The property of major cellulases from the guts of Anoplophora glabripennis larvae have been characterized. The optimal temperatures of both β 1,4 glucosidase (β glucosidase) and endo β 1,4 endoglucanase (...The property of major cellulases from the guts of Anoplophora glabripennis larvae have been characterized. The optimal temperatures of both β 1,4 glucosidase (β glucosidase) and endo β 1,4 endoglucanase (endoglucanase, Cx) are 40℃. The β glucosidase was optimally active at pH 4\^8, while the optimal activity of the endoglucanase occurred at pH 4 4 5 6 The endoglucanase was active with a wide range of pH and temperature, the levels of activity from 25℃ to 50℃ were more than 80%, and the activity remained 60% between pH 3 2 and pH 7 2. The endoglucanase exhibited higher thermal stability than β glucosidase. Both enzymes lose their activities by heat treatment at 60℃. Two isozymes of endoglucanase were detected in sodium carboxymethylcellulose polyacrymide gels (CMC gel) by chemical colorization, and purified by elution from the gel slices. The molecular weights of the two isozymes were estimated as 26kD and 39kD respectively. Moreover molecular characteristics of the two isozymes are currently underway.展开更多
One of the latest sources of alternative energy, bioethanol, has been the focus of modem research, The production of bioethanol is commonly restricted by the activity of cellulase. Therefore, cellulase has become one ...One of the latest sources of alternative energy, bioethanol, has been the focus of modem research, The production of bioethanol is commonly restricted by the activity of cellulase. Therefore, cellulase has become one of the critical issues in the conversion of lignocelluloses to bioethanol. This article is an overview of the sources and factors affecting enzyme activity, as well as methods of evaluation and utilizations of cellulase. We conclude that a combination of cellulases from various strains can enhance hydrolysis of substrates. Large enough amounts of cellobiase or sufficient cellobiase activity can reduce the inhibition to exoglucanase activity of cellobiose. Characterization and exploitation of cellulase should focus on a definite substrate. Promotion and mixed incubation of strains can reduce the cost of industrial utilization of cellulase.展开更多
Lignocellulosic substrates are a good carbon source and provide rich growth media for a variety of microorganisms which prodLuce industrially important enzymes. Cellulases are a group of hydrolytic enzymes such as fil...Lignocellulosic substrates are a good carbon source and provide rich growth media for a variety of microorganisms which prodLuce industrially important enzymes. Cellulases are a group of hydrolytic enzymes such as filter paperase (FPase), carboxymethyl cellulase(CMCase) andβ-glucosidase-responsible for release of sugars in the bioconversion of the lignocellulosic biomass into a variety of value-added products. This study examined cellulase production by a newly isolated Aspergillus unguis on individual lignocellulosic substrates in solid state fermentation (SSF). The maximum peak production of enzymes varied from one substrate to another, however,based on the next best solid support and local availability of groundnut fodder supported maximum enzyme yields compared with other solid supports used in this study.Groundnut fodder supported significant production of FPase (5.9 FPU/g of substrate), CMCase (1.1 U/g of substrate) andβ-glucosidase activity (6.5 U/g of substrate) in SSF. Considerable secretion of protein (27.0 mg/g of substrate) on groundnut fodder was recorded. Constant increment of protein content in groundnut fodder due to cultivation of A. unguis is an interesting observation and it has implications for the improvement of nutritive value of groundnut fodder for cattle.展开更多
In order to investigate different kinds of cellulase in insect pests, we selected male and female adults of Anoplophora glabripennis （Motschulsky） emerging from four different host species （Populus alba var. pyrami...In order to investigate different kinds of cellulase in insect pests, we selected male and female adults of Anoplophora glabripennis （Motschulsky） emerging from four different host species （Populus alba var. pyramidalis, P. nigra var. thevestina （Dode） Bean., P. simonii × P. pyramidliscr cv. Opera 8277 Hsu. and Salix matsudana f. lobato-glandulosa） as our research material. The enzyme activitives of three kinds of cellulase in the intestines of the adult insects were measured. The Cx-cellulase isozymes were detected with a CMC-incorporated polyacrylamide gel. The results show that： there are no statistically significant differences between the enzyme activities of males and females emerging from the different host species. The order of magnitude in activity is： Cx-cellulase 〉 β-glycosidase 〉 C1-cellulase. When the adults emerge from the same host species, there are no statistically significant differences between male and female enzyme activities of β-glycosidase and C1-cellulase, but the enzyme activity of Cx-cellulase of males is clearly higher than that of females. The patterns and migration of Cx-cellulase isozymes of males and females emerging from differ-ent hosts trees are clearly not different, and neither are they different when emerging from the same hosts.展开更多
In this study,a high-throughput screening method was established through the 24-square deep-well microliter plate(MTP) fermentation and micro-plate detection for large-scale screening of the mutants.It was suitable fo...In this study,a high-throughput screening method was established through the 24-square deep-well microliter plate(MTP) fermentation and micro-plate detection for large-scale screening of the mutants.It was suitable for screening a large number of mutants and improving the breeding efficiency after heavy-ion beam irradiation.Seventeen strains showed higher cellulase activity compared with the initial strain after the screening of plate and MTP fermentation.The filter paper activity and β-glucosidase activity of Aspergillus niger H11201 had increased 38.74 and 63.23%separately compared with A.niger H11 by shaking flask fermentation,and it was genetically stable after being passaged to nine generations.The results indicate that the high-throughput screening method can be used for the quick breeding of A.niger with high cellulase activity.展开更多
Due to high cost and relatively low efficiency of cellulase enzymes used for the saccharification of pretreated lignocelluloses, the improvement of cellulase secreting microorganisms is of vital importance. Trichoderm...Due to high cost and relatively low efficiency of cellulase enzymes used for the saccharification of pretreated lignocelluloses, the improvement of cellulase secreting microorganisms is of vital importance. Trichoderma reesei QM 6a, an excellent source of cellulase was selected in the late 1960’s. at Natick Laboratories by its performance on pure cellulose (Solka Floc, Avicel) . QM 6a is the wild parent strain of best existing hypercellulolytic mutants such as Rut C30, VTT-D-80133, L27, CL-847 and others. Utilization of cheaper carbon sources (e.g., pretreated wood or straw) both in enzyme production and in hydrolysis necessitates to investigate fungal species other than T.reesei. A screening program was initiated to test 150 wild-type Trichoderma strains in shake flask for cellulase production on SO 2-impregnated and steam pretreated spruce and willow, candidate substrates for bioalcohol program in Sweden. Filter paper activity (FPA) method was used to determine the overall cellulase activity. Strain TUB F-1505 was selected as promising candidate for mutagenesis. This wild strain was isolated from a tropical rain forest area near Manaus, Brazil. Isolate F-1505 was subjected to NTG-mutation to select catabolite (glucose, glycerol) resistant mutants. A Petri plate clearing assay using Walseth cellulose, glycerol or glucose and Triton X100 (colony size inhibitor) was applied for pre-screening of the colonies. Over 6000 colonies were evaluated. Best colonies were tested in shake flask fermentation on pretreated spruce and willow as carbon sources. Mutants producing higher levels of cellulase (FPA) were further mutated by either NTG or UV-light. At least 4 mutants were obtained and freeze-dried exhibiting equivalent or higher cellulase production as compared to Trichoderma reesei Rut C30.展开更多
利用木质纤维素生产燃料和化学品是解决现阶段能源危机的有效途径之一。尽管木质纤维素的预处理技术受到广泛研究,但传统预处理技术的高能源需求、高成本以及设备抗腐蚀技术的限制催生了新型预处理工艺的发展。低共熔溶剂(deep eutectic...利用木质纤维素生产燃料和化学品是解决现阶段能源危机的有效途径之一。尽管木质纤维素的预处理技术受到广泛研究,但传统预处理技术的高能源需求、高成本以及设备抗腐蚀技术的限制催生了新型预处理工艺的发展。低共熔溶剂(deep eutectic solvents,DES)的出现有效地解决了这些问题,并大大提高了木质纤维素的转化效率。目前,二元低共熔溶剂(binary deep eutectic solvents,BDES)的应用与研究较为广泛,效率更高且具有针对性的三元低共熔溶剂(ternary deep eutectic solvents,TDES)也在逐渐发展中。本研究对TDES进行了系统的分类(包括含醇和酸的TDES、含有机分子化合物的TDES、含金属卤化物的TDES和含有两种不同酸的TDES),探讨了上述TDES在木质纤维素预处理后酶解性能改善方面的研究进展,同时对联合预处理方法进行了综述,并展望了DES在不同研究方向的应用潜力。展开更多
目的利用生物软件和数据库对耐高温解淀粉芽孢杆菌BA-DES4的基因组数据进行处理、注释和分析,并在基因水平上对菌株特征进行功能注释。方法通过京都基因和基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)和基因本体论(ge...目的利用生物软件和数据库对耐高温解淀粉芽孢杆菌BA-DES4的基因组数据进行处理、注释和分析,并在基因水平上对菌株特征进行功能注释。方法通过京都基因和基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)和基因本体论(gene ontology,GO)等代谢信号通路,蛋白质同源组(clusters of orthologous groups of proteins,COG)、非冗余蛋白质(non-redundant protein,NR)数据库等分析,分别与预测得到的基因序列做比对,获得基因功能注释表。将预测得到基因的蛋白序列与COG、KEGG和GO数据库进行蛋白质序列对蛋白质序列库比对(basic local alignment search tool:protein,BLASTP)比对分析,从而实现基因注释信息预测及功能预测。结果通过数据库分析可知其碱基数目为4188731,GC含量占比为46.18%,编码的蛋白基因4445条,基因总长度为3696380 bp。对基因组蛋白编码基因进行功能注释可知,COG注释基因中占比最高的功能为G(碳水化合物的转运和代谢),其次为K(转录),表明COG数据库编码的蛋白基因主要参与细胞的基本功能。GO注释中,基因种类和数目最多的为生物学过程;碳水化合物活性酶(carbohydrate-active enzymes,CAZy)注释中,水解酶占比最高;KEGG注释中,碳水化合物代谢数目最多;在环境信息中,信息转导占比最多。耐高温解淀粉芽孢杆菌BA-DES4共11条编码纤维素酶的基因,β-葡萄糖苷酶和内切葡聚糖酶为基因编码的酶。结论本研究对耐高温解淀粉芽孢杆菌BA-DES4的基因组数据进行处理、注释和分析,进一步探索菌株研究潜力,以便更好地探究菌株产纤维素的调控机制,为后续实验提供理论基础。展开更多
文摘The property of major cellulases from the guts of Anoplophora glabripennis larvae have been characterized. The optimal temperatures of both β 1,4 glucosidase (β glucosidase) and endo β 1,4 endoglucanase (endoglucanase, Cx) are 40℃. The β glucosidase was optimally active at pH 4\^8, while the optimal activity of the endoglucanase occurred at pH 4 4 5 6 The endoglucanase was active with a wide range of pH and temperature, the levels of activity from 25℃ to 50℃ were more than 80%, and the activity remained 60% between pH 3 2 and pH 7 2. The endoglucanase exhibited higher thermal stability than β glucosidase. Both enzymes lose their activities by heat treatment at 60℃. Two isozymes of endoglucanase were detected in sodium carboxymethylcellulose polyacrymide gels (CMC gel) by chemical colorization, and purified by elution from the gel slices. The molecular weights of the two isozymes were estimated as 26kD and 39kD respectively. Moreover molecular characteristics of the two isozymes are currently underway.
文摘One of the latest sources of alternative energy, bioethanol, has been the focus of modem research, The production of bioethanol is commonly restricted by the activity of cellulase. Therefore, cellulase has become one of the critical issues in the conversion of lignocelluloses to bioethanol. This article is an overview of the sources and factors affecting enzyme activity, as well as methods of evaluation and utilizations of cellulase. We conclude that a combination of cellulases from various strains can enhance hydrolysis of substrates. Large enough amounts of cellobiase or sufficient cellobiase activity can reduce the inhibition to exoglucanase activity of cellobiose. Characterization and exploitation of cellulase should focus on a definite substrate. Promotion and mixed incubation of strains can reduce the cost of industrial utilization of cellulase.
文摘Lignocellulosic substrates are a good carbon source and provide rich growth media for a variety of microorganisms which prodLuce industrially important enzymes. Cellulases are a group of hydrolytic enzymes such as filter paperase (FPase), carboxymethyl cellulase(CMCase) andβ-glucosidase-responsible for release of sugars in the bioconversion of the lignocellulosic biomass into a variety of value-added products. This study examined cellulase production by a newly isolated Aspergillus unguis on individual lignocellulosic substrates in solid state fermentation (SSF). The maximum peak production of enzymes varied from one substrate to another, however,based on the next best solid support and local availability of groundnut fodder supported maximum enzyme yields compared with other solid supports used in this study.Groundnut fodder supported significant production of FPase (5.9 FPU/g of substrate), CMCase (1.1 U/g of substrate) andβ-glucosidase activity (6.5 U/g of substrate) in SSF. Considerable secretion of protein (27.0 mg/g of substrate) on groundnut fodder was recorded. Constant increment of protein content in groundnut fodder due to cultivation of A. unguis is an interesting observation and it has implications for the improvement of nutritive value of groundnut fodder for cattle.
基金the National Natural Science Foundation of China （Grant No. 30571503） the Program for Changjiang Scholars and Innovative Research Team in Universities （IRT0607）.
文摘In order to investigate different kinds of cellulase in insect pests, we selected male and female adults of Anoplophora glabripennis （Motschulsky） emerging from four different host species （Populus alba var. pyramidalis, P. nigra var. thevestina （Dode） Bean., P. simonii × P. pyramidliscr cv. Opera 8277 Hsu. and Salix matsudana f. lobato-glandulosa） as our research material. The enzyme activitives of three kinds of cellulase in the intestines of the adult insects were measured. The Cx-cellulase isozymes were detected with a CMC-incorporated polyacrylamide gel. The results show that： there are no statistically significant differences between the enzyme activities of males and females emerging from the different host species. The order of magnitude in activity is： Cx-cellulase 〉 β-glycosidase 〉 C1-cellulase. When the adults emerge from the same host species, there are no statistically significant differences between male and female enzyme activities of β-glycosidase and C1-cellulase, but the enzyme activity of Cx-cellulase of males is clearly higher than that of females. The patterns and migration of Cx-cellulase isozymes of males and females emerging from differ-ent hosts trees are clearly not different, and neither are they different when emerging from the same hosts.
基金supported by the National Natural Science Foundation of China(No.11305225)the Science and Technology Service Network Initiative(KFJ-EW-STS-086)
文摘In this study,a high-throughput screening method was established through the 24-square deep-well microliter plate(MTP) fermentation and micro-plate detection for large-scale screening of the mutants.It was suitable for screening a large number of mutants and improving the breeding efficiency after heavy-ion beam irradiation.Seventeen strains showed higher cellulase activity compared with the initial strain after the screening of plate and MTP fermentation.The filter paper activity and β-glucosidase activity of Aspergillus niger H11201 had increased 38.74 and 63.23%separately compared with A.niger H11 by shaking flask fermentation,and it was genetically stable after being passaged to nine generations.The results indicate that the high-throughput screening method can be used for the quick breeding of A.niger with high cellulase activity.
文摘Due to high cost and relatively low efficiency of cellulase enzymes used for the saccharification of pretreated lignocelluloses, the improvement of cellulase secreting microorganisms is of vital importance. Trichoderma reesei QM 6a, an excellent source of cellulase was selected in the late 1960’s. at Natick Laboratories by its performance on pure cellulose (Solka Floc, Avicel) . QM 6a is the wild parent strain of best existing hypercellulolytic mutants such as Rut C30, VTT-D-80133, L27, CL-847 and others. Utilization of cheaper carbon sources (e.g., pretreated wood or straw) both in enzyme production and in hydrolysis necessitates to investigate fungal species other than T.reesei. A screening program was initiated to test 150 wild-type Trichoderma strains in shake flask for cellulase production on SO 2-impregnated and steam pretreated spruce and willow, candidate substrates for bioalcohol program in Sweden. Filter paper activity (FPA) method was used to determine the overall cellulase activity. Strain TUB F-1505 was selected as promising candidate for mutagenesis. This wild strain was isolated from a tropical rain forest area near Manaus, Brazil. Isolate F-1505 was subjected to NTG-mutation to select catabolite (glucose, glycerol) resistant mutants. A Petri plate clearing assay using Walseth cellulose, glycerol or glucose and Triton X100 (colony size inhibitor) was applied for pre-screening of the colonies. Over 6000 colonies were evaluated. Best colonies were tested in shake flask fermentation on pretreated spruce and willow as carbon sources. Mutants producing higher levels of cellulase (FPA) were further mutated by either NTG or UV-light. At least 4 mutants were obtained and freeze-dried exhibiting equivalent or higher cellulase production as compared to Trichoderma reesei Rut C30.
文摘利用木质纤维素生产燃料和化学品是解决现阶段能源危机的有效途径之一。尽管木质纤维素的预处理技术受到广泛研究,但传统预处理技术的高能源需求、高成本以及设备抗腐蚀技术的限制催生了新型预处理工艺的发展。低共熔溶剂(deep eutectic solvents,DES)的出现有效地解决了这些问题,并大大提高了木质纤维素的转化效率。目前,二元低共熔溶剂(binary deep eutectic solvents,BDES)的应用与研究较为广泛,效率更高且具有针对性的三元低共熔溶剂(ternary deep eutectic solvents,TDES)也在逐渐发展中。本研究对TDES进行了系统的分类(包括含醇和酸的TDES、含有机分子化合物的TDES、含金属卤化物的TDES和含有两种不同酸的TDES),探讨了上述TDES在木质纤维素预处理后酶解性能改善方面的研究进展,同时对联合预处理方法进行了综述,并展望了DES在不同研究方向的应用潜力。
文摘目的利用生物软件和数据库对耐高温解淀粉芽孢杆菌BA-DES4的基因组数据进行处理、注释和分析,并在基因水平上对菌株特征进行功能注释。方法通过京都基因和基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)和基因本体论(gene ontology,GO)等代谢信号通路,蛋白质同源组(clusters of orthologous groups of proteins,COG)、非冗余蛋白质(non-redundant protein,NR)数据库等分析,分别与预测得到的基因序列做比对,获得基因功能注释表。将预测得到基因的蛋白序列与COG、KEGG和GO数据库进行蛋白质序列对蛋白质序列库比对(basic local alignment search tool:protein,BLASTP)比对分析,从而实现基因注释信息预测及功能预测。结果通过数据库分析可知其碱基数目为4188731,GC含量占比为46.18%,编码的蛋白基因4445条,基因总长度为3696380 bp。对基因组蛋白编码基因进行功能注释可知,COG注释基因中占比最高的功能为G(碳水化合物的转运和代谢),其次为K(转录),表明COG数据库编码的蛋白基因主要参与细胞的基本功能。GO注释中,基因种类和数目最多的为生物学过程;碳水化合物活性酶(carbohydrate-active enzymes,CAZy)注释中,水解酶占比最高;KEGG注释中,碳水化合物代谢数目最多;在环境信息中,信息转导占比最多。耐高温解淀粉芽孢杆菌BA-DES4共11条编码纤维素酶的基因,β-葡萄糖苷酶和内切葡聚糖酶为基因编码的酶。结论本研究对耐高温解淀粉芽孢杆菌BA-DES4的基因组数据进行处理、注释和分析,进一步探索菌株研究潜力,以便更好地探究菌株产纤维素的调控机制,为后续实验提供理论基础。
