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Purification and characterization of an extracellular xylanase from Thermomyces lanuginosus
绵毛嗜热丝孢菌木聚糖酶的纯化与性质

CHEN Wei-Wei JIANG Zheng-Qiang,WANG Rui-Jun,
陈威威 江正强
,王瑞君

菌物学报 , 2009,
Abstract: 研究了绵毛嗜热丝孢菌Thermomyces lanuginosus W205胞外木聚糖酶的纯化与性质.粗酶液经硫酸铵沉淀和Q-Sepharose FF离子交换层析即可得到电泳纯木聚糖酶,回收率为46.6%,比酶活为1396.9U/mg.该酶的最适pH和最适温度分别为pH 7.0和75℃,pH稳定范围为5.5-10.8,70℃处理30min残存酶活在70%以上.薄层层析结果显示该酶水解桦木木聚糖的主要产物是木二糖和木三糖,并且能够通过转糖苷作用将木三糖转化为木二糖.该木聚糖酶易于纯化并且具有较宽的pH稳定性及良好的热稳定性,具有较大的潜在工业应用价值.
Cloning of a gene encoding lipase from Thermomyces lanuginosus and effective expression of the gene in Pichia pastoris
疏棉状嗜热丝孢菌脂肪酶基因的克隆及其在毕赤酵母中的高效表达

ZHENG Yan,ZHOU Bo,SONG Ning-Ning,LI Duo-Chuan,
郑艳
,周波,宋宁宁,李多川

菌物学报 , 2009,
Abstract: Thermomyces lanuginosus produces an extracellular lipase that is important for industrial applications. Based on published DNA sequence, we cloned the full length genomic DNA and cDNA of the lipase using PCR and RT-PCR methods. DNA sequencing revealed that the genomic DNA of the T. lanuginosus lipase gene has an open reading frame of 876bp encoding a protein lipase precursor of 292 amino acid residues, and three introns. And the cDNA of lipase gene we cloned is identical to the registered lipase gene of The...
β-xylanase from Thermomyces lanuginosus and its Biobleaching Application
K. Khucharoenphaisan,K. Sinma
Pakistan Journal of Biological Sciences , 2010,
Abstract: Thermomyces lanuginosus is thermophilic fungus in which was isolated from widespread material. A high number of this fungus was found in composts especially mushroom composts. This fungus has been reported to produce a high level xylanase when cultivated in the medium containing xylan and corn cob as a carbon source. Various strains of T. lanuginosus produced a single xylanase with molecular masses in range of 22.0 to 29.0 kDa. Pure β-xylanase obtained from various strains of this fungus exhibited highly stability at high temperature and wide pH range. The optimal temperature and optimal pH of pure β-xylanase from various strains of T. lanuginosus have been reported in range of 60-75°C and pH 6.0-7.0, respectively. The great thermal stability was resulting from the present of hydrophilic amino acid on beta sheet of the surface of xylanase structure. Moreover, the relatedness between high and low xylanase producing strains can be distinguish by random amplification of polymorphic DNA (RAPD). Based on nucleotide sequences and T. lanuginosus xylanase gene has been classified to be a member of family 11 (formerly known as cellulase family G) glycosyl hydrolases. This enzyme was endo-type xylanase having main product are xylose and xylobiose. The expression of xylanase gene from T. lanuginosus was achieved in Escherichia coli and methylotrophic yeast Pichia pastoris. The ability of T. lanuginosus in which produced large amount of high thermos stable xylanase has made this fungus to be a source of xylanase production for biobleaching in pulp and paper process.
Cell surface display of Thermomyces lanuginosus lipase in Pichia pastoris and its characterization
疏棉状嗜热丝孢菌脂肪酶在毕赤酵母中的表面展示及酶学性质

Min Dai,Changtao Ji,Xiaofeng Wang,Xiaoyan Zhi,Hua Shao,Li Xu,Yunjun Yan,
代敏
,纪昌涛,汪小锋,智晓燕,邵化,徐莉,闫云君

微生物学报 , 2012,
Abstract: 【目的】构建疏棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus lipase,TLL)在毕赤酵母GS115中的细胞表面展示体系,筛选展示成功且酶活力及展示率较高的重组子作为全细胞催化剂,并研究其酶学性质。【方法】克隆TLL基因tll,以酿酒酵母细胞壁蛋白Sed1p为锚定蛋白,构建表面展示载体pPICZαA-TLS。重组载体经SacⅠ线性化后转入毕赤酵母GS115中,经三丁酸甘油酯平板检测及摇甁发酵筛选获得高酶活力的毕赤酵母重组子,采用抗FLAG标签一抗和R-PE荧光素标记的二抗处理细胞后,进行荧光显微镜检测和流式细胞仪分析,并考察全细胞催化剂的最适反应温度和pH、金属离子耐受性等酶学性质。【结果】成功构建TLL毕赤酵母细胞表面展示体系,筛选到1株具有三丁酸甘油酯和橄榄油水解活力的克隆子,经1%的甲醇诱导发酵120 h后,水解橄榄油酶活力达257.8 U/g干细胞。经抗体处理后的重组菌发酵细胞在荧光显微镜下呈现强烈的红色荧光,流式细胞仪分析结果也证实脂肪酶被成功展示在酵母细胞表面,展示率达98.36%。展示的TLL作为全细胞催化剂水解对硝基苯酚丁酸酯(pNPB)的最适温度为30℃,最适pH为8.0,且具备良好的热稳定性和有机溶剂耐受性;K+、Ca2+、Mg2+对其有微弱的激活作用,Mn2+、Ni2+则有微弱的抑制作用,Cu2+的抑制作用较强,而EDTA、SDS、Tween 20对酶活力影响不明显。【结论】首次将TLL脂肪酶成功展示在毕赤酵母细胞表面,获得具有较高水解活力和良好酶学特性的全细胞催化剂,为表面展示TLL脂肪酶的规模化应用奠定了技术基础。
Induction and Repression of β-Xylanase of Thermomyces lanuginosus TISTR 3465  [PDF]
K. Khucharoenphaisan,S. Tokuyama,K. Ratanakhanokchai,V. Kitpreechavanich
Pakistan Journal of Biological Sciences , 2010,
Abstract: The effect of carbon sources on the production of β-xylanase by Thermomyces lanuginosus TISTR 3465 was investigated. Xylan showed the highest inductive effect on the enzyme formation whereas xylobiose and xylooligosaccharides resulted in lesser effect. β-Xylanase was also produced at low level with xylose as well as other sugars tested. Xylan concentration at 15 g L-1 gave the maximum inductive effect on β-xylanase formation, whereas xylooligosaccharides and xylose were effective at a lower concentration of 2.5 g L-1. High concentrations of these sugars significantly repressed the enzyme formation. Crude enzyme from the supernatants, without and with other sugars produced a single xylanase band on non-denaturing PAGE gels. However, an intense xylanase activity band was observed from the supernatant of media with xylan, xylobiose and xylooligosaccharides as the carbon sources. An intense protein band of 24.9 kDa from the culture filtrate of xylan medium was observed. Xylan increased β-xylanase production by the fungus 16-fold when it was added to the xylose medium after cultivation for 3 days. In contrast, addition of xylose to the xylan medium decreased β-xylanase production 3-fold. A distinct appearance and disappearance of a 24.9 kDa protein and the activity band coincided with an increase and decrease of xylanase activity, respectively. This indicated the synthesis of xylanase by this strain was most induced by xylan. Moreover, the level of xylanase induction has no related to amino acid sequence of the enzyme.
Purification and properties of a thermostable chitinase from thermophilic fungus Thermomyces lanuginosus
疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

GUO Run-fang,LI Duo-chuan,WANG Rong,
郭润芳
,李多川,王荣

微生物学报 , 2005,
Abstract: A thermostable extracellular chitinase from culture supernatant of a thermophilic fungus Thermomyces lanuginosus was purified to SDS-PAGE homogeneity, by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography, Phenyl-Sepharose Fast Flow chromatography. A molecular mass of the purified enzyme was between 48-49.8 kD determined by SDS-PAGE and gel filtration chromatography. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55 degrees C respectively. It was thermostable at 50 degrees C and retained 24% activity after 20 min at 70 degrees C. The half life time of the enzyme at 65 degrees C was 25 min. Different metal ions showed different effects on the chitinase activity. Ca2+, Ba2+, Na+, K+ enhanced the enzyme activity, whereas Fe2+, Ag+, Hg2+, Cu2+ caused obvious inhibition. The Km and Vmax values of chitinase on colloidal chitin were 9.56 mg/mL and 22.12 micromol/min respectively. The chitinase showed antifungal activity aginst tested fungi to different degree.
A comparative study of Thermomyces lanuginosus strains on thermostable xylanase production
K Khucharoenphaisan, S Tokuyama, K Ratanakhanokchai, V Kitpreechavanich
African Journal of Biotechnology , 2009,
Abstract: Strains of Thermomyces lanuginosus could be differentiated into two groups based on their ability to produce xylanase using xylan or xylose as sole of carbon source. One group of these strains produced high xylanase activity either in the medium using xylan or xylose as a sole of carbon source. The xylanase production by T. lanuginosus ATCC 44008, THKU-11, and THKU-25, which were the representative members of this group, increased when xylose was added to the xylan medium. In contrast, there was another group that produced high xylanase activity only in the xylan medium. Addition of xylose to the xylan medium resulted decreasing of xylanase production in T. lanuginosus ATCC 46882, TISTR 3465 and THKU-85 that belonged to this group. The finding indicated that induction and repression mechanism of xylose on xylanase expression was different among the strains of T. lanuginosus. In addition, phylogenetic analysis obtained from random amplified polymorphic DNA (RAPD) pattern using primer UBC 241 pointed to greater diversity of high and low xylanase producing strains using xylose as a carbon source.
IN SILICO STUDIES FOR EVALUATING CONSERVATION HOMOLOGY AMONG FAMILY 11 XYLANASES FROM Thermomyces lanuginosus  [PDF]
SMRITI SHRIVASTAVA,PRATYOOSH SHUKLA,RAJU PODDAR
Journal of Applied Sciences in Environmental Sanitation , 2007,
Abstract: Xylanases are unique hydrolytic enzymes with potential applications in industries. In the present study a complete comparative analysis of DNA and amino acid sequences from the xylanase precursor from Thermomyces lanuginosus has been carried out with respect to other fungal xylanases. A study was made on phylogenetic trees showing genetic and morphological distance from different xylanase producing fungi. Further analysis was conducted using multiple sequence alignment and conserved domains responsible for xylanolytic activity of the enzyme. Comparative study of structure of xylanase from Thermomyces lanuginosus with other closely related xylanase producing fungi like Aspergillus kawachii, Trichoderma harzianum was reported and conserved catalytic domains were marked. It has been found that xylanase producing gene sequences from fungi related to Thermomyces lanuginosus coded for proteins which were all structurally similar. All these structurally similar xylanases belonged to the family 11 of glycoside hydrolase.
Immobilization of Thermomyces lanuginosus Lipase in PVA-alginate Beads  [cached]
Brenda Rogelina Cruz-Ortiz,Leopoldo Javier Ríos-González,Yolanda Garza García,José Antonio Rodríguez de la Garza
Revista de la Sociedad Química de México , 2011,
Abstract: Se inmovilizó la lipasa de Thermomyces lanuginosus en esferas de PVA-alginato, obteniendo un porcentaje de inmovilización del 94.4% al 98.4% utilizando concentraciones de PVA del 11% al 12.5%, y con tiempos de entrecruzamiento con ácido bórico de 45 y 60 min. Se determinó la velocidad inicial de forma libre e inmovilizada mediante la hidrólisis del p-nitrofenol palmitato. Se llevó a cabo un estudio de estabilidad operacional a diferente pH (4-7), velocidad de agitación (100-500 r.p.m.) y temperatura (40-80 °C). Los resultados mostraron que a pH 6 y 7 no se observó una considerable pérdida de actividad enzimática o de enzima. A temperaturas arriba de los 70 °C el biocatalizador sufrió da o físico y la enzima mostró una considerable pérdida de actividad enzimática. En la velocidad de agitación no se observó una diferencia significativa cuando se varió la velocidad de agitación de 100 a 500 r.p.m.
Localization and partial characterization of thermostable glucoamylase produced by newly isolated Thermomyces lanuginosus TO3 in submerged fermentation
Gon?alves, Aline Zorzetto Lopes;Carvalho, Ana Flávia Azevedo;Silva, Roberto da;Gomes, Eleni;
Brazilian Archives of Biology and Technology , 2008, DOI: 10.1590/S1516-89132008000400024
Abstract: thermophilic thermomyces lanuginosus strain to3 was isolated from compost pile samples and was used for its ability to produce considerable glucoamylase activity when growing in liquid medium at 45oc with starch as the sole carbon source. enzyme productivity was high in submerged fermentation (smf) with maximum activity of 13 u/ml after 168 h of fermentation. higher quantities of glucose were released when the substrate for enzyme was soluble starch than maltose or maltooligosaccharides were used. the distribution of glucoamylase between the extracellular and cell-associated fractions varied according to fermentation time. glucoamylase produced from t. lanuginosus to3 had optimum activity at 65 oc and good thermostability in the absence of substrate, with a half-life of 6 h at 60 oc. the enzyme was stable over a wide ph range (4.0-10.0).
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