从海洋中分离的弧菌QY102褐藻胶裂解酶的纯化和性质研究

从马尾藻(Sargassum)表面分离到一株产生高效胞外褐藻胶裂解酶的海洋弧菌（Vibrio sp.） QY102。以褐藻胶为唯一碳源发酵培养后,发酵液上清通过0.22μm滤膜过滤、DEAESepharose离子交换和Superdex75凝胶过滤得到电泳纯的褐藻胶裂解酶。酶的性质研究表明：其分子量约为28.5kD（SDSPAGE）,反应最适温度为40℃,最适pH为7.1,Ca2+、Mg2+对酶活有促进作用,而Ni2+、Al3+、Zn2+、Ba2+对酶活有抑制作用。该酶的活性明显高于已报道的褐藻胶裂解酶,pH稳定范围广（5～10）,并且对聚甘露糖醛酸的活性高于对聚古罗糖醛酸的活性。     

重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究

将产热稳定性过氧化氢酶的重组大肠杆菌培养后菌体破碎得到的粗酶液经热处理、硫酸铵分级沉淀、DEAE\|Sephadex A\|50离子交换层析、HiPrep16/10 Phenyl疏水作用层析、Superdex200 HR 10/30凝胶层析提纯后得到电泳纯的酶,比酶活达到15629U/mg。此酶的最适温度为70℃,最适pH70,在60℃保温60min酶活力基本不变,在pH3～8的范围内比较稳定。此酶的Km和Vmax分别为775mmol/L和278mmol\5min\+\{-1\}·mg-1。1mmol/L的Zn2+、Ba2+、Mn2+可使该酶完全失活,KCN、NaN\-3、Na\-2S\-2O\-4、巯基乙醇对酶活力有抑制作用,50mmol/L的EDTA不影响酶活性。     

Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

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.     

An extracellular--pepstatin insensitive acid protease produced by Thermoplasma volcanium

In this study, some parameters for the production and caseinolytic activity of an extracellular thermostable acid protease from a thermoacidophilic archaeon Thermoplasma volcanium were determined. The highest level of growth and enzyme production were detected at pH 3.0 over an incubation period of 192 h at 60 degrees C. The pH optimum for the acid protease activity was 3.0 and the enzyme was fairly stable over a broad pH range (pH 3.0-8.0). The temperature for maximum activity of the enzyme was 55 degrees C and activity remained stable between 50 degrees C and 70 degrees C. These features could be of relevance for various biotechnological applications of this enzyme. Serine-(PMSF), cysteine-(DTT), metallo-(EDTA) and aspartate-(pepstatin) protease inhibitors did not inhibit the caseinolytic activity of the enzyme. Therefore, Tp. volcanium acid protease could be a member of the pepstatin-insensitive carboxyl proteinases.     

重组超耐热酸性α-淀粉酶的分离纯化及其性质研究

基因工程菌所产生的重组超耐热酸性α-淀粉酶,通过超滤浓缩、脱盐和聚丙烯酰胺垂直板凝胶电泳进行纯化,得到电泳纯的超耐热酸性α-淀粉酶,纯化倍数为11.7,活力回收率为29.8%。用SDSPAGE测得该酶的分子量为55kD,酶的等电点pI(室温)为5.0,以可溶性淀粉为底物的Km值为1.12gL,用硫酸酚法测得其含糖量为15.4%。该酶的最适反应温度为95℃,最适反应pH值为4.5。在pH4.0～7.0室温放置48h酶活没有变化,110℃保温1h残留60%活力。Cr3 、Fe2 、Cu2 抑制酶的活性,Ca2 对酶活无影响。EDTA和DTT对酶的活性无影响。     

Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1

A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH(2)-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85 degrees C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co(2+) and Mn(2+). The k(cat)/K(m) for D-alaninamide was measured as 544.4 +/- 5.5 mM(-1) min(-1) at 50 degrees C with 1 mM Co(2+).     

Characterization of a thermostable Bacillus stearothermophilus alpha-amylase

Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized. The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B. stearothermophilus, B. subtilis, and Escherichia coli. Properties of the purified enzyme were similar irrespective of the host. Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0. The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C. The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin. About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C. Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect. The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min. Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C. The Km value for soluble starch was 14 mg/ml. Mr is 59,000 and pI 8.8. The only difference between the enzymes produced in different hosts was in signal peptide processing.     

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.     

Purification and characteristics of alkaline proteinase from alkalophylic Bacillus sp.]

Alkaline protease was purified from Bacillus sp. isolated from soil. The pH optimum was 11.5 at 37 degrees C. Calcium divalent cation was effective to stabilize the enzyme especially at higher temperatures. The proteolytic activity was inhibited by active site inhibitors of PMSF (Phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, Hg. The enzyme was stable in the presence of some detergents, such as Triton-X-100, Tween-80, SDS (sodium dodecyl sulfate) and EDTA (ethylendiaminetetraacetic acid), pH 11.5 and 37 degrees C for 30 min. The optimum pH was 11.5 at 37 degrees C and the optimum temperature was 62 degrees C at pH 11.5.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96

A beta-galactosidase isoenzyme, beta-Gall, from Bifidobacterium infantis HL96, was expressed in Escherichia coli and purified to homogeneity. The molecular mass of the beta-Gall subunit was estimated to be 115 kDa by SDS-PAGE. The enzyme appeared to be a tetramer, with a molecular weight of about 470 kDa by native PAGE. The optimum temperature and pH for o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose were 60 degrees C, pH 7.5, and 50 degrees C, pH 7.5, respectively. The enzyme was stable over a pH range of 5.0-8.5, and remained active for more than 80 min at pH 7.0, 50 degrees C. The enzyme activity was significantly increased by reducing agents. Maximum activity required the presence of both Na+ and K+, at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, divalent metal cations, and Cr3+, and to a lesser extent by EDTA and urea. The hydrolytic activity using lactose as a substrate was significantly inhibited by galactose. The Km, and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. beta-Gall possesses strong transgalactosylation activity. The production rate of galactooligosaccharides from 20% lactose at 30 and 60 degrees C was 120 mg/ml, and this rate increased to 190 mg/ml when 30% lactose was used.     

L-asparaginase activity in Aeromonas sp. isolated from freshwater mussel

Aeromonas sp. from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C. The optimum enzyme activity was at pH 9 when temperature was 45 degrees C. Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively. Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively. The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1. Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations. Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.     

枯草芽孢杆菌中性β—甘露聚糖酶的产生及性质

由土壤中分离出一株产中性β甘露聚糖酶的枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）,编号ＢＭ９６０２。该菌在液体培养条件下,产生中性β甘露聚糖酶。多糖能作为碳源,而单糖不能作为碳源;有机氮源优于无机氮源。产酶最适培养基组成：魔芋粉４％,牛肉蛋白胨和酵母膏各１％。产酶最适培养条件：培养基起始ｐＨ８５,３５℃,振荡培养３６ｈ。以槐豆胶为底物,培养滤液中性β甘露聚糖酶活力为９６ＩＵ／ｍＬ。酶在ｐＨ５０～１００和５０℃下稳定;作用最适条件为ｐＨ６０和５０℃;水解魔芋粉和槐豆胶均产生寡聚糖。     

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.     

The effect of lead on the calcium-handling capacity of rat heart mitochondria.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.     

Purification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn

Syncephalastrum racemosum Cohn. produces an extracellular xylanase that was shown to potentially bleach pulp at pH 10 and 50 degrees C. The enzyme was found to be a dimer with an apparent molecular weight of 29 kDa as determined by SDS-PAGE. The optimum activity was found at two pH values 8.5 and 10.5; however the activity sharply decreased below pH 6 and above pH 10.5. The enzyme was stable for 72 h at pH 10.5 and at 50 degrees C. Kinetic experiments at 50 degrees C gave V(max) and K(m) of 1,400 U/ml min(-1) mg(-1) protein and 0.05 mg/ml respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by group II b metal ions like Zn2+, Hg2+, etc. Xylan completely protected the enzyme from being inactivated by N-bromosuccinimide.     

Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T. saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference.     

Characterization of D-Aminoacylase from Alcaligenes denitrificans DA181

The D-aminoacylase produced by Alcaligenes denitrificans DA181 was a new type of aminoacylase which had both high stereospecificity and specific activity. The molecular weight and isoelectric point of this enzyme were 58,000 and 4.4, respectively. The apparent K_m and k_cat values of this enzyme for N-acetyl-D-methionine were estimated to be 0.48 him and 6.24 × 10⁴ min respectively. The optimum temperature was 45°C. The enzyme was stable up to 55°C for 1 hr in the presence of 0.2 mg/ml bovine serum albumin. The enzyme was stable in the pH range of 6.0 to 11.0 with an optimum pH of 7.5. This enzyme contained about 2.1 g atom of zinc per mole of enzyme. Enzyme activity was inhibited by incubation with EDTA. The inhibition by EDTA was fully reversed by Co²⁺ and partially by Zn²⁺.     

Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.