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
pH is a measure of the hydrogen ion activity in a solution,which is a function of temperature.Under normal seawater conditions,it is well constrained.Nowadays,with an increasing interest in complex environments(e.g.,s...pH is a measure of the hydrogen ion activity in a solution,which is a function of temperature.Under normal seawater conditions,it is well constrained.Nowadays,with an increasing interest in complex environments(e.g.,sea ice),a better understanding of the temperature change on pH under extreme conditions is needed.The objective of this paper was to investigate the temperature coefficient of the seawater pH(△pH/△T)over a wide range of temperature,pH,dissolved inorganic carbon(DIC)and salinity by a method of continuous pH measurement with the temperature change,and to verify the application of CO2SYS for pH conversion under extreme conditions(on the National Bureau of Standards(NBS)scale and the total proton scale).Both experimental results and CO2SYS calculations showed that△pH/△T was slightly affected by temperature over the range of 0℃ to 40℃ and by pH(at 25℃)from 7.8 to 8.5.However,when pH was out of this range,△pH/△T varied greatly with pH value.According to the experimental results,changes in DIC from 1 mmol/kg to 5 mmol/kg and salinity from 20 to 105 had no significant effect on△pH/△T.CO2SYS calculations showed a slight increase in△pH/△T with DIC on both the NBS scale and the total proton scale;and underestimated△pH/△T at high salinity(i.e.,beyond the oceanographic range)on the NBS scale.Nevertheless,CO2SYS is still suitable for pH conversion even under extreme conditions by simply setting the input values of DIC and salinity in CO2SYS within the oceanographic range(e.g.,DIC=2 mmol/kg and S=35).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.
基金The National Natural Science Foundation of China under contract No.41806094the Young Scholars Program of Shandong University under contract No.2018WLJH43。
文摘pH is a measure of the hydrogen ion activity in a solution,which is a function of temperature.Under normal seawater conditions,it is well constrained.Nowadays,with an increasing interest in complex environments(e.g.,sea ice),a better understanding of the temperature change on pH under extreme conditions is needed.The objective of this paper was to investigate the temperature coefficient of the seawater pH(△pH/△T)over a wide range of temperature,pH,dissolved inorganic carbon(DIC)and salinity by a method of continuous pH measurement with the temperature change,and to verify the application of CO2SYS for pH conversion under extreme conditions(on the National Bureau of Standards(NBS)scale and the total proton scale).Both experimental results and CO2SYS calculations showed that△pH/△T was slightly affected by temperature over the range of 0℃ to 40℃ and by pH(at 25℃)from 7.8 to 8.5.However,when pH was out of this range,△pH/△T varied greatly with pH value.According to the experimental results,changes in DIC from 1 mmol/kg to 5 mmol/kg and salinity from 20 to 105 had no significant effect on△pH/△T.CO2SYS calculations showed a slight increase in△pH/△T with DIC on both the NBS scale and the total proton scale;and underestimated△pH/△T at high salinity(i.e.,beyond the oceanographic range)on the NBS scale.Nevertheless,CO2SYS is still suitable for pH conversion even under extreme conditions by simply setting the input values of DIC and salinity in CO2SYS within the oceanographic range(e.g.,DIC=2 mmol/kg and S=35).
