里氏木霉纤维素酶的液态深层发酵生产及其应用的研究
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摘要
随着能源危机的加剧,天然纤维质原料酶解生产燃料酒精成为当前研究的热点,然而,纤维素酶的使用成本是束缚此项研究的瓶颈所在。本实验旨在通过优化里氏木霉突变株WX-112液态发酵产纤维素酶的培养条件,寻求能更好降低纤维素酶使用成本的有效途径。用经过预处理的蔗渣代替微晶纤维素作为主要碳源用于纤维素酶的生产,既解决了环境污染问题,又可有效降低生产成本。
通过机械粉碎、酸水解、碱水解、微波与碱联合处理、活性乳酸菌处理等方法预处理蔗渣后用于里氏木霉液态发酵生产纤维素酶。单因素试验结果显示:浓度为1%(w/v)的NaOH溶液水解蔗渣效果较好。在此基础上,结合微波辐射处理,由正交试验得出了最佳的预处理工艺为:用0.30mol/L的NaOH溶液浸泡蔗渣,在微波功率为160W下处理5min,在此条件下单位能耗的酶活净增值最大,后续发酵结束后,酶活较未经处理的蔗渣发酵后所得酶活有显著提高。其中,滤纸酶活和CMC酶活分别由原来的1.73IU/mL和60.2IU/mL提高到3.54IU/mL和134.8 IU/mL,提高了104.6%和123.4%。
利用葡萄糖阻遏效应筛选抗阻遏的高产菌株。在平板上添加4%的葡萄糖筛选得到的菌株产酶最高,滤纸酶活和CMC酶活分别可达4.3IU/mL和165.0IU/mL左右,比原始菌株的产酶量分别提高了33.5%和31.0%。在此基础上,通过单因素试验和L16(45)的正交试验得到了该菌株摇瓶液态发酵的最佳培养基组成为(g/L):蔗渣30,麸皮25,豆饼粉40,乳清粉10,KH2PO4 2.0,胰蛋白胨3.0,硫酸铵2.0,CaCl2 0.5,MgSO4 0.5;对该菌株摇瓶液态发酵的条件进行了优化,得到的最佳培养条件为:种龄48h,培养基的起始pH5.0~5.5,培养温度26～28℃,接种量10%,装液量25ml/250ml,摇床转速180r/min,培养144h。在此条件下最有利于菌体产酶,β-葡萄糖苷酶活、滤纸酶活和CMC酶活最高可分别达到4.5IU/mL、10.1IU/mL、294.6IU/mL。
对酶学性质进行了研究。β-葡萄糖苷酶活、滤纸酶活及CMC酶活的最适pH值均在5.0左右,滤纸酶活和CMC酶活的最适反应温度为55℃,β-葡萄糖苷酶的最适反应温度为50℃。当酶液在低于50℃,环境pH在5.0左右时保存可以较好的保持酶活力。Zn2+、Mg2+、Na+、Ca2+、Mn2+等对酶活有一定的激活作用,不同浓度的酒精可以不同程度的抑制酶活,且抑制程度随酒精浓度的升高而增加,随温度的升高而加强。
蔗渣经碱与微波联合处理后纤维素的相对含量提高了11.0%,并且比未处理的蔗渣更易被纤维素酶水解糖化。研究了经过预处理的蔗渣酶解的最佳条件为:纤维素酶用量8-10FPU/mL,底物浓度2%,反应温度55℃,反应体系pH4.8,并适当添加一定浓度的表面活性剂Tween-80,在此条件下,得到的最终糖化率为35%左右。酒精对酶解糖化反应的抑制作用表现为:一方面,随着酒精浓度的升高,抑制作用逐步加强;另一方面,温度越高,酒精的抑制作用也越显著。
Currently, the research of using the natural fiber raw material to produce fuel alcohol has been paid more and more attention because of the aggravating of energy crisis. At the same time, the key point of this research is how to reduce the cost of cellulase. The optimal culture conditions of the submerged fermentation of the mutant strain Trichoderma reesei WX-112 were studied. The pretreated bagasse was used to produce cellulase as main materials replacing pure cellulose so as to alleviate the pollution of enviroment and reduce the cost of cellulase effectively.
The bagasse pretreated with the metheds such as smash, acids hydrolysis, alkalis hydrolysis, microwave and EM were used to produce cellulase by the mutant strain Trichoderma reesei WX-112 submerged fermentation. The result of single factor test showed: bagasse pretreated with NaOH(1%w/v) was better for producing cellulase. Based on this, the optimal conditions of pretreatement of bagasse gained by orthogonal experiment were as follows: alkali with 0.30 mol/L NaOH, the microwave radiation with power 160W, 5 minutes. Under these conditions, the cellulase yield can get the highest increase per unit in the view of energy consumption. The results showed that the maximum cellulase yield using pretreated bagasses as raw material was higher than that of using non-pretreated bagasses. The highest yield of FPA and CMCase reached 3.54IU/mL and 134.8 IU/mL from1.73IU/mL and 60.2IU/mL after 5 days’culturing, which increased by 104.6% and 123.4% respectively.
Based on the theory of glucose repression, the anti-repression strains were obtained by using agar plate screening techniques, which can synthenize cellulase when high concentation glucose exists. The strain can produce more cellulase than others by submerged fermentation , which was screened from the plate containing 4% glucose. The yield of FPA and CMCase reached 4.3IU/mL and 165.0IU/mL, which increased by 33.5% and 31.0% respectively. Then, the optimal culture conditions of this screening strain were studied by single factor test and L16(45) orthogonal experiment. The optimal fermentation medium was determined (g/L): bagasse 30, wheat bran 25, bean cake flour 40, whey powder 10, KH2PO4 2.0, tryptone 3.0, (NH4)2SO4 2.0, CaCl2 0.5, MgSO4 0.5. The sutiable fermentation conditions: plants age was 48h, the initial pH of culture medium was 5.0-5.5, the culture temperature was 26-28℃, the inoculum size was 10%, volume of medium was 25ml/250ml and the speed of shaker was 180r/min, culturing 144h. Under these conditions, the highest yield ofβ-glucosidase, FPA and CMCase reached 4.5IU/mL, 10.1IU/mL and 294.6IU/mL respectively.
The crude enzyme properties were investigated preliminarily. The optimum pH ofβ-glucosidase, FPA and CMCase were all 5.0 and the temperature were 50-55℃. The stable pH ranger, the thermal stability were obtained at the same time. Influences on enzymatic activity of many kinds of ions were studied. Zn2+, Mg2+, Na+, Ca2+, Mn2+ and so on could promoted its catalytic activity significantly. At last, the effects of alcohol on cellulase production were investigated. The results showed: alcohol with different concentration could all restrained the activity of cellulase, and this kind of effect would be strengthened with the increasing of tempreature and the concentration of alcohol.
The cellulose content of bagasse pretreated by alkali and microwave increased by 11.0%, which could be hydrolysised easier than non-pretreated bagasse. The optimal enzymatic hydrolysis conditions were determined: 8-10FPU cellulase/mL, the substrate concentration 2%, the reaction temperature 55℃, pH4.8. Then, the final hydrolysis efficiency could reach to 35%. Alcohol could restrained the enzymatic hydrolysis of bagasse: the influence would be strengthened with the increasing of tempreature and the concentration of alcohol.
通过机械粉碎、酸水解、碱水解、微波与碱联合处理、活性乳酸菌处理等方法预处理蔗渣后用于里氏木霉液态发酵生产纤维素酶。单因素试验结果显示:浓度为1%(w/v)的NaOH溶液水解蔗渣效果较好。在此基础上,结合微波辐射处理,由正交试验得出了最佳的预处理工艺为:用0.30mol/L的NaOH溶液浸泡蔗渣,在微波功率为160W下处理5min,在此条件下单位能耗的酶活净增值最大,后续发酵结束后,酶活较未经处理的蔗渣发酵后所得酶活有显著提高。其中,滤纸酶活和CMC酶活分别由原来的1.73IU/mL和60.2IU/mL提高到3.54IU/mL和134.8 IU/mL,提高了104.6%和123.4%。
利用葡萄糖阻遏效应筛选抗阻遏的高产菌株。在平板上添加4%的葡萄糖筛选得到的菌株产酶最高,滤纸酶活和CMC酶活分别可达4.3IU/mL和165.0IU/mL左右,比原始菌株的产酶量分别提高了33.5%和31.0%。在此基础上,通过单因素试验和L16(45)的正交试验得到了该菌株摇瓶液态发酵的最佳培养基组成为(g/L):蔗渣30,麸皮25,豆饼粉40,乳清粉10,KH2PO4 2.0,胰蛋白胨3.0,硫酸铵2.0,CaCl2 0.5,MgSO4 0.5;对该菌株摇瓶液态发酵的条件进行了优化,得到的最佳培养条件为:种龄48h,培养基的起始pH5.0~5.5,培养温度26～28℃,接种量10%,装液量25ml/250ml,摇床转速180r/min,培养144h。在此条件下最有利于菌体产酶,β-葡萄糖苷酶活、滤纸酶活和CMC酶活最高可分别达到4.5IU/mL、10.1IU/mL、294.6IU/mL。
对酶学性质进行了研究。β-葡萄糖苷酶活、滤纸酶活及CMC酶活的最适pH值均在5.0左右,滤纸酶活和CMC酶活的最适反应温度为55℃,β-葡萄糖苷酶的最适反应温度为50℃。当酶液在低于50℃,环境pH在5.0左右时保存可以较好的保持酶活力。Zn2+、Mg2+、Na+、Ca2+、Mn2+等对酶活有一定的激活作用,不同浓度的酒精可以不同程度的抑制酶活,且抑制程度随酒精浓度的升高而增加,随温度的升高而加强。
蔗渣经碱与微波联合处理后纤维素的相对含量提高了11.0%,并且比未处理的蔗渣更易被纤维素酶水解糖化。研究了经过预处理的蔗渣酶解的最佳条件为:纤维素酶用量8-10FPU/mL,底物浓度2%,反应温度55℃,反应体系pH4.8,并适当添加一定浓度的表面活性剂Tween-80,在此条件下,得到的最终糖化率为35%左右。酒精对酶解糖化反应的抑制作用表现为:一方面,随着酒精浓度的升高,抑制作用逐步加强;另一方面,温度越高,酒精的抑制作用也越显著。
Currently, the research of using the natural fiber raw material to produce fuel alcohol has been paid more and more attention because of the aggravating of energy crisis. At the same time, the key point of this research is how to reduce the cost of cellulase. The optimal culture conditions of the submerged fermentation of the mutant strain Trichoderma reesei WX-112 were studied. The pretreated bagasse was used to produce cellulase as main materials replacing pure cellulose so as to alleviate the pollution of enviroment and reduce the cost of cellulase effectively.
The bagasse pretreated with the metheds such as smash, acids hydrolysis, alkalis hydrolysis, microwave and EM were used to produce cellulase by the mutant strain Trichoderma reesei WX-112 submerged fermentation. The result of single factor test showed: bagasse pretreated with NaOH(1%w/v) was better for producing cellulase. Based on this, the optimal conditions of pretreatement of bagasse gained by orthogonal experiment were as follows: alkali with 0.30 mol/L NaOH, the microwave radiation with power 160W, 5 minutes. Under these conditions, the cellulase yield can get the highest increase per unit in the view of energy consumption. The results showed that the maximum cellulase yield using pretreated bagasses as raw material was higher than that of using non-pretreated bagasses. The highest yield of FPA and CMCase reached 3.54IU/mL and 134.8 IU/mL from1.73IU/mL and 60.2IU/mL after 5 days’culturing, which increased by 104.6% and 123.4% respectively.
Based on the theory of glucose repression, the anti-repression strains were obtained by using agar plate screening techniques, which can synthenize cellulase when high concentation glucose exists. The strain can produce more cellulase than others by submerged fermentation , which was screened from the plate containing 4% glucose. The yield of FPA and CMCase reached 4.3IU/mL and 165.0IU/mL, which increased by 33.5% and 31.0% respectively. Then, the optimal culture conditions of this screening strain were studied by single factor test and L16(45) orthogonal experiment. The optimal fermentation medium was determined (g/L): bagasse 30, wheat bran 25, bean cake flour 40, whey powder 10, KH2PO4 2.0, tryptone 3.0, (NH4)2SO4 2.0, CaCl2 0.5, MgSO4 0.5. The sutiable fermentation conditions: plants age was 48h, the initial pH of culture medium was 5.0-5.5, the culture temperature was 26-28℃, the inoculum size was 10%, volume of medium was 25ml/250ml and the speed of shaker was 180r/min, culturing 144h. Under these conditions, the highest yield ofβ-glucosidase, FPA and CMCase reached 4.5IU/mL, 10.1IU/mL and 294.6IU/mL respectively.
The crude enzyme properties were investigated preliminarily. The optimum pH ofβ-glucosidase, FPA and CMCase were all 5.0 and the temperature were 50-55℃. The stable pH ranger, the thermal stability were obtained at the same time. Influences on enzymatic activity of many kinds of ions were studied. Zn2+, Mg2+, Na+, Ca2+, Mn2+ and so on could promoted its catalytic activity significantly. At last, the effects of alcohol on cellulase production were investigated. The results showed: alcohol with different concentration could all restrained the activity of cellulase, and this kind of effect would be strengthened with the increasing of tempreature and the concentration of alcohol.
The cellulose content of bagasse pretreated by alkali and microwave increased by 11.0%, which could be hydrolysised easier than non-pretreated bagasse. The optimal enzymatic hydrolysis conditions were determined: 8-10FPU cellulase/mL, the substrate concentration 2%, the reaction temperature 55℃, pH4.8. Then, the final hydrolysis efficiency could reach to 35%. Alcohol could restrained the enzymatic hydrolysis of bagasse: the influence would be strengthened with the increasing of tempreature and the concentration of alcohol.
引文
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