A highly thermostable and alkaline amylase from a Bacillus sp. PN5

A highly thermostable alkaline amylase producing Bacillus sp. PN5 was isolated from soil, which yielded 65.23 U mL(-1) of amylase in medium containing (%) 0.6 starch, 0.5 peptone and 0.3 yeast extract at 60 degrees C, pH 7.0 after 60 h of incubation. Maximum amylase activity was at pH 10.0 and 90 degrees C. The enzyme retained 80% activity after 1 h at pH 10.0. It exhibited 65% activity at 105 degrees C and had 100% stability in the temperature range between 80 and 100 degrees C for 1 h. In addition, there was 86.36% stability after 1-h incubation with sodium dodecylsulphate. These properties indicated possible use of this amylase in starch saccharification and detergent formulation.     

Production of laccase by a newly isolated strain of Trametes modesta

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by Trametes modesta were evaluated using the one-factor-at-a-time method. The final optimisation was done using a central composite design resulting in a four-fold increase of the laccase activity to 178 nkat ml(-1). Response-surface analysis showed that 7.34 g l(-1) wheat bran, 0.87 g l(-1) glucose, 2.9 g l(-1) yeast extract, 0.25 g l(-1) ammonium chloride, an initial pH of 6.95 and an incubation temperature of 30.26 degrees C were the optimal conditions for laccase production. Laccase produced by T. modesta was fully active at pH 4 and at 50 degrees C. The laccase was very stable at pH 4.5 and at 40 degrees C but half-lives decreased to 120 and 125 min at higher temperature (60 degrees C) and lower pH (pH 3).     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump.

The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temperature-dependence measurements of the stationary pump current yielded an activation energy of approximately 100 kJ mol-1. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Na+ release. Simulating the data with a simple three-state model enabled us to estimate the turnover rate under saturating substrate concentrations, yielding rates (at pH 7.4) of approximately 60 s-1 and 200 s-1 at 24 degrees C and 36 degrees C, respectively.     

Thermal stability of beta-xylanases produced by different Thermomyces lanuginosus strains

The thermostability of beta-xylanases produced by nine thermophilic Thermomyces lanuginosus strains in a coarse corn cob medium was assessed. The xylanase produced by T. lanuginosus strain SSBP retained 100% of its activity after 6 h at temperatures up to 65 degrees C. In comparison seven ATCC strains and the DSM 5826 strain of T. lanuginosus only retained 100% xylanase activity at temperatures up to 60 degrees C. Culture filtrates of T. lanuginosus strain SSBP grown on coarse corn cobs, oatspelts xylan, birchwood xylan, wheatbran, locust beangum, and sugar cane bagasse, retained 100% xylanase activity at temperatures up to 60 degrees C. The xylanase produced on corn cobs was the most thermostable and showed an increase of approximately 6% from 70 degrees C to 80 degrees C. The T(1/2) of all strains at 70 degrees C at pH 6.5 varied greatly from 63 min for strain ATCC 28083 to 340 min for strain SSBP. The xylanase of strain SSBP was much less thermostable at pH 5.0 and pH 12.0 with T(1/2) values of 11.5 min and 15 min, respectively at 70 degrees C. At 50 degrees C, the enzyme of T. lanuginosus strain SSBP produced on coarse corn cobs was stable within the pH range of 5.5-10.0. Furthermore, the enzyme retained total activity at 60 degrees C for over 14 days and at 65 degrees C for over 48 h. The xylanase of T. lanuginosus strain SSBP possesses thermo- and pH stability properties that may be attractive to industrial application.     

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.     

A novel thermostable xylanase from Thermomonospora sp.: influence of additives on thermostability

An alkalothermophilic Thermomonospora sp. producing high levels of xylanase was isolated from self-heating compost. The culture produced 125 IU/ml of xylanase when grown in shake flasks at pH 9 and 50 degrees C for 96 h. The culture filtrate also contained cellulase (23 IU/ml), mannanase (1 IU/ml) and beta-xylosidase (0.1 IU/ml) activities. The xylanase was active at a broad range of pH (5-9) and temperature (40-90 degrees C). The optimum pH and temperature were 7 and 70 degrees C, respectively. The enzyme was stable in the pH range 5-8 and was thermostable with half-lives of 8 and 4 h at 60 degrees C and 70 degrees C, respectively, but only 9 min at 80 degrees C. The effects of a variety of compounds to enhance the stability of xylanase at 80 degrees C was studied. Addition of sorbitol, mannitol and glycerol increased the thermostability of xylanase in proportion to the number of hydroxyl groups per polyol molecule. Glycine also offered protection against thermoinactivation. Xylan, trehalose, gelatin and trehalose-gelatin mixture had marginal effect on the thermostability of xylanase at 80 degrees C.     

The possible existence of a heat-stable alkaline proteinase (HAP) in rat skeletal muscle

1. A unique caseinolytic activity was found in the crude extract from chicken and rat skeletal muscle. Hardly any activity was detected at physiological assay temperatures at pH 8.0 but did well at around 60 degrees C. 2. The activity partially purified from rat skeletal muscle showed optimum pH at around 8.0 at 60 degrees C. It hardly hydrolyzed casein below 50 degrees C, but in the presence of 5 M urea it showed relatively high activity at 30 degrees C. The activity was completely stable at 50 degrees C for 1 hr. 3. The activity seems to be contained in a high mol. wt (450,000) protein from the elution volume and is due to cysteine proteinase from the effect of inhibitors. 4. The above properties agreed with those of the heat-stable alkaline proteinase (HAP) of fish purified homogeneously by electrophoresis. This seems to suggest that HAP may also exist in rat skeletal muscle.     

萌发绿豆中水解大豆胰蛋白酶抑制子蛋白酶的纯化及固定化

大豆是豆类植物中最早发现存在蛋白酶抑制子的 ,由于其存在影响了豆类的利用价值 ,因此研究人员一直在寻找着解决办法。采用加热处理方法不能彻底钝化豆类蛋白的蛋白酶抑制子活性 ,且豆类蛋白的含硫氨基酸主要存在于各类蛋白酶抑制子中 ,从豆类蛋白中除去抑制子蛋白将大大降低其营养效价。本研究的目的是试图寻找一种可在常温下降解豆类胰蛋白酶抑制子的蛋白酶 ,从而钝化豆类的胰蛋白酶抑制活性。在前期工作中 ,我们发现枯草杆菌蛋白酶 (Ｓｕｂ ｔｉｌｉｓｉｎ)可在在常温下降解花生及大豆胰蛋白酶抑制剂[1] ,近期我们的研究表明 ,Ａｌｃａ…     

The isolation and use of iron-oxidizing,moderately thermophilic acidophiles from the Collie coal mine for the generation of ferric iron leaching solution

Moderately thermophilic, iron-oxidizing acidophiles were enriched from coal collected from an open-cut mine in Collie, Western Australia. Iron-oxidizers were enriched in fluidized-bed reactors (FBR) at 60 degrees C and 70 degrees C; and iron-oxidation rates were determined. Ferrous iron oxidation by the microbiota in the original coal material was inhibited above 63;C. In addition to four iron-oxidizers, closely related to Sulfobacillus spp that had been earlier isolated from the 60 degrees C FBR, one heterotroph closely related to Alicyclobacillus spp was isolated. The Alicyclobacillus sp. isolated from the Collie coal mine tolerated a lower pH than known Alicyclobacillus spp and therefore may represent a new species. The optimum temperature for growth of the iron-oxidizing strains was approximately 50 degrees C and their maximum temperatures were approximately 60 degrees C. The FBR was adjusted to operate at 50 degrees C and was inoculated with all of the isolated iron-oxidizing strains. At 60 degrees C, an iron-oxidation rate of 0.5 g Fe(2+) l(-1) x h(-1) was obtained. At 50 degrees C, the iron-oxidation rate was only 0.3 g Fe(2+) l(-1) x h(-1). These rates compare favourably with the iron-oxidation rate of Acidianus brierleyi in shake-flasks, but are considerably lower than mesophilic iron-oxidation rates.     

Binding strength of calcium ion to bovine alpha-lactalbumin

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.     

Purification and kinetics of a raw starch-hydrolyzing,thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae

The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degrees C and pH 7.0, with a t(1/2) of 12 h at 60 degrees C and 7 h at 80 degrees C. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degrees C (K(m) 0.50 mg mL(-1) and V(max) 109 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.40 and V(max) 143 micromol mg(-1) protein min(-1)). The experimental K(m) and V(max) values are in agreement with the predicted values at 50 degrees C (K(m) 0.45 mg mL(-1) and V(max) 111.11 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.36 mg mL(-1)and V(max) 142.85 micromol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degrees C, and thereafter, inactivation. The enzyme was strongly stimulated by Co(2+), Fe(2+), Ag(2+), and Ca(2+), slightly stimulated by Cu(2+) and Mg(2+), and inhibited by Hg(2+), Zn(2+), Ni(2+), and Mn(2+). Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K(m) 2.4 mg mL(-1), V(max) 50 micromol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification.     

Purification and characterization of a novel pectinase from Acrophialophora nainiana with emphasis on its physicochemical properties

An extracellular pectinase (PECI) was purified to apparent homogeneity from liquid state cultures of the thermophilic fungus Acrophialophora nainiana by ultrafiltration and a combination of gel filtration and ion-exchange chromatographic procedures. The molecular masses of PECI were 35,500 and 30,749 Da, as determined by SDS-PAGE and mass spectrometry, respectively. It was more active at 60 degrees C and pH 8.0 and showed high stability at 50 degrees C with half-life of 7 days. However at 60 and 70 degrees C, PECI was much less stable with half lives of approximately 20 and 3 min, respectively. The thermostability of purified PECI was also investigated by fluorescence and circular dichroism spectroscopy. Fluorescence revealed that the unfolding transition region was observed between 45 and 70 degrees C. A major decrease in the stability was found at 70 degrees C. Circular dichroism measurements at pH between 5.0 and 9.0 showed a transition temperature (T(m)) range of 50-55 degrees . The thermodynamic analysis of these results showed that EPGI is thermal stable protein exhibiting maximum stability (DeltaG(25)) of 22.65 and 19.19 kcal/mol at pH 8.0 and 9.0, respectively. The apparent K(m) value on pectin from citrus fruits was 4.22 mgml(-1). PECI exhibited no detectable activity of pectin methylesterase, endo-polygalacturonase, mannanase, xylanase and cellulase. However, it showed exo-polygalacturonase and pectin lyase activities. The presence of carbohydrate was detected in the pure PECI. It was activated by l-tryptophan, DEPC, DTT, DTNB, DTP, l-cystein and beta-mercaptoethanol and inhibited by NBS, Fe(2+), Cu(2+), Zn(2+), Mn(2+), Al(3+) and Ca(2+). The enzyme showed homology with a pectin lyases from Xanthomonas campestris and Bacillus licheniformis.     

链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２株脂肪酶产生菌中,链霉菌Ｚ９４－２产脂肪酶活力为５９６ｕ／ｍＬ,其最适培养基（ｇ／Ｌ）为：糊精１０、黄豆饼粉３０、尿素１０、Ｋ２ＨＰＯ４０．５、ＭｇＳＯ４０．５、ＮａＣｌ１和ＡＥＯ９０．５,产酶的最适条件为：初始ｐＨ９．５～１０．０,在２６℃培养４８ｈ。用ＰＶＡ橄榄油乳化系统测定该酶的最适ｐＨ９．８,最适温度３７℃,在ｐＨ８．６～１０．２于５℃存放２４ｈ,酶活力不变。０．１４ｍｏｌ／Ｌ的氯     

Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces

A novel laccase from the ascomycete Melanocarpus albomyces was purified and characterised. The enzyme was purified using anion exchange chromatography, hydrophobic interaction chromatography and gel filtration, and the purified laccase was biochemically characterised. It had activity towards typical substrates of laccases including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), dimethoxyphenol, guaiacol, and syringaldazine. The laccase showed good thermostability and it had a pH optimum at neutral pH, both unusual properties for most known fungal laccases. The activity of the laccase from M. albomyces was highest at 60-70 degrees C. With guaiacol and syringaldazine the pH optima were rather broad: 5-7.5 and 6-7, respectively. It retained 50% of its activity after 5 h incubation at 60 degrees C. The molecular weight of the laccase was about 80 kDa and the isoelectric point 4.0. The ultraviolet-visible absorption and electron paramagnetic resonance spectra of the purified laccase indicated that the typical three types of copper were present.     

嗜热毛壳菌内切β-葡聚糖酶的分离纯化及特性

探讨了液体发酵嗜热毛壳菌（Chaetomium thermophile)产生的内切β－葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀,DEAE－Seplharose Fast Flow阴离子层析,Pheny1-Sepha-rose疏水层析,Sephacry1 S-100分子筛层析等步骤便可获得凝胶电泳均一的内切β－葡聚糖酶,经12．5％SDS－PAGE和凝胶过滤层析法分离纯化酶蛋白的分子量约为67．8kD的69．8kD。该酶反应的最适温度和pH分别为60℃和4．0－4．5在pH5.0条件下,该酶在60℃下稳定：70℃保温1h后,仍保留30％的活性;在80摄氏度的半衰期为25min,金属离子内切β－葡聚糖酶的活性影响较大,其中Na^ 对酶有激活作用;Fe^2 ,Ag^ ,Cu^2 ,Ba^2 ,Zn^2 等对酶有抑制作用。该酶对结晶纤维素有没水解能力。     

A novel ribonuclease from fresh fruiting bodies of the portabella mushroom Agaricus bisporus.

A 14 kDa ribonuclease with a novel N-terminal sequence was isolated from fresh fruiting bodies of the portabella mushroom. It was adsorbed on DEAE-cellulose and carboxymethyl-cellulose, and demonstrated the highest ribonucleolytic potency toward poly (A), 60% as much activity toward poly (C), 40% as much activity toward poly (U), and the least activity (7% as much) toward poly (G). It exhibited a pH optimum at pH 4.5 and a temperature optimum at 60 degrees C. Its activity at 100 degrees C was higher than that at 20 degrees C.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86.

A thermostable D-xylose-glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by gamma-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE-Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12 columns. The N-terminal amino acid sequence and amino acid analysis shows 73-92% homology with xylose-glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12 column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60-85 degrees C at pH 7.0, using D-glucose, and up to 50-60 degrees C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ x mol(-1)) and the critical temperature (Tc = 60 degrees C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59 degrees C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 k x mol(-1). The specific activity (km values) for D-xylose-glucose isomerase at 70 degrees C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, respectively.     

Optimization of culture conditions for the production of thermostable polygalacturonase by <Emphasis Type="Italic">Penicillium</Emphasis> SPC-F 20

Penicillium strain isolated from citrus fruit was found to produce thermostable polygalacturonases. Optimization of process parameters resulted in high levels of enzyme production after 3 days of incubation at a pH of 5.0 at 30 degrees C in the presence of 1% pectin. The optimum temperature for enzyme activity was 60 degrees C and a pH of 5.5 was found to be the optimal pH. The enzyme showed a high level of thermostability in the presence of substrate with a residual activity of 48% after 2 h of incubation at 60 degrees C. A thermostable nature with a high pH range for activity makes it an industrially important enzyme.