Ultrasonic extraction （UE） was employed for the extraction of bamboo leaf polysaccharides （BLP）. The influential parameters of UE procedure including extraction time, ultrasonic power and solid/liquid ratio were o...Ultrasonic extraction （UE） was employed for the extraction of bamboo leaf polysaccharides （BLP）. The influential parameters of UE procedure including extraction time, ultrasonic power and solid/liquid ratio were optimized by orthogonal experiments. DEAE-cellulose col- umn chromatography was applied to purify BLP and then the radical scavenging activity of BLP was also evaluated. Optimal extraction con- ditions were： extraction time .of 15 min, ultrasonic power of 300 W, and solid/liquid ratio of 1：15. Four kinds of polysaccharides were obtained by DEAE-cellulose colunm chromatography; the maximum superoxide radical scavenging rate （20.4%） of BLP was inferior to that of vitamin C （Vc, the control） and the hydroxyl radical scavenging rate （50%） was equivalent to that of Vc.展开更多
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
Vibrio pacini synthesizes multiple chitinases, of which three have been purified in this study by ammonium sulphate fractionation, chitin affinity chromatography and gel chromatography. Molecular weights of the three ...Vibrio pacini synthesizes multiple chitinases, of which three have been purified in this study by ammonium sulphate fractionation, chitin affinity chromatography and gel chromatography. Molecular weights of the three chitinases, Chi1, Chi2 and Chi3 are 27×103, 39×103 and 46×103 respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzymes have optimal activity at pH 7–8, and retain 50% enzymatic activity pH 4–9. The activities of chitinases are inhibited by Pb2+, Fe3+ and Cu2+, and increased by Ca2+, Mg2+ and Mn2+. Chi3 is found to inhibit the growth of six species of fungi. Such characters of chitinase are different from those of any other chitinase that were reported before. Key words chitinase - Vibrio pacini - purification - inhibit CLC number Q 55 Foundation item: Supported by the Key Technologies Research and Development Programme of the Tenth Five-Year Plan of the Nation Scientific and Technological Development (2001 BA708B04-07)Biography: HAN Bao-qin (1963-), female, Professor.展开更多
Twelve samples derived from different locations in south central area of China are treated by enrichment and spread-plate technique for initial screening. Seven chitinase-producing strains are isolated. The chitinase ...Twelve samples derived from different locations in south central area of China are treated by enrichment and spread-plate technique for initial screening. Seven chitinase-producing strains are isolated. The chitinase present in the culture supernatant of strain CS-01 possesses the maximum activity of 0.118 U/mL. Analysis of the morphological feature and the ITS rDNA sequence reveals that strain CS-01 belongs to Aspergillus fumigatus. Production of the chitinase is regulated by a inducible way and the maximum activity appears at 36 h in colloidal chitin culture. Purification of the chitinase is carried out by salting out, gel filtrate chromatography and anion exchange chromatography sequentially. Native-PAGE and SDS-PAGE indicate that the chitinase from A. fumigatus CS-01 is a monomer with the relative molecular mass estimated to be 4.50×104. Its maximum activity appears at pH 5 and 55 ℃ . The chitinase is stable at pH 4.0?7.5 and below 45 ℃.展开更多
Chinese black sleeper(Bostrychus sinensis)is a fish that lives both in seawater and freshwater,feeds on crustaceans,aquatic insects and occasionally shellfish.The existence of digestive enzyme in viscera to act on chi...Chinese black sleeper(Bostrychus sinensis)is a fish that lives both in seawater and freshwater,feeds on crustaceans,aquatic insects and occasionally shellfish.The existence of digestive enzyme in viscera to act on chitinous exoskeleton of the prey is of interest.In this study,a chitinase was purified to homogeneity using ammonium sulfate precipitation,DEAE-Sephacel ion exchange,Sephacryl S-200 HR and Superdex 200 gel filtration columns.The purified protein presents a molecular mass of 58 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and results in a single band on native PAGE.According to peptide mass fingerprinting,two peptides containing a total of 20 amino acid residues,were 95%identical to a chitinase from yellow perch(Perca flavescens)and 100%identical to the chitinase from greater amberjack(Seriola dumerili).The purified chitinase showed optimum activity at pH 6.0,and was stable at acidic conditions and temperature below 55℃.The enzymatic activity was quite stable in the presence of NaCl,even at 1 mol/L.The chitinase was capable of degrading chitosan into low molecular mass chitooligosaccharides(COS)with sizes in a range of 200-700 Da,and the circular dichroism profile of the COS greatly differed from native chitosan.Full-length cDNA encoding the present chitinase was cloned and the transcript levels of chitinase in various tissues were determined by quantitative real-time PCR.The results showed that the transcript level of chitinase was highest in esophagus and hepatopancreas.展开更多
Bacillus sp.SL-13 produced antifungal proteins.The growth of the plant-pathogenic fungi Rhizoctonia solani was considerably inhibited by the presence of the SL-13 culture supernatant.The proteins were purified by DEAE...Bacillus sp.SL-13 produced antifungal proteins.The growth of the plant-pathogenic fungi Rhizoctonia solani was considerably inhibited by the presence of the SL-13 culture supernatant.The proteins were purified by DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration,and the main antifungal protein was purified to be chitinase.The molecular weight of chitinase was estimated to be 36 kD by 12%SDS PAGE.The optimal pH and temperature for the chitinase was 7.0 and 50℃.It demonstrated that the enzyme was stable from pH 5 to 9 and form 40?C to 60℃.The enzyme still kept 70%activity when incubated at 121℃,0.11MPa up to 20 minutes and the enzyme is also not lost the activity when treated with protease K and ultraviolet radiation for 1.5hours.It is very suitable for the use in a relatively unstable environment,exhibiting effective biological control.展开更多
为优化紫苏叶总黄酮的大孔树脂分离纯化工艺,以静态吸附率和解吸率为指标,比较了5种不同大孔树脂对紫苏叶总黄酮的吸附和解吸能力,筛选最优树脂,确定了最佳工艺参数。结果显示,D101型大孔树脂对紫苏叶总黄酮有较好的吸附和解吸效果,最...为优化紫苏叶总黄酮的大孔树脂分离纯化工艺,以静态吸附率和解吸率为指标,比较了5种不同大孔树脂对紫苏叶总黄酮的吸附和解吸能力,筛选最优树脂,确定了最佳工艺参数。结果显示,D101型大孔树脂对紫苏叶总黄酮有较好的吸附和解吸效果,最佳条件为样品质量浓度3 mg·mL^(-1)、树脂质量3 g、pH4、50 mL 70%乙醇洗脱。此条件下,紫苏叶总黄酮的纯度为70.90%。展开更多
文摘Ultrasonic extraction （UE） was employed for the extraction of bamboo leaf polysaccharides （BLP）. The influential parameters of UE procedure including extraction time, ultrasonic power and solid/liquid ratio were optimized by orthogonal experiments. DEAE-cellulose col- umn chromatography was applied to purify BLP and then the radical scavenging activity of BLP was also evaluated. Optimal extraction con- ditions were： extraction time .of 15 min, ultrasonic power of 300 W, and solid/liquid ratio of 1：15. Four kinds of polysaccharides were obtained by DEAE-cellulose colunm chromatography; the maximum superoxide radical scavenging rate （20.4%） of BLP was inferior to that of vitamin C （Vc, the control） and the hydroxyl radical scavenging rate （50%） was equivalent to that of Vc.
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Vibrio pacini synthesizes multiple chitinases, of which three have been purified in this study by ammonium sulphate fractionation, chitin affinity chromatography and gel chromatography. Molecular weights of the three chitinases, Chi1, Chi2 and Chi3 are 27×103, 39×103 and 46×103 respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzymes have optimal activity at pH 7–8, and retain 50% enzymatic activity pH 4–9. The activities of chitinases are inhibited by Pb2+, Fe3+ and Cu2+, and increased by Ca2+, Mg2+ and Mn2+. Chi3 is found to inhibit the growth of six species of fungi. Such characters of chitinase are different from those of any other chitinase that were reported before. Key words chitinase - Vibrio pacini - purification - inhibit CLC number Q 55 Foundation item: Supported by the Key Technologies Research and Development Programme of the Tenth Five-Year Plan of the Nation Scientific and Technological Development (2001 BA708B04-07)Biography: HAN Bao-qin (1963-), female, Professor.
基金Projects(50621063, 50674101) supported by the National Natural Science Foundation of China
文摘Twelve samples derived from different locations in south central area of China are treated by enrichment and spread-plate technique for initial screening. Seven chitinase-producing strains are isolated. The chitinase present in the culture supernatant of strain CS-01 possesses the maximum activity of 0.118 U/mL. Analysis of the morphological feature and the ITS rDNA sequence reveals that strain CS-01 belongs to Aspergillus fumigatus. Production of the chitinase is regulated by a inducible way and the maximum activity appears at 36 h in colloidal chitin culture. Purification of the chitinase is carried out by salting out, gel filtrate chromatography and anion exchange chromatography sequentially. Native-PAGE and SDS-PAGE indicate that the chitinase from A. fumigatus CS-01 is a monomer with the relative molecular mass estimated to be 4.50×104. Its maximum activity appears at pH 5 and 55 ℃ . The chitinase is stable at pH 4.0?7.5 and below 45 ℃.
基金Supported by the National Natural Science Foundation of China（20776017） the Xinjiang Uygur Autonomous Region High-tech Research and Development Project（20081108）+1 种基金 the Fok Ying Tung Education Foundation（101071） the Xinjiang Bingtuan Key Science and Technology Industry Project（2008GG24）
基金The National Key R&D Program of China under contract No.2018YFD0901004the National Natural Science Foundation of China under contract Nos 31772049,31702372。
文摘Chinese black sleeper(Bostrychus sinensis)is a fish that lives both in seawater and freshwater,feeds on crustaceans,aquatic insects and occasionally shellfish.The existence of digestive enzyme in viscera to act on chitinous exoskeleton of the prey is of interest.In this study,a chitinase was purified to homogeneity using ammonium sulfate precipitation,DEAE-Sephacel ion exchange,Sephacryl S-200 HR and Superdex 200 gel filtration columns.The purified protein presents a molecular mass of 58 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and results in a single band on native PAGE.According to peptide mass fingerprinting,two peptides containing a total of 20 amino acid residues,were 95%identical to a chitinase from yellow perch(Perca flavescens)and 100%identical to the chitinase from greater amberjack(Seriola dumerili).The purified chitinase showed optimum activity at pH 6.0,and was stable at acidic conditions and temperature below 55℃.The enzymatic activity was quite stable in the presence of NaCl,even at 1 mol/L.The chitinase was capable of degrading chitosan into low molecular mass chitooligosaccharides(COS)with sizes in a range of 200-700 Da,and the circular dichroism profile of the COS greatly differed from native chitosan.Full-length cDNA encoding the present chitinase was cloned and the transcript levels of chitinase in various tissues were determined by quantitative real-time PCR.The results showed that the transcript level of chitinase was highest in esophagus and hepatopancreas.
文摘Bacillus sp.SL-13 produced antifungal proteins.The growth of the plant-pathogenic fungi Rhizoctonia solani was considerably inhibited by the presence of the SL-13 culture supernatant.The proteins were purified by DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration,and the main antifungal protein was purified to be chitinase.The molecular weight of chitinase was estimated to be 36 kD by 12%SDS PAGE.The optimal pH and temperature for the chitinase was 7.0 and 50℃.It demonstrated that the enzyme was stable from pH 5 to 9 and form 40?C to 60℃.The enzyme still kept 70%activity when incubated at 121℃,0.11MPa up to 20 minutes and the enzyme is also not lost the activity when treated with protease K and ultraviolet radiation for 1.5hours.It is very suitable for the use in a relatively unstable environment,exhibiting effective biological control.
文摘为优化紫苏叶总黄酮的大孔树脂分离纯化工艺,以静态吸附率和解吸率为指标,比较了5种不同大孔树脂对紫苏叶总黄酮的吸附和解吸能力,筛选最优树脂,确定了最佳工艺参数。结果显示,D101型大孔树脂对紫苏叶总黄酮有较好的吸附和解吸效果,最佳条件为样品质量浓度3 mg·mL^(-1)、树脂质量3 g、pH4、50 mL 70%乙醇洗脱。此条件下,紫苏叶总黄酮的纯度为70.90%。
