Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum

A thermostable superoxide dismutase (SOD) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum strain CT2 was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-sepharose, phenyl-sepharose hydrophobic interaction chromatography. The pure SOD had a specific activity of 115.77 U/mg of protein and was purified 7.49-fold, with a yield of 14.4%. The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD was found to be inhibited by NaN3, but not by KCN and H2O2. Atomic absorption spectrophotometric analysis showed that the content of Mn was 2.05 microg/mg of protein and Fe was not detected in the purified enzyme. These results suggested that the SOD in C. thermophilum was the manganese superoxide dismutase type. N-terminal amino acid sequencing (10 residues) was KX (X is uncertain) TLPDLKYD. The N-terminal amino acid sequencing homologies to other MnSod also indicated that it was a manganese-containing superoxide dismutase. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60 C. It was thermostable at 50 and 60 C and retained 60% activity after 60 min at 70 C. The half-life of the SOD at 80 C was approximately 25 min and even retained 20% activity after 30 min at 90 C.     

Purification and characterization of two thermostable proteases from the thermophilic fungus Chaetomium thermophilum

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and PhenylSepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65 degrees C. The PRO33 had a K(m) value of 6.6 mM and a V(max) value of 10.31 micromol/l/min, and PRO63 17.6 mM and 9.08 micromol/l/min, with casein as substrate. They were thermostable at 60 degrees C. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at 70 degrees C for 1 h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70 degrees C after 60 min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.     

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

AIMS: Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. METHODS AND RESULTS: First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4.5-5.0 and 65 degrees C. The enzyme was stable at 60 degrees C, the enzyme activity kept 80% after 60 min incubation at 70 degrees C. The half-life was 40 and 10 min under incubation at 80 and 90 degrees C respectively. CONCLUSIONS: A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.     

Purification and characterization of a thermostable glucoamylase from a Myrothecium isolate

Two glucoamylases, gluc I and gluc II, were purified to homogeneity from the culture filtrate of a Myrothecium strain M1 by chromatography on DEAE-cellulose and concanavalin A-sepharose. Molecular masses deduced by SDS-PAGE were 72000 ± 2500 for gluc I and 96 000 ± 4000 for glue II. The temperature optima of the enzymes were both about 70°C and their pH optima were around 4.0. Both enzymes were glycoprotein and preferentially hydrolysed high molecular mass substrates. Hg²⁺ was a potent inhibitor of both glucoamylases. Glue II had higher debranching activity than gluc I.     

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

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。     

Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus.

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.     

嗜热毛壳菌热稳定脂肪酶基因(lm)的克隆及其在毕赤酵母中的表达(英文)

研究从嗜热毛壳菌Chaetomium thermophilum中克隆了一个新的脂肪酶基因(lm)。其中DNA序列包含一个由870个碱基构成的开放阅读框,编码289个氨基酸,含有4个内含子,没有信号肽序列。序列提交Gen Bank,登录号为GU338248。将该基因在毕赤酵母中表达。在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,蛋白达到0.428mg/m L,菌物学报酶活力为19.77U/mg。SDS-PAGE检测该蛋白的分子量为35k Da。该脂肪酶的最适反应温度为60℃,具有热稳定性,在40–80℃热稳定,80℃处理60min仍有65%的相对酶活。该酶最适反应p H值为10.0,在p H 9.0–12.0酶活相对稳定。该酶具有较好的热稳定性和耐碱性,具有良好的工业应用价值。     

Cloning of a gene encoding thermostable cellobiohydrolase from the thermophilic fungus Chaetomium thermophilum and its expression in Pichia pastoris

Aims:  A new cellobiohydrolase (CBH) gene ( cbh3 ) from Chaetomium thermophilum was cloned, sequenced and expressed in Pichia pastoris .
Methods and Results:  Using RACE-PCR, a new thermostable CBH gene ( cbh3 ) was cloned from C. thermophilum . The cDNA of the CBH was 1607 bp and contained a 1356 bp open reading frame encoding a protein CBH precursor of 451 amino acid residues. The mature protein structure of C. thermophilum CBH3 only comprises a catalytic domain and lacks cellulose-binding domain and a hinge region. The gene was expressed in P. pastoris . The recombinant CBH purified was a glycoprotein with a size of about 48·0 kDa, and exhibited optimum catalytic activity at pH 5·0 and 60 °C. The enzyme was more resistant to high temperature. The CBH could hydrolyse microcrystalline cellulose and filter paper.
Conclusions:  A new thermostable CBH gene of C. thermophilum was cloned, sequenced and overexpressed in P. pastoris .
Significance and Impact of the Study:  This CBH offers an interesting potential in saccharification steps in both cellulose enzymatic conversion and alcohol production.     

嗜热毛壳菌热稳定脂肪酶基因(Im)的克隆及其在毕赤酵母中的表达

研究从嗜热毛壳菌Chaetomium thermophilum中克隆了一个新的脂肪酶基因(lm).其中DNA序列包含一个由870个碱基构成的开放阅读框,编码289个氨基酸,含有4个内含子,没有信号肽序列.序列提交GenBank,登录号为GU338248.将该基因在毕赤酵母中表达.在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,蛋白达到0.428mg/mL,酶活力为19.77U/mg.SDS-PAGE检测该蛋白的分子量为35kDa.该脂肪酶的最适反应温度为60℃,具有热稳定性,在40-80℃热稳定,80℃处理60min仍有65％的相对酶活.该酶最适反应pH值为10.0,在pH 9.0--12.0酶活相对稳定.该酶具有较好的热稳定性和耐碱性,具有良好的工业应用价值.     

Purification and characterization of a thermostable xylanase from Fomitopsis pinicola

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular weight of F. pinicola xylanase was determined to be 58 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of 70 degreesC. The enzyme showed t(1/2) value of 33 h at 70 degrees C and catalytic efficiency (k(cat) = 77.4 s?1, k(cat)/K(m) = 22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.     

Purification and characterization of a thermostable cellobiohydrolase from Fomitopsis pinicola

A screening for cellobiohydrolase (CBH) activity was performed and Fomitopsis pinicola KMJ812 was selected for further characterization as it produced a high level of CBH activity. An extracellular CBH was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants. The molecular mass of the F. pinicola CBH was determined to be 64 kDa by SDS-PAGE and by size-exclusion chromatography, indicating that the enzyme is a monomer. The F. pinicola CBH showed a t1/2 value of 42 h at 70 degrees C and catalytic efficiency of 15.8 mM-1 S-1 (kcat/ Km) for p-nitrophenyl-beta-D-cellobioside, one of the highest levels seen for CBH-producing microorganisms. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, the F. pinicola CBH is distinguished from other CBHs by its high catalytic efficiency and thermostability.     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4

A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues. The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography. The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 °C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 °C for 6 h. TtcGA can hydrolyze both α-1, 4- and α-1, 6-glycosidic linkages in various α-glucans. It showed preference for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca²⁺, Mn²⁺, Co²⁺, Mg²⁺, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn²⁺, Pb²⁺, Cu²⁺, and EDTA.     

Production and characterization of a thermostable glucoamylase from Streptosporangium sp. endophyte of maize leaves

Thermostable amylolytic enzymes are currently investigated to improve industrial processes of starch degradation. Streptosporangium sp. an endophytic actinomycete isolated from leaves of maize (Zea mays L.) showed glucoamylase production, using starch-Czapek medium, and the highest rate was obtained in the initial growth phase, after incubation for 24 h at pH 8.0. Maximum glucoamylase activity (158 U mg(-1) protein) was obtained at pH 4.5 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C for 30 min with total inhibition at 100 degrees C. Extracellular enzyme from Streptosporangium sp. was purified by fractionated precipitation with ammonium sulphate. After 60% saturation produced 421 U mg(-1) protein, and yield was 74% with purification 2.7 fold. The enzyme produced by Streptosporangium sp. has potential for industrial applications.     

Cloning,heterologous expression,and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii

The gene encoding a thermostable glucoamylase from Talaromyces emersonii was cloned and, subsequently, heterologously expressed in Aspergillus niger. This glucoamylase gene encodes a 618 amino acid long protein with a calculated molecular weight of 62,827Da. T. emersonii glucoamylase fall into glucoside hydrolase family 15, showing approximately 60% sequence similarity to glucoamylase from A. niger. The expressed enzyme shows high specific activity towards maltose, isomaltose, and maltoheptaose, having 3-6-fold elevated k(cat) compared to A. niger glucoamylase. T. emersonii glucoamylase showed significantly improved thermostability with a half life of 48h at 65 degrees C in 30% (w/v) glucose, compared to 10h for glucoamylase from A. niger. The ability of the glucoamylase to hydrolyse amylopectin at 65 degrees C is improved compared to A. niger glucoamylase, giving a significant higher final glucose yield at elevated temperatures. The increased thermal stability is thus reflected in the industrial performance, allowing T. emersonii glucoamylase to operate at a temperature higher than the A. niger enzyme.     

Purification and characterization of a novel thermostable lipase from Bacillus sp

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.     

Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus.

A highly thermostable beta-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus. The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The enzyme exhibited the highest affinity towards p-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82 degrees C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T(opt), 5 min, the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93 degrees C; however at 65 and 82 degrees C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82 degrees C (pH 5.0 to 5.5). The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates. An incubation for 3 h at 82 degrees C in the absence of substrate reduced the activity to around 75%. At 86 degrees C the half-life was around 15 min. With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined. The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences.     

嗜热毛壳菌多糖单加氧酶的氧化特性及协同作用

以嗜热毛壳菌Chaetomium thermophilum多糖单加氧酶CtPMO1为研究对象,利用薄层层析色谱法(TLC)、高效液相色谱-示差折光检测法(HPLC-RID)和飞行时间质谱(MALDI-TOF-MS)检测CtPMO1的酶活性,并根据定点突变的原理,将CtPMO1第1位的组氨酸(His1)和第166位的谷氨酰胺(Gln166)突变为H1A、Q166A和Q166E,研究两个突变位点是否参与CtPMO1的氧化作用;另外,采用3,5-二硝基水杨酸(DNS)法检测CtPMO1与纤维素酶(EGⅡ、BGLⅠ和CBHⅠ)的协同效应。研究发现CtPMO1在温度为50 ℃、pH为5.0的条件下降解磷酸膨胀纤维素(PASC),其酶解产物中不仅存在纤维二糖至纤维五糖,还存在C1氧化寡糖和C4氧化寡糖;此外,发现突变酶H1A完全丧失了酶活,Q166A丧失了C1和C4氧化活性,而Q166E保留了部分C1氧化活性;通过对CtPMO1与纤维素酶协同作用的探究,发现利用CtPMO1预处理磷酸膨胀纤维素(PASC),分别添加EGⅡ、BGLⅠ和CBHⅠ,使还原糖产量分别提高2.10倍、2.08倍和2.16倍,协同度分别是1.022、0.799和0.875。研究结果表明CtPMO1对底物具有C1和C4氧化的功能,其反应的最适温度为50 ℃、最适pH为5.0;CtPMO1活性中心氨基酸His1和平坦表面氨基酸Gln166均是关键性位点;CtPMO1预处理PASC,使纤维素酶的降解效率发生不同程度的提高。