A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular...A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer （pH 2.5）. The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2＋, Ba^2＋, Na^＋, and K^＋ enhanced the enzyme activity, whereas Fe^2＋, Ag^＋, Hg^2＋, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.展开更多
A partial phase diagram characterizing the conformational change that occurs in Thermomyces lanuginosus xylanase as it is slowly heated in 150 mM sodium phosphate (pH = 7.0) has been con-structed from slow-scan-rate d...A partial phase diagram characterizing the conformational change that occurs in Thermomyces lanuginosus xylanase as it is slowly heated in 150 mM sodium phosphate (pH = 7.0) has been con-structed from slow-scan-rate differential scanning calorimetry measurements. The Clausius-Clapeyron equation was applied to determine an associated volume change of -205 L·mol-1 at 24°C, the equilibrium transition temperature at 1.0 atm pressure. This value is in excellent agreement with that predicted using a previously published [1] empirical equation for calculating the hydro-dynamic radius if the transition is regarded as from a random coil to a functional, folded state and with the assumption that the hydrodynamic radius is a good approximation of the true random coil radius. The existence of a low-temperature random coil is confirmed by circular dichroism and dynamic light scattering measurements. Thus, at 24°C and 1.0 atm pressure the enzyme appears to fold from a random coil to a functional, folded form as it is slowly heated.展开更多
The gene of xylanase （xynA） was amplified by RT-PCR from the total RNA of a themophilic fungus Thermomyces lanuginosus SY2. The sequence analysis showed that gene coding region of mature peptide contained 0.585 kb, ...The gene of xylanase （xynA） was amplified by RT-PCR from the total RNA of a themophilic fungus Thermomyces lanuginosus SY2. The sequence analysis showed that gene coding region of mature peptide contained 0.585 kb, which coded 194 amino acids. The putative amino acid sequence and DNA sequence of xylanase from T. lanuginosus SY2 （GenBank no.： GU166389） were 98.97 and 99.49% identical to the other T. lanuginosus （GenBank no.： U35436）. A recombinant plasmid pPIC9K-xynA was constructed by inserting gene xynA into Pichia pastoris secretory vector pPIC9K. Linearized pPIC9K-xynA was transformed into P. pastoris GS115 with the method of electroporation. The recombinant strain was identified by G418 selection and confirmed by PCR analysis. It was induced by 1.0% methanol at 28°C to express the recombinant xylanase. The results showed that the recombinant xylanase was secreted into extracellular fermentation liquid. The highest enzyme activity of 113.5 IU mL-1 and protein content of 889.7 μg mL-1 were detected for 216 h of induction. The optimal pH value and temperature of the enzyme activity was 5.5 and 65°C, respectively. The xylanase activity retained above 80% from pH value 2.5 to 8.5 for 48 h. The enzyme activity was above 85% at incubation temperature of 55°C.展开更多
基金the Science Technology Plan Foundation of Hebei Province, China (07225533)the Doctor Foundation from Agricultural University of Hebei (050031)
文摘A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer （pH 2.5）. The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2＋, Ba^2＋, Na^＋, and K^＋ enhanced the enzyme activity, whereas Fe^2＋, Ag^＋, Hg^2＋, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.
文摘A partial phase diagram characterizing the conformational change that occurs in Thermomyces lanuginosus xylanase as it is slowly heated in 150 mM sodium phosphate (pH = 7.0) has been con-structed from slow-scan-rate differential scanning calorimetry measurements. The Clausius-Clapeyron equation was applied to determine an associated volume change of -205 L·mol-1 at 24°C, the equilibrium transition temperature at 1.0 atm pressure. This value is in excellent agreement with that predicted using a previously published [1] empirical equation for calculating the hydro-dynamic radius if the transition is regarded as from a random coil to a functional, folded state and with the assumption that the hydrodynamic radius is a good approximation of the true random coil radius. The existence of a low-temperature random coil is confirmed by circular dichroism and dynamic light scattering measurements. Thus, at 24°C and 1.0 atm pressure the enzyme appears to fold from a random coil to a functional, folded form as it is slowly heated.
基金supported by the Scientific & Technological Support Project of Hebei Province,China(07225553)
文摘The gene of xylanase （xynA） was amplified by RT-PCR from the total RNA of a themophilic fungus Thermomyces lanuginosus SY2. The sequence analysis showed that gene coding region of mature peptide contained 0.585 kb, which coded 194 amino acids. The putative amino acid sequence and DNA sequence of xylanase from T. lanuginosus SY2 （GenBank no.： GU166389） were 98.97 and 99.49% identical to the other T. lanuginosus （GenBank no.： U35436）. A recombinant plasmid pPIC9K-xynA was constructed by inserting gene xynA into Pichia pastoris secretory vector pPIC9K. Linearized pPIC9K-xynA was transformed into P. pastoris GS115 with the method of electroporation. The recombinant strain was identified by G418 selection and confirmed by PCR analysis. It was induced by 1.0% methanol at 28°C to express the recombinant xylanase. The results showed that the recombinant xylanase was secreted into extracellular fermentation liquid. The highest enzyme activity of 113.5 IU mL-1 and protein content of 889.7 μg mL-1 were detected for 216 h of induction. The optimal pH value and temperature of the enzyme activity was 5.5 and 65°C, respectively. The xylanase activity retained above 80% from pH value 2.5 to 8.5 for 48 h. The enzyme activity was above 85% at incubation temperature of 55°C.
