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产纤维素酶细菌的筛选鉴定与特性分析
Screening, Identification and Characteristic Analysis of Cellulase-Producing Bacteria
为了筛选获得产纤维素酶细菌,为新型饲料添加剂的开发提供材料基础。本研究以玉米田土壤为样品,使用刚果红平板法筛选产纤维素酶细菌并分析其所产酶的特性。通过试验,筛选获得产纤维素酶菌株,经形态学和分子生物学法将其鉴定为巨大芽孢杆菌(Bacillus megaterium)。对巨大芽孢杆菌XT2所产纤维素酶进行酶学特性分析,发现其最适反应条件为50℃,pH 6.0,具有一定的热稳定性,K+对纤维素酶具有激活作用,Mg2+、Ca2+和Mn2+对酶活具有抑制作用。该菌生长至20 h时,所产纤维素酶活力最高,达到0.774 U/mL。该产纤维素酶芽孢杆菌的成功分离获取为饲料新资源的开发以及新型饲料添加剂的研制提供了菌种材料。
To screen cellulase-producing bacteria and provide a material basis for the development of new feed additives, in this study, soil samples from corn fields were used to screen cellulase-producing bacteria by congo red plate method, and the characteristics of cellulase produced by it were analyzed. Through the test, the cellulase-producing strain was screened and identified as Bacillus megaterium by morphological and molecular biological methods. The enzymatic characteristics of cellulase produced by Bacillus megaterium XT2 was analyzed and the optimal reaction condition was found to be 50℃ and pH 6.0, and the cellulase showed certain thermal stability, K+ could activate the cellulase activity, and Mg2+, Ca2+ and Mn2+ had an inhibitory effect on enzyme activity. When the strain grew to 20 h, the cellulase activity reached the highest (0.774 U/mL). The successful isolation of cellulase-producing Bacillus could provide a strain material for the development of new feed resources and new feed additives.
纤维素酶 / 芽孢杆菌 / 筛选 / 鉴定 / 酶学特性 {{custom_keyword}} /
cellulase / Bacillus / screening / identification / enzymatic characteristics {{custom_keyword}} /
[1] |
Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.
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[2] |
One of the defining features of plants is a body plan based on the physical properties of cell walls. Structural analyses of the polysaccharide components, combined with high-resolution imaging, have provided the basis for much of the current understanding of cell walls. The application of genetic methods has begun to provide new insights into how walls are made, how they are controlled, and how they function. However, progress in integrating biophysical, developmental, and genetic information into a useful model will require a system-based approach.
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[3] |
More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei.
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[4] |
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[5] |
Biologically mediated processes seem promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Although processes featuring a step dedicated to the production of cellulase enzymes have been the focus of most research efforts to date, consolidated bioprocessing (CBP)--featuring cellulase production, cellulose hydrolysis and fermentation in one step--is an alternative approach with outstanding potential. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer, and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase system enabling cellulose utilization. Recent studies of the fundamental principles of microbial cellulose utilization support the feasibility of CBP.
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[6] |
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[7] |
Lignocellulose is a complex substrate which requires a variety of enzymes, acting in synergy, for its complete hydrolysis. These synergistic interactions between different enzymes have been investigated in order to design optimal combinations and ratios of enzymes for different lignocellulosic substrates that have been subjected to different pretreatments. This review examines the enzymes required to degrade various components of lignocellulose and the impact of pretreatments on the lignocellulose components and the enzymes required for degradation. Many factors affect the enzymes and the optimisation of the hydrolysis process, such as enzyme ratios, substrate loadings, enzyme loadings, inhibitors, adsorption and surfactants. Consideration is also given to the calculation of degrees of synergy and yield. A model is further proposed for the optimisation of enzyme combinations based on a selection of individual or commercial enzyme mixtures. The main area for further study is the effect of and interaction between different hemicellulases on complex substrates.Copyright © 2012 Elsevier Inc. All rights reserved.
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[8] |
{{custom_citation.content}}
{{custom_citation.annotation}}
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[9] |
{{custom_citation.content}}
{{custom_citation.annotation}}
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[10] |
{{custom_citation.content}}
{{custom_citation.annotation}}
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[11] |
Cellulases have many useful applications in industry and biotechnology. So, identification of new bacterial strains expressing cellulases with better properties is desired. Five soil bacterial strains screened for high carboxymethyl cellulase (CMCase) activities were characterized and identified by 16S rRNA analysis as Bacillus amyloliquefaciens (FAY088), B. velezensis (FAY0103), B. tequilensis (FAY0117), B. subtilis (FAY0136), and B. subtilis (FAY0182). Their CMCase activities were 1.49, 1.26, 1.21, 1.21, and 1.24 U/ml, respectively. The maximum CMCase production was attained by growth at 35 °C, pH 6, and 180 rpm for 5 days. Residual activities of CMCases from FAY088 and FAY0117 were 88% or more after growth at 40 °C, which is same as FAY0182 CMCase at 40 and 45 °C. Additionally, FAY0182 retained 73% residual activity at 50 °C. FAY088 and FAY0182 retained more than 85% at pH 7 and 8. Conversely, residual activities from FAY0103 and FAY0136 declined a lot by increasing growth temperature beyond 40 °C and pH beyond 7. The maximum CMCase stability in all isolates was observed at pH 7, 3-h incubation, and 40 °C except for FAY0103 CMCase showed optimum temperature at 30 °C. More than 70% CMCase stability was retained in case of FAY088 at 50 °C, FAY0117 at 50-70 °C, and FAY0136 at 50-60 °C. FAY088 CMCase seemed to be the lest sensitive to temperature variation as it displayed residual activities 67, 72, 78, 84, 77, 74, and 72% at pH 3, 4, 5, 6, 8, 9, and 10, respectively. Finally, the five CMCase-producing isolates are recommended further enzyme applications in biotechnology and industry.
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[12] |
In the current study, an extracellular cellulase belonging to symbiotic Bacillus subtilis Bc1 of the leopard moth is purified and characterized. The molecular mass of enzyme was 47.8 kDa using SDS-PAGE. The purified enzyme had optimum activity in temperature and pH around 60 °C and 8, respectively. The purified cellulase was introduced as a stable enzyme in a wide variety of temperature (20-80 °C) and pH (4-10) and remained active to more than 74% at 80 °C for 1 h. Moreover, the cellulase extremely was stabled in the presence of metal ions and organic solvents and its activity was increased by acetone (20% v/v), CaCl and CoCl and inhibited by MnCl and NiCl. The values of enzyme's K and V were found to be 1.243 mg/mL and 271.3 µg/mL/min, respectively. The purified cellulase hydrolyzed cellulose, avicel and carboxymethyl cellulose (CMC) and the final product of CMC hydrolysis was cellobiose using thin-layer chromatography analysis. Consequently, owing to exo/endoglucanase activity and organic solvent, temperature and pH stability of the purified cellulase belong to B. subtilis BC1, it can be properly employed for various industrial purposes.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[13] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[14] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[15] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[16] |
赵龙妹. 青藏高原土壤微生物多样性研究进展[J]. 江苏农业科学, 2019, 47(14):6-12.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[17] |
王静娅, 王明亮, 张凤华. 干旱区典型盐生植物群落下土壤微生物群落特征[J]. 生态学报, 2016, 36(8):2363-2372.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[18] |
孔维栋. 极地陆域微生物多样性研究进展[J]. 生物多样性, 2013, 21(4):457-468.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[19] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[20] |
刘刚, 余少文, 孔舒, 等. 碱性纤维素酶及其应用的研究进展[J]. 生物加工过程, 2005(2):9-14.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[21] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[22] |
谷笑笑, 王振华, 潘康成. 益生芽孢杆菌对动物免疫功能影响研究进展[J]. 微生物学通报, 2016, 43(9):2079-2085.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[23] |
韩学易, 唐自钟, 胡云龙, 等. 响应面法优化巨大芽孢杆菌产纤维素酶发酵条件[J]. 四川农业大学学报, 2013, 31(03):319-321,334.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[24] |
刘露, 李丽, 闫洪雪, 张鹏鹏, 梁文辉, 赵宏涛. 巨大芽孢杆菌的应用研究进展[J]. 北方农业学报, 2016, 44(4):117-120.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[25] |
王金革. 植物蛋白质饲料中添加巨大芽孢杆菌对杂交鲟幼鱼生长、免疫的影响及机理探讨[D]. 上海:上海海洋大学, 2017:15-26.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[26] |
丁从文, 冯群, 李春焕. 特殊环境巨大芽孢杆菌LB01抗菌活性成分的分离鉴定及抗病机理[J]. 食品科学, 2020, 41(17):75-82.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
{{custom_ref.label}} |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
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