高活性纤维素酶菌株的筛选鉴定和秸秆降解特性的研究
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摘要
农作物秸秆是世界上最丰富的纤维素类物质之一。由于纤维素分子结构的特殊性及纤维素物质在自然界的存在状态(以木质纤维素的形式存在)的特殊性,目前这一巨大资源不仅没有得到充分利用,反而因大部分被焚烧或弃置在自然环境而成了一大污染源。如何利用纤维素酶将这些秸秆纤维素废弃物大规模地转化为简单糖类或蛋白质等产品,进一步转化酒精、饲料等已成为全世界研究的热点,也是缓解当今世界所面临的粮食短缺、饲料资源紧张、能源危机和环境污染等问题的有效途经之一。
纤维素酶是一类能够水解纤维素的β-D-糖苷键生成葡萄糖的多组分酶的总称。自然界中能分解纤维素的生物主要为真菌类及部分细菌,以木霉、曲霉、青霉的能力最突出,但目前研究得最多的是一些真菌对经物理、化学处理(辐射、蒸汽爆破、膨化、碾磨、酸化、碱化等)的不溶性纤维素的作用,生产成本高,对环境的污染严重,限制了农作物秸秆的高效利用。而筛选性能稳定的高产纤维素酶菌株,研究其对未处理的天然纤维素的降解作用对解决上述这些问题至关重要。我们通过大规模的菌株筛选和测试,取得了以下主要结果:
1.采用CMC-Na平板进行菌种分离,筛选出具有较高降解纤维素酶能力的细菌35株,真菌11株。
2.以天然水稻秸秆为碳源,进行液体摇瓶培养复筛得到一株能高效降解秸秆纤维素的丝状真菌,经18SrDNA基因序列分析和菌株形态特性分析,初步确定该菌株为藤仓赤霉(Gibberella fujikuroi)。
3.以未处理的水稻秸秆为唯一碳源,研究了氮源、接种量、发酵时间、初始发酵温度、培养基初始pH等对藤仓赤霉产酶的影响.结果表明该菌株产纤维素酶的最适宜的氮源为CO(NH_2)_2,接种量为5%,培养时间为120 h,培养温度为28～37℃,培养基初始pH为5～6。
4.SEM分析表明,藤仓赤霉作用水稻秸秆是从内表面向外表面进行的;作用前后的稻草成分测定结果显示,纤维素降解率、半纤维素降解率和木质素降解率分别达38.9%,57.9%和26.5%;该菌对未处理秸秆作用8 d,秸秆失重率可达53%;对该菌的酶成分进行分析,结果表明该菌可产生多种降解酶,其CMCase、FPase、xylanase酶活分别可达1.723 IU/mL、0.344 IU/mL、0.532 IU/mL。且该菌产生的CMC酶的反应温度以50℃左右为宜,pH值以6左右为适,Mg~(2+)与Ca~(2+)对酶反应有促进作用,Pb~(2+)和Cu~(2+)对酶反应有抑制作用。
本实验所获得的藤仓赤霉对水稻秸秆的这些作用特性未见其他作者报道,因此,为进一步利用该菌进行改良、提高酶活以应用于生产奠定了基础。
Crop straw is the most abundant collulose in the world.The special structure of the collulose molecule,along with the particularity of the existence state of a majority of collulose materials(exist in the form of wood)in nature,so,the ratio of using the resourse is low now,what is more,most of it have become the source of pollution for being discarded in natural environment or being burnt.How to transform the discarded straw fibre into monosaccharide or proteins on a large scale and transform alcohol,feed even food ulteriorly has become a hotspot in worldwide study,also a effective way of resolving the short-commons,the lack of feed resource and the environmental pollution.
Cellulase is the kind of enzyme including multi-component which can hydrolyze theβ-D-indican bond of cellulose to glucose.The major organism that can decompose cellulose are fungi:Trichoderma,Aspergillus,Penicillium and some bacteria in nature. But at present most studies are focused on the effect of Trichoderma,Aspergillus, Penicillium on insoluble cellulose treated by physics and chemical methods including the radiation,steam demolition,expansion,milling,acidification,alkalize and so on.Because of high cost of production and serious pollution to the environment,these methods limit the efficient utilization of crop straw.Screening the natural microbial organism with collulose degradation ability is a basic way to solve the problem above.By high though screening(HTS) technology,we obtained following result.
In this research,35 bacterial strains and 11 fungal strains that have efficient cellulose degradation ability have been obtained.
With natural straw as carbon source,we obtained a strain of filamentous fungi that could effectively degenerate crop straw cellulose.This strain was preliminarily identified as Gibberella fujikuroi according to morphologic taxonomy and the analysis of its 18S rDNA gene sequences.
With unsettled straw as only carbon source,various factors including nitrogen source, inoculating quantities,incubating time,initial temperature,initial pH et al that can affect the production of cellulase have been studied.The results showed that the best nitrogen source was CO(NH_2)_2,inoculating volume was 5%,incubating time was 120 h,initial temperature was 28～37℃and initial pH of culture medium was 5～6.
SEM analysis showed that Gibberella fujikuroi degradated the cellulose from the inner surface to the external surface.Comparison with the unsettled rice straw,the ratio of degradation of cellulose,hemicellulose and xylogen reached 38.9%,57.9%and 26.5%. The ratio of straw weightlessness reached over 53%via the fungis action on natural straw for 8 d.The analysis of its enzymatic compositions showed that Gibberella fujikuroi could product a variety of degrading enzymes,the activity of cellulose(CMCase)、natural cellulase and FPAase reached 1.723 IU/mL、0.368 IU/mL and 0.344 IU/mL.The optimal conditions for the enzymatic reaction were about 50℃and pH 6;Under 50℃,at pH 5～6,the enzyme showed high activity and good stability;Mg~(2+),Ca~(2+) were the activators and Pb~(2+),Cu~(2+) were the inhibitors of the enzyme.
These characterization have not being reported,so it established bases for improving the strain and increasing its enzymatic activity in order to meet application in industrial production.
纤维素酶是一类能够水解纤维素的β-D-糖苷键生成葡萄糖的多组分酶的总称。自然界中能分解纤维素的生物主要为真菌类及部分细菌,以木霉、曲霉、青霉的能力最突出,但目前研究得最多的是一些真菌对经物理、化学处理(辐射、蒸汽爆破、膨化、碾磨、酸化、碱化等)的不溶性纤维素的作用,生产成本高,对环境的污染严重,限制了农作物秸秆的高效利用。而筛选性能稳定的高产纤维素酶菌株,研究其对未处理的天然纤维素的降解作用对解决上述这些问题至关重要。我们通过大规模的菌株筛选和测试,取得了以下主要结果:
1.采用CMC-Na平板进行菌种分离,筛选出具有较高降解纤维素酶能力的细菌35株,真菌11株。
2.以天然水稻秸秆为碳源,进行液体摇瓶培养复筛得到一株能高效降解秸秆纤维素的丝状真菌,经18SrDNA基因序列分析和菌株形态特性分析,初步确定该菌株为藤仓赤霉(Gibberella fujikuroi)。
3.以未处理的水稻秸秆为唯一碳源,研究了氮源、接种量、发酵时间、初始发酵温度、培养基初始pH等对藤仓赤霉产酶的影响.结果表明该菌株产纤维素酶的最适宜的氮源为CO(NH_2)_2,接种量为5%,培养时间为120 h,培养温度为28～37℃,培养基初始pH为5～6。
4.SEM分析表明,藤仓赤霉作用水稻秸秆是从内表面向外表面进行的;作用前后的稻草成分测定结果显示,纤维素降解率、半纤维素降解率和木质素降解率分别达38.9%,57.9%和26.5%;该菌对未处理秸秆作用8 d,秸秆失重率可达53%;对该菌的酶成分进行分析,结果表明该菌可产生多种降解酶,其CMCase、FPase、xylanase酶活分别可达1.723 IU/mL、0.344 IU/mL、0.532 IU/mL。且该菌产生的CMC酶的反应温度以50℃左右为宜,pH值以6左右为适,Mg~(2+)与Ca~(2+)对酶反应有促进作用,Pb~(2+)和Cu~(2+)对酶反应有抑制作用。
本实验所获得的藤仓赤霉对水稻秸秆的这些作用特性未见其他作者报道,因此,为进一步利用该菌进行改良、提高酶活以应用于生产奠定了基础。
Crop straw is the most abundant collulose in the world.The special structure of the collulose molecule,along with the particularity of the existence state of a majority of collulose materials(exist in the form of wood)in nature,so,the ratio of using the resourse is low now,what is more,most of it have become the source of pollution for being discarded in natural environment or being burnt.How to transform the discarded straw fibre into monosaccharide or proteins on a large scale and transform alcohol,feed even food ulteriorly has become a hotspot in worldwide study,also a effective way of resolving the short-commons,the lack of feed resource and the environmental pollution.
Cellulase is the kind of enzyme including multi-component which can hydrolyze theβ-D-indican bond of cellulose to glucose.The major organism that can decompose cellulose are fungi:Trichoderma,Aspergillus,Penicillium and some bacteria in nature. But at present most studies are focused on the effect of Trichoderma,Aspergillus, Penicillium on insoluble cellulose treated by physics and chemical methods including the radiation,steam demolition,expansion,milling,acidification,alkalize and so on.Because of high cost of production and serious pollution to the environment,these methods limit the efficient utilization of crop straw.Screening the natural microbial organism with collulose degradation ability is a basic way to solve the problem above.By high though screening(HTS) technology,we obtained following result.
In this research,35 bacterial strains and 11 fungal strains that have efficient cellulose degradation ability have been obtained.
With natural straw as carbon source,we obtained a strain of filamentous fungi that could effectively degenerate crop straw cellulose.This strain was preliminarily identified as Gibberella fujikuroi according to morphologic taxonomy and the analysis of its 18S rDNA gene sequences.
With unsettled straw as only carbon source,various factors including nitrogen source, inoculating quantities,incubating time,initial temperature,initial pH et al that can affect the production of cellulase have been studied.The results showed that the best nitrogen source was CO(NH_2)_2,inoculating volume was 5%,incubating time was 120 h,initial temperature was 28～37℃and initial pH of culture medium was 5～6.
SEM analysis showed that Gibberella fujikuroi degradated the cellulose from the inner surface to the external surface.Comparison with the unsettled rice straw,the ratio of degradation of cellulose,hemicellulose and xylogen reached 38.9%,57.9%and 26.5%. The ratio of straw weightlessness reached over 53%via the fungis action on natural straw for 8 d.The analysis of its enzymatic compositions showed that Gibberella fujikuroi could product a variety of degrading enzymes,the activity of cellulose(CMCase)、natural cellulase and FPAase reached 1.723 IU/mL、0.368 IU/mL and 0.344 IU/mL.The optimal conditions for the enzymatic reaction were about 50℃and pH 6;Under 50℃,at pH 5～6,the enzyme showed high activity and good stability;Mg~(2+),Ca~(2+) were the activators and Pb~(2+),Cu~(2+) were the inhibitors of the enzyme.
These characterization have not being reported,so it established bases for improving the strain and increasing its enzymatic activity in order to meet application in industrial production.
引文
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