酵结果显示,除了5个与淀粉酶适配性很低的信号肽,其它信号肽均有不同的引导淀粉酶细胞外分泌的能力,其中bgls引导淀粉酶细胞外分泌的能力最强,上清酶活的峰值达1 393.3 U/m L。     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.     

Cadmium ion biosorption by the thermophilic bacteria Geobacillus stearothermophilus and G. thermocatenulatus

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl2 (as high as 400 to 3,200 microM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd2+ adsorption experiments to characterize Cd2+ complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd2+ adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.     

Tagatose production by immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant in a packed-bed bioreactor

Using immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant (Gali 152), we found that the galactose isomerization reaction was maximal at 70 degrees C and pH 7.0. Manganese ion enhanced galactose isomerization to tagatose. The immobilized cells were most stable at 60 degrees C and pH 7.0. The cell and substrate concentrations and dilution rate were optimal at 34 g/L, 300 g/L, and 0.05 h(-1), respectively. Under the optimum conditions, the immobilized cell reactor with Mn2+ produced an average of 59 g/L tagatose with a productivity of 2.9 g/L.h and a conversion yield of 19.5% for the first 20 days. The operational stability of immobilized cells with Mn2+ was demonstrated, and their half-life for tagatose production was 34 days. Tagatose production was compared for free and immobilized enzymes and free and immobilized cells using the same mass of cells. Immobilized cells produced the highest tagatose concentration, indicating that cell immobilization was more efficient for tagatose production than enzyme immobilization.     

Dual production of amylase and δ-endotoxin by Bacillus thuringiensis subsp. kurstaki during biphasic fermentation

This study examined the efficacy of a Bacillus thuringiensis (Bt) strain in producing amylase (EC 3.2.1.1) as a by-product without affecting its unique ability for producing δ-endotoxin, thus to establish a cultivation strategy for the dual production and recovery of both δ-endotoxin and amylase from the fermented medium with an industrial perspective. LB medium was individually supplemented (5 to 100%, wt/vol) with flour from six naturally available starchy stored foods (banana, Bengal gram, jack seed, potato, taro or tapioca); after initial fermentation (12 h), the supernatant in the medium obtained by centrifugation (1000 g, 10 min) was used for harvesting amylase and the resultant pellet was further incubated aseptically for the production of endospores and δ-endotoxin by solid-state fermentation. Maximum crude amylase activity (867 U/gram dry substrate, 12 h) was observed in potato flour-supplemented medium (10% wt/vol, 12 h), while the activity in LB control was only 4.36 U/mL. SDS-PAGE profile of the crude (supernatant), as well as partially purified (40–60% (NH₄)₂SO₄ fractionation) amylase showed that its apparent molecular mass was 51 kDa, which was further confirmed by native PAGE. The harvest of industrially significant extracellular amylase (probably α-amylase) produced as a byproduct during early growth phase would boost the economics of the Bt-based bio-industry engaged in δ-endotoxin production.     

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.     

Intestinal lactase and other disaccharidase activities of a suckling crabeater seal (Lobodon carcinophagus)

1. The intestinal disaccharidase activities of a suckling crabeater seal were investigated. 2. Lactase, maltase, isomaltase and cellobiase activities were readily detected but trehalase and sucrase activities were absent. 3. The intestinal homogenates were separated into a soluble (S2) fraction and a particulate brush border (P2) fraction. The lactase activities of the two fractions had different properties corresponding to those of an acid and a neutral beta-galactosidase respectively. Approximately two-thirds of the total lactase activity measured at pH 6.0 was due to the acid beta-galactosidase. 4. The isomaltase and cellobiase activities were found almost exclusively in the particulate fractions but about one third of the maltase activity was in the S2 fraction. This soluble maltase activity appeared to be due to an acid maltase.     

The S-layer from Bacillus stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase.

The S-layer lattice from Bacillus stearothermophilus DSM 2358 completely covers the cell surface and exhibits oblique symmetry. During growth of B. stearothermophilus DSM 2358 on starch medium, three amylases with molecular weights of 58,000, 98,000, and 184,000 were secreted into the culture fluid, but only the high-molecular-weight enzyme was found to be cell associated. Studies of interactions between cell wall components and amylases revealed no affinity of the high-molecular-weight amylase to isolated peptidoglycan. On the other hand, this enzyme was always found to be associated with S-layer self-assembly products or S-layer fragments released during preparation of spheroplasts by treatment of whole cells with lysozyme. The molar ratio of S-layer subunits to the bound amylase was approximately 8:1, which corresponded to one enzyme molecule per four morphological subunits. Immunoblotting experiments with polyclonal antisera against the high-molecular-weight amylase revealed a strong immunological signal in response to the enzyme but no cross-reaction with the S-layer protein or the smaller amylases. Immunogold labeling of whole cells with anti-amylase antiserum showed that the high-molecular-weight amylase is located on the outer face of the S-layer lattice. Because extraction of the amylase was possible without disintegration of the S-layer lattice into its constituent subunits, it can be excluded that the enzyme is incorporated into the crystal lattice and participates in the self-assembly process. Affinity experiments strongly suggest the presence of a specific recognition mechanism between the amylase molecules and S-layer protein domains either exposed on the outermost surface or inside the pores. In summary, results obtained in this study confirmed that the S-layer protein from B. stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase.     

Human small-intestinal β-galactosidases. Separation and characterization of three forms of an acid β-galactosidase

1. An acid beta-galactosidase, optimum pH4.0-4.5, in the human small-intestinal mucosa was separated and characterized. 2. Autolysis of mucosal homogenates at acid pH inactivated the lactase and hetero beta-galactosidase; the total activity of the acid beta-galactosidase was only slightly depleted, but a greater proportion of the enzyme was solubilized by this treatment. 3. Separation on a Sephadex G-200 column revealed that the acid beta-galactosidase could occur in at least three different forms, probably representing monomer, dimer and octamer or polymer of the enzyme. 4. The properties of the different forms of the acid beta-galactosidase were studied with regard to pH optimum, K(m), rate of hydrolysis of different substrates, and sensitivity to p-chloromercuribenzoate and tris as inhibitors. All these properties were the same for the different forms of the enzyme. 5. The acid beta-galactosidase hydrolyses lactose as well as hetero beta-galactosides and contributes to the lactase activity of intestinal biopsies also when measured at pH 6. This enzyme may therefore be responsible for a considerable part of the residual lactase activity found in lactose-intolerant patients.     

高温乳糖酶基因在大肠杆菌中的高效表达

来源于嗜热脂肪芽孢杆菌（Bacillus stearothermophilus)的β-半乳糖苷酶基因bgaB经克隆,测序后,转入大肠杆菌高效表达载体pET-20(b)中,重组菌在IPTG诱导下,表达出的重组蛋白比酶活量为6.66U/mg。比出发菌株高50倍。     

Inhibition of beta-glycosidases by acarbose analogues containing cellobiose and lactose structures

Acarbose analogues, containing cellobiose and lactose structures, were prepared by reaction of the two disaccharides with acarbose and Bacillus stearothermophilus maltogenic amylase. The kinetics for the inhibition by the two analogues was studied for beta-glucosidase, beta-galactosidase, cyclomaltodextrin glucanosyltransferase (CGTase), and alpha-glucosidase. Both analogues were potent competitive inhibitors for beta-glucosidase, with K(I) values in the range of 0.04-2.44 microM, and the lactose analogues were good uncompetitive inhibitors for beta-galactosidase, with K(I) values in the range of 159-415 microM, while acarbose was not an inhibitor for either enzyme at 10 and 5 mM, respectively. Both analogues were also potent mixed inhibitors for CGTase, with K(I) values in the range of 0.1-9.3 microM. The lactose analogue was a 6.4-fold better competitive inhibitor for alpha-glucosidase than was acarbose.     

Application of pheB as a reporter gene for Geobacillus spp., enabling qualitative colony screening and quantitative analysis of promoter strength

The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene for Geobacillus spp. The gene product, catechol 2,3-dioxygenase (C23O), catalyzes the formation of 2-hydroxymuconic semialdehyde, which can be readily assayed. The reporter was used to examine expression from the ldh promoter associated with fermentative metabolism.