糠醛渣特定底物产酶培养及其水解研究
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摘要
纤维素酶是由几种不同酶组成的复合酶系,由于酶催化反应底物的复杂性,从不同霉菌和不同底物发酵分离得到的纤维素酶组分和酶活力有着很大差别。本论文选择不同菌种针对特定底物进行产纤维素酶活力比较评价,并进行糠醛渣酶解过程研究。木论文选取了产纤维素酶的四种霉菌绿色木霉CGMCC3.2941、康宁木霉CICC13007、里氏木霉CICC40360和黑曲霉ACCC30557,采用液体发酵法在微晶纤维素和糠醛渣两种底物上进行产酶培养,在选定时间测定所产纤维素酶的纤维二糖酶活力、内切葡聚糖酶活力、外切葡聚糖酶活力以及滤纸酶活力。其中两种滤纸酶活比较高的菌株康宁木霉CICC13007和绿色木霉CGMCC3.2941以糠醛渣为特定底物的滤纸酶活比微晶纤维素特定底物的滤纸酶活分别高0.64U/mL和6.31U/mL。使用拟康氏木霉、康宁木霉、里氏木霉所产纤维素酶及诺维信纤维素酶水解不同纤维素含量的糠醛渣。糠醛渣纤维素含量为94.92%时,诺维信纤维素酶得到的底物糖化率最高,为92.06%,而自产的纤维素酶中拟康氏木霉达到了83.76%。结果表明糠醛渣中的木质素脱除后,纤维素酶对纤维素的降解效率提高了,说明在水解反应中木素的存在对纤维素酶有无效吸附作用。去除了木素对纤维素酶的影响后,纤维素酶能更好的与纤维素进行有效的吸附解吸,从而提高了水解效率将绿色木霉、拟康氏木霉、康宁木霉三种菌种生产的纤维素酶复配,并补充由黑曲霉生产的纤维二糖酶,对糠醛渣原料进行水解,结果发现绿色木霉与拟康氏木霉的混合酶液对糠醛渣的降解效果最好,糖化率达80%。不同菌种所产的纤维素酶系组分之间产生了协同增效作用。一株产纤维素酶的粗糙脉胞菌作为出发菌株,经过不同的紫外线照射时间,确定了菌株的致死率。以糠醛渣为底物培养粗糙脉胞菌的突变菌株,获得的滤纸酶活力比对照菌种高出30%。该诱变菌株优化后可用于纤维素乙醇直接转化工艺过程中。
Cellulase is composed of several different enzymes. The component and enzyme activity of cellulase which is isolated from the different fungi fermentation, because the substrate of enzyme-catalyzed reaction is complex. The aim of the experiment is to evaluate cellulase production from different fungi.This experiment picks up four kinds of fungi which produce cellulase, which are CGMCC3.2941、 CICC13007、CICC40360. and ACCC30557. These fungi were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. After cellulase production, the filter paper activity (FPA) of the supernatant from each fungus was measured to compare the performance of the enzyme from different fungal strains. Moreover, the individual activities of β-glucosidase, endoglucanase, and exoglucanase were also evaluated. In the case of CGMCC3.2941and CICC13007, furfural residues supported a better environment for cellulase production than MCC.The FPAs of CGMCC3.2941and CICC13007in furfural residues were6.31U/mL and0.64U/mL higher than that in MCC, respectively.Furfural residues with different cellulose contents were hydrolysed by a mixture of cellulase which are from CGMCC3.3002, CICC13007. CICC13052and Celluclast1.5L and β-glucosidase. The glucose yield of furfural residues increased incrementally according to the cellulose content of the substrate. The highest glucose yield (92.06%) in the digest with Celluclast1.5L preparation was obtained from the enzymatic hydrolysate of the furfural residue with a cellulose content of94.92%. The glucose yield in the digest with cellulase preparation from CGMCC3.3002is83.76%. After removing of ligin. the efficiency of degradation of cellulose increased, which indicated that there was non-productive adsorption between the enzyme and the lignin in the furfural residues. The increase of hydrolysis efficiency due to removing the impact of ligin on cellulase and thus the better adsorption and desorption between the cellulase and cellulose.Furfural residues were hydrolysed by a mixture of cellulase, which contain the cellulase from CGMCC3.2941, CGMCC3.3002and CICC1007. During the hydrolysis, β-glucosidase from ACCC30557was added into the digest. The result showed the highest glucose yield in the digest with the cellulase mixture of CGMCC3.2941and CGMCC3.3002is80%. There is synergistic action among the different constituents.In the experiment, after different UV exposure time, the death rate of the Neurospora crassa was determined. Compared to the initial Neurospora crassa, the cellulase production from Neurospora crassa after ultraviolet mutagenesis was improved by30%. After optimizing, Neurospora crassa after ultraviolet mutagenesis can be used drectly in the production of ethanol.
Cellulase is composed of several different enzymes. The component and enzyme activity of cellulase which is isolated from the different fungi fermentation, because the substrate of enzyme-catalyzed reaction is complex. The aim of the experiment is to evaluate cellulase production from different fungi.This experiment picks up four kinds of fungi which produce cellulase, which are CGMCC3.2941、 CICC13007、CICC40360. and ACCC30557. These fungi were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. After cellulase production, the filter paper activity (FPA) of the supernatant from each fungus was measured to compare the performance of the enzyme from different fungal strains. Moreover, the individual activities of β-glucosidase, endoglucanase, and exoglucanase were also evaluated. In the case of CGMCC3.2941and CICC13007, furfural residues supported a better environment for cellulase production than MCC.The FPAs of CGMCC3.2941and CICC13007in furfural residues were6.31U/mL and0.64U/mL higher than that in MCC, respectively.Furfural residues with different cellulose contents were hydrolysed by a mixture of cellulase which are from CGMCC3.3002, CICC13007. CICC13052and Celluclast1.5L and β-glucosidase. The glucose yield of furfural residues increased incrementally according to the cellulose content of the substrate. The highest glucose yield (92.06%) in the digest with Celluclast1.5L preparation was obtained from the enzymatic hydrolysate of the furfural residue with a cellulose content of94.92%. The glucose yield in the digest with cellulase preparation from CGMCC3.3002is83.76%. After removing of ligin. the efficiency of degradation of cellulose increased, which indicated that there was non-productive adsorption between the enzyme and the lignin in the furfural residues. The increase of hydrolysis efficiency due to removing the impact of ligin on cellulase and thus the better adsorption and desorption between the cellulase and cellulose.Furfural residues were hydrolysed by a mixture of cellulase, which contain the cellulase from CGMCC3.2941, CGMCC3.3002and CICC1007. During the hydrolysis, β-glucosidase from ACCC30557was added into the digest. The result showed the highest glucose yield in the digest with the cellulase mixture of CGMCC3.2941and CGMCC3.3002is80%. There is synergistic action among the different constituents.In the experiment, after different UV exposure time, the death rate of the Neurospora crassa was determined. Compared to the initial Neurospora crassa, the cellulase production from Neurospora crassa after ultraviolet mutagenesis was improved by30%. After optimizing, Neurospora crassa after ultraviolet mutagenesis can be used drectly in the production of ethanol.
引文
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