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疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究 总被引:6,自引:1,他引:6
采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8~ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。 相似文献
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嗜热毛壳菌内切β-葡聚糖酶的分离纯化及特性 总被引:5,自引:1,他引:5
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。 相似文献
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[目的]建立疏绵状嗜热丝孢菌的稳定遗传转化体系并获得插入突变体.[方法]利用农杆菌介导的方法建立疏绵状嗜热丝孢菌的遗传转化体系 ;分别通过Southern杂交、克隆转移DNA(T-DNA)侧翼序列来确定T-DNA在疏绵状嗜热丝孢菌基因组中的拷贝数和插入位点.[结果]成功建立了可靠的疏绵状嗜热丝孢菌的遗传转化体系.共培养过程中使用萌发孢子是成功建立疏绵状嗜热丝孢菌遗传转化体系的必要条件.疏绵状嗜热丝孢菌萌发的孢子与农杆菌在28℃共培养48h时,转化效率最高.乙酰丁香酮(AS)在农杆菌预培养及疏绵状嗜热丝孢菌萌发的孢子与农杆菌的共培养阶段都是必需的,且在共培养阶段当AS浓度为500 μM时转化效率最高.Southern杂交验证表明,79.2%的转化子为T-DNA单拷贝插入,且通过热不对称PCR (TAIL-PCR)分析得出T-DNA在该菌基因组中的插入位点是随机的.通过该转化系统筛选到部分表型突变体.[结论]我们首次报道了利用ATMT技术成功转化嗜热真菌-疏绵状嗜热丝孢菌,证明了该方法是一种简单有效的获得插入突变体的方法,并为该嗜热真菌进行基因定位提供了工具. 相似文献
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嗜热毛壳菌外切葡聚糖纤维二糖水解酶的纯化和部分性质研究 总被引:3,自引:0,他引:3
研究液体发酵嗜热毛壳菌(Chaetomium thermophilum)产生的一种外切葡聚糖纤维二糖水解酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Sephacryl S-100分子筛层析、Q Sepharose Fast Flow强阴离子层析等步骤后获得凝胶电泳均一的外切葡聚糖纤维二糖水解酶。经12.5%SDS-PAGE和凝胶过滤层析方法测得该酶的分子量大小约为66.3kDa和67.1kDa。该酶反应的最适温度和pH值分别为65℃和5.0。在60℃以下酶比较稳定,在70℃酶的半衰期为1h,在80℃下保温20min仍具有20%的活性,该酶的热稳定性较中温真菌的同类酶高,与国外报道的嗜热真菌的同类酶热稳定性接近。以pNPC为底物的Km值为0.956mmol/L。 相似文献
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嗜热毛壳菌一种β-葡萄糖苷酶的分离纯化及特性 总被引:2,自引:0,他引:2
研究了嗜热毛壳菌Chaetomiumthermophilum液体发酵产生的一种胞外β-葡萄糖苷酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-SepharoseFastFlow阴离子层析、Phenyl-Sepharose疏水层析、SephacrylS-100分子筛层析等步骤后获得凝胶电泳均一的β-葡萄糖苷酶。经10%SDS-PAGE和凝胶过滤层析方法分别测得该酶的分子量大小约为118.0kDa和120.1kDa。该酶反应的最适温度为70℃,最适pH值为4.0~5.0。有高的热稳定性,在60℃保温1小时酶活性不丧失,在70℃时的半衰期为16min,在90℃保温10min仍具有7.6%的活性。且能在pH4.0~11.0之间保持稳定。金属离子对β-葡萄糖苷酶的活性影响较大,其中Ca2 、Ba2 对酶有激活作用,而Zn2 、Cu2 、Al3 、Ag 、Hg2 对酶有显著的抑制作用。 相似文献
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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\|Sepharose Fast Flow阴离子层析、Pheny1\|Sepharose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125%SDS\|PAGE和凝胶过滤层析法分别测得所分离纯化酶蛋白的分子量约为67.8kD和69.8kD。该酶反应的最适温度和pH分别为60℃和40~45在pH50条件下,该酶在60℃下稳定;70℃保温1h后,仍保留30%的活性;在80℃的半衰期为25min。金属离子对内切β\|葡聚糖酶的活性影响较大,其中Na+对酶有激活作用;Fe2+、Ag+、Cu2+、Ba2+、Zn2+等对酶有抑制作用。该酶对结晶纤维素没有水解能力。 相似文献
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一株嗜热毛壳菌β-葡萄糖苷酶的分离纯化及特性 总被引:5,自引:2,他引:5
研究了液体发酵嗜热毛壳菌Chaetomium thermophile产生的β-葡萄糖苷酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose 疏水层析、Sephacryl S-100分子筛层析等步骤后获得凝胶电泳均一的β-葡萄糖苷酶。经12.5%SDS-PAGE和凝胶过滤层析方法分别测得该酶的分子量大小约为78.4kDa和81kDa。该酶反应的最适温度和pH值分别为60℃和4.5-5.0。有较好的酸稳定性和热稳定性。金属离子对β-葡萄糖苷酶的活性影响较大, 其中Ca2+对酶有激活作用, 而Ag+、Cu2+ 、Hg2+对酶有显著的抑制作用。该酶对水杨苷具有很强的底物特异性。 相似文献
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An extracellular protease produced by the filamentous fungus Thermomyces lanuginosus has been purified and characterized. The results indicate that the enzyme, which we have called humicolin, is a thiol-containing serine protease with a molecular mass of 38,000 kilodaltons. Secretion of humicolin, which is glycosylated, is tightly regulated by protein substrates. Kinetic characterization has revealed that humicolin activity is highly dependent upon the deprotonation of a group with a pKa of 6.6 and that the enzyme has a specificity for phenylalanine in the P1 position of the substrate. 相似文献
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A polygalacturonase was purified from the thermophilic fungus, Thermomyces lanuginosus to apparent homogeneity by ultrafiltration, acetone precipitation and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60 °C. The apparent KM with potassium pectate was 0.67 mg/ml and the Vmax was 7.2 × 105 mol/min/mg protein. The apparent molecular weight of the enzyme was 59 kDa and it contained approximately 10% carbohydrate. The enzyme was completely stable at room temperature (32 ± 3 °C) and retained about 50% activity at 50 °C for 6 h. The zymogram of the purified enzyme revealed two activity bands, one of which was a major one. Polyclonal antibodies raised against the enzyme did not show any immunological relatedness with other mesophilic polygalacturonases. 相似文献
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Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus. 总被引:1,自引:0,他引:1 下载免费PDF全文
A new inducible intracellular beta-galactosidase (EC 3.2.1.23) of the thermophilic fungus Thermomyces lanuginosus was purified by fractional salt precipitation, hydrophobic interaction, and anion exchange chromatography. The first 22 amino acid residues were determined by N-terminal sequencing. Electrophoretic investigations revealed a dimeric enzyme with a molecular mass of 75 to 80 kDa per identical subunit and an isoelectric point of 4.4 to 4.5. The native beta-galactosidase was identified as a glycoprotein by the enzyme-linked immunosorbent assay technique. The beta-galactosidase activity was optimal at pH 6.7 to 7.2, and the enzyme displayed stability between pH 6 and 9. It was completely stable at pH 6.8 and 47 degrees C for 2 h. After 191 h at 50 degrees C, the remaining beta-galactosidase activity of an enzyme fraction after salt precipitation was 58%. The beta-galactosidase hydrolyzed p- and o-NO2-phenyl-beta-D-galactopyranoside, lactose, lactulose, MeOH-beta-D-galactopyranoside, phenyl-beta-D-galactopyranoside, and p-NO2-phenyl-alpha-L-arabinopyranoside. The kinetic constants (Km) measured for p- and o-NO2-phenyl-beta-D-galactopyranoside and beta-lactose were 4.8, 11.3, and 18.2 mM, respectively. 相似文献
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An extracellular alpha-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl alpha-D-galactosides but efficiently hydrolyzed alpha-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl alpha-galactopyranoside and galactosyl(1)mannotriose, followed by (1)H NMR spectroscopy, pointed out the alpha-anomer of D-galactose was the primary product of hydrolysis from which the beta-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl alpha-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases. 相似文献
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The thermostability of the endo-beta-1,4-xylanase from Thermomyces lanuginosus (xynA) was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were first selected on 0.4% Remazol Brilliant Blue-xylan and then exposed to 80 degrees C. Whereas the wild type XynA lost 90% activity after 10 min at 80 degrees C, five mutants displayed both higher stabilities and activities than XynA. Four mutants were subjected to further mutagenesis to improve the stability and activity of the xylanase. Subsequent screening revealed three mutants with enhanced thermostability. Mutant 2B7-10 retained 71% of its activity after treatment at 80 degrees C for 60 min and had a half-life of 215 min at 70 degrees C, which is higher than that attained by XynA. Sequence analysis of second generation mutants revealed that mutations were not concentrated in any particular region of the protein and exhibited much variation. The best mutant obtained from this study was variant 2B7-10, which had a single substitution (Y58F) in beta-sheet A of the protein, which is the hydrophilic, solvent-accessible outer surface of the enzyme. Most of the mutants obtained in this study displayed a compromise between stability and activity, the only exception being mutant 2B7-10. This variant showed increased activity and thermostability. 相似文献
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Mark Gaffney Stephen Carberry Sean Doyle Richard Murphy 《Enzyme and microbial technology》2009,45(5):348-354
A xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100 °C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production. 相似文献
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Singh S Reddy P Haarhoff J Biely P Janse B Pillay B Pillay D Prior BA 《Journal of biotechnology》2000,81(2-3):119-128
Properties of an endo-beta-xylanase produced by a locally isolated Thermomyces lanuginosus strain SSBP was compared to seven other T. lanuginosus strains isolated from different geographical regions. Strain SSBP produced the highest xylanase activity of 59600 nkat ml(-1) when cultivated on corn cobs (maize) medium, whereas the seven other strains produced xylanase activities ranging from 6000 to 32000 nkat ml(-1). No cellulase activity was produced by the strains. Despite the variability in the production of xylanase, little difference in the other characteristics of the strains could be found. The optimal temperature and pH for xylanase production by the strains was either 40 or 50 degrees C and between pH 6 and 7, respectively. Optimal xylanase activity of the strains was observed at 70 degrees C and at pH 6 or 6.5. Culture supernatant analysis by SDS-PAGE and isoelectric focusing PAGE of all strains revealed the presence of a single 24.7 kDa and pI 3.9 xylanase. Phylogenetic analysis by PCR amplification and sequencing of the internal transcribed spacer of nuclear rRNA repeat units and 5.8S rDNA revealed no strain diversity. However, random amplified polymorphic DNA analysis pointed to greater diversity and with one primer (5'-GCCCGACGCG-3'), a relationship was established between xylanase levels and the RAPD pattern. 相似文献
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嗜热真菌DSM10635生产耐热木聚糖酶的小试研究 总被引:3,自引:0,他引:3
应用嗜热真菌Thermomyces lanuginosus DSM10635,采用固体发酵的方法探索耐热木聚糖酶的优化生产条件。在研究玉米芯,玉米皮,玉米秆,麸皮,松树屑,桦树屑等不同底物,在不同温度、玉米芯颗粒大小以及料水比条件下培养比较酶产量后,发现该嗜热真菌产耐热木聚糖酶的最佳底物为玉米芯或玉米皮,最佳培养温度为50℃--55℃,在加水量为1份玉米芯:2.8份水,玉米芯的颗粒直径大约为1mm时产酶量最高。实验结果显示,嗜热真菌DSM10635在优化后的培养条件下木聚糖酶产量可达到12525.80IU/g玉米芯。 相似文献
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Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus. 总被引:1,自引:0,他引:1 下载免费PDF全文
Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was purified from the culture filtrates of the thermophilic fungus Thermomyces lanuginosus and was established to be homogeneous by a number of criteria. The enzyme was a glycoprotein with an average molecular weight of about 57 000 and a carbohydrate content of 10-12%. The enzyme hydrolysed successive glucose residues from the non-reducing ends of the starch molecule. It did not exhibit any glucosyltransferase activity. The enzyme appeared to hydrolyse maltotriose by the multi-chain mechanism. The enzyme was unable to hydrolyse 1,6-alpha-D-glucosidic linkages of isomaltose and dextran. It was optimally active at 70 degrees C. The enzyme exhibited increase in the Vmax. and decreased in Km values with increasing chain length of the substrate molecule. The enzyme was inhibited by the substrate analogue D-glucono-delta-lactone in a non-competitive manner. The enzyme inhibited remarkable resistance towards chemical and thermal denaturation. 相似文献