[Objective] This study aimed to investigate the sporulation conditions of Tri- choderma reesei by solid fermentation. [Method] With sporulation yield as the response value, single-factor test, Plackett-Burmam design, ...[Objective] This study aimed to investigate the sporulation conditions of Tri- choderma reesei by solid fermentation. [Method] With sporulation yield as the response value, single-factor test, Plackett-Burmam design, steepest ascent test, BoxBehnken design and response surface analysis were employed to optimize the con- ditions for sporulation of Trichoderma reesei by solid fermentation. [Result] Based on single-factor test, the most appropriate carbon source for Trichoderma reesei was straw stalk powder and wheat bran with the ratio of 3：2 and optimal amount of 15 g/L; the most appropriate inorganic nitrogen was （NH4）2O4 with the optimal amount of 3 g/L. According to Plackett-Burmam design, moisture content, initial pH and incubation temperature were identified as significant factors affecting the sporulation yield of Trichoderma reeseL The maximum sporulation yield area was approached by steepest ascent test. Based on Box-Behnken design and response surface analysis, the optimal fermentation conditions for the maximum sporulation yield were determined as： straw stalk powder of 6 g/L, wheat bran of 9 g/L, （NH4）2SO4 of 3 g/L, moisture content of 65%, incubation temperature of 29 ℃, fermentation period of 72 h and initial pH of 5.5, under these conditions, the sporulation yield reached 2×10^10 spores/g, which was improved by 1.4 times compared with that before optimization. [Conclusion] In this study, the conditions for sporulation of Trichoderma reesei by solid fermentation were optimized with low-cost straw stalk powder and wheat bran as carbon sources, which was conducive to reducing the production cost of Trichoderma reesei and increasing the sporulation yield, showing certain social and economic significance.展开更多
[Objective] The study was to construct a exogenous expression vector for Trichoderma reesei.[Method] Using CBHI promoter and terminator of T.reesei strain 40359,we constructed an expression vector of T.reesei strain 4...[Objective] The study was to construct a exogenous expression vector for Trichoderma reesei.[Method] Using CBHI promoter and terminator of T.reesei strain 40359,we constructed an expression vector of T.reesei strain 40359 for expressing Hpt gene and got six strains capable of growing on basic medium containing 175 mg/L of hygromycin B,further conducted hygromycin resistance test.[Results] In comparison with the original strain（wild type）,hygromycin resistance the six engineered strains was increased by 75%;the hygromycin resistance could inherit stably.[Conclusion] Our results laid basis for biological study on T.reesei at molecular and genetically engineering levels.展开更多
Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by p...Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by plasmid pAB4-1. A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934―939 oligo dC position of the pyrG coding region, resulted in a frameshift mutation. Transformation efficiency was approximately 200―300 transformants per microgram of DNA with plasmid pAB4-1. Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation. Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of 26.1% with pAN7-1. Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA. The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system.展开更多
glucanase was purified from a solid\|state culture of \%Trichoderma reesei \%on wheat bran in three steps which comprised ammonium sulfate precipitation, Sephadex G\|100 chromatography, and DEAE\|Sephadex A\|50 chroma...glucanase was purified from a solid\|state culture of \%Trichoderma reesei \%on wheat bran in three steps which comprised ammonium sulfate precipitation, Sephadex G\|100 chromatography, and DEAE\|Sephadex A\|50 chromatography. The molecular mass was determined to be 35.21 kilodaltons by sodium dodecyl sulfate\|12.5% polyacrylamide gel electrophoresis. The \%β\%\|glucanase at low pHs was more stable than that at high pHs, and optimum pH was 5.0. The optimum temperature was 60 ℃, and \%β\%\|glucanase was relatively stable at below 40 ℃ for 60 min. The \%K\%\-m of the enzyme on \%β\%\|glucan was 10.86 mg/ml, and the \%V\%\-\{max\} on \%β\%\|glucan was 14286 μmol of glucose equivalents per mg of the pure enzyme per min. The \%β\%\|glucanase activity was significantly inhibited by Fe\+\{3+\} ions, and was reduced in the presence of Cu\+\{2+\} ions, Mn\+\{2+\} ions and Mg\+\{2+\} ions at 5 mmol/L and 10 mmol/L, respectively. The \%β\%\|glucanase activity was stimulated by Co\+\{2+\} ions, Ca\+\{2+\} ions, Zn\+\{2+\} ions, and Fe\+\{2+\} ions at 1 mmol/L and 5 mmol/L, respectively.展开更多
Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck of the production process of bioethanol from lignocellulose. It is generally admitted that the most efficient organism for...Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck of the production process of bioethanol from lignocellulose. It is generally admitted that the most efficient organism for the production ofcellulolytic enzymes is the fungus Trichoderma reesei, mostly due to its high secretion capacity. Unfortunately, this fungus secretes very low concentrations of β-glucosidase, thereby often requiring β-glucosidase supplementation for complete cellulose hydrolysis. It is especially important to have sufficient quantities of β-glucosidase in order to prevent inhibition of cellobiohydrolases by cellobiose. In order to optimize the produced cocktail, a more efficient β-glucosidase was cloned into T. reesei CL847 strain. The new strain, called CL847 TR3002, secretes the evolved β-glucosidase and was tested for cellulase production in laboratory-scale reactors. Its growth kinetics and cellulase production were characterized using fed-batch and chemostat modes under various culture conditions. The cellulase activities of the evolved strain were compared with activities of the parent strain. In addition, hydrolysis of a steam exploded wheat straw was performed at three different enzyme-loading levels （5 mg/g, 10 mg/g and 20 mg/g of dry matter） and a new kinetic model was developed.展开更多
基金Supported by General Project of Science and Technology in Hunan Province(No.2012NK3080)~~
文摘[Objective] This study aimed to investigate the sporulation conditions of Tri- choderma reesei by solid fermentation. [Method] With sporulation yield as the response value, single-factor test, Plackett-Burmam design, steepest ascent test, BoxBehnken design and response surface analysis were employed to optimize the con- ditions for sporulation of Trichoderma reesei by solid fermentation. [Result] Based on single-factor test, the most appropriate carbon source for Trichoderma reesei was straw stalk powder and wheat bran with the ratio of 3：2 and optimal amount of 15 g/L; the most appropriate inorganic nitrogen was （NH4）2O4 with the optimal amount of 3 g/L. According to Plackett-Burmam design, moisture content, initial pH and incubation temperature were identified as significant factors affecting the sporulation yield of Trichoderma reeseL The maximum sporulation yield area was approached by steepest ascent test. Based on Box-Behnken design and response surface analysis, the optimal fermentation conditions for the maximum sporulation yield were determined as： straw stalk powder of 6 g/L, wheat bran of 9 g/L, （NH4）2SO4 of 3 g/L, moisture content of 65%, incubation temperature of 29 ℃, fermentation period of 72 h and initial pH of 5.5, under these conditions, the sporulation yield reached 2×10^10 spores/g, which was improved by 1.4 times compared with that before optimization. [Conclusion] In this study, the conditions for sporulation of Trichoderma reesei by solid fermentation were optimized with low-cost straw stalk powder and wheat bran as carbon sources, which was conducive to reducing the production cost of Trichoderma reesei and increasing the sporulation yield, showing certain social and economic significance.
文摘[Objective] The study was to construct a exogenous expression vector for Trichoderma reesei.[Method] Using CBHI promoter and terminator of T.reesei strain 40359,we constructed an expression vector of T.reesei strain 40359 for expressing Hpt gene and got six strains capable of growing on basic medium containing 175 mg/L of hygromycin B,further conducted hygromycin resistance test.[Results] In comparison with the original strain（wild type）,hygromycin resistance the six engineered strains was increased by 75%;the hygromycin resistance could inherit stably.[Conclusion] Our results laid basis for biological study on T.reesei at molecular and genetically engineering levels.
基金the National Natural Science Foundation of China(No.30470052)the National Basic Research Program of China(Nos.2003CB716006 and 2004CB719702)the Natural Science Research Foundation for the Doctoral Program of Edu-cation Ministry of China(No.20040422042).
文摘Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by plasmid pAB4-1. A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934―939 oligo dC position of the pyrG coding region, resulted in a frameshift mutation. Transformation efficiency was approximately 200―300 transformants per microgram of DNA with plasmid pAB4-1. Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation. Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of 26.1% with pAN7-1. Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA. The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system.
文摘glucanase was purified from a solid\|state culture of \%Trichoderma reesei \%on wheat bran in three steps which comprised ammonium sulfate precipitation, Sephadex G\|100 chromatography, and DEAE\|Sephadex A\|50 chromatography. The molecular mass was determined to be 35.21 kilodaltons by sodium dodecyl sulfate\|12.5% polyacrylamide gel electrophoresis. The \%β\%\|glucanase at low pHs was more stable than that at high pHs, and optimum pH was 5.0. The optimum temperature was 60 ℃, and \%β\%\|glucanase was relatively stable at below 40 ℃ for 60 min. The \%K\%\-m of the enzyme on \%β\%\|glucan was 10.86 mg/ml, and the \%V\%\-\{max\} on \%β\%\|glucan was 14286 μmol of glucose equivalents per mg of the pure enzyme per min. The \%β\%\|glucanase activity was significantly inhibited by Fe\+\{3+\} ions, and was reduced in the presence of Cu\+\{2+\} ions, Mn\+\{2+\} ions and Mg\+\{2+\} ions at 5 mmol/L and 10 mmol/L, respectively. The \%β\%\|glucanase activity was stimulated by Co\+\{2+\} ions, Ca\+\{2+\} ions, Zn\+\{2+\} ions, and Fe\+\{2+\} ions at 1 mmol/L and 5 mmol/L, respectively.
文摘Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck of the production process of bioethanol from lignocellulose. It is generally admitted that the most efficient organism for the production ofcellulolytic enzymes is the fungus Trichoderma reesei, mostly due to its high secretion capacity. Unfortunately, this fungus secretes very low concentrations of β-glucosidase, thereby often requiring β-glucosidase supplementation for complete cellulose hydrolysis. It is especially important to have sufficient quantities of β-glucosidase in order to prevent inhibition of cellobiohydrolases by cellobiose. In order to optimize the produced cocktail, a more efficient β-glucosidase was cloned into T. reesei CL847 strain. The new strain, called CL847 TR3002, secretes the evolved β-glucosidase and was tested for cellulase production in laboratory-scale reactors. Its growth kinetics and cellulase production were characterized using fed-batch and chemostat modes under various culture conditions. The cellulase activities of the evolved strain were compared with activities of the parent strain. In addition, hydrolysis of a steam exploded wheat straw was performed at three different enzyme-loading levels （5 mg/g, 10 mg/g and 20 mg/g of dry matter） and a new kinetic model was developed.
