纤维素酶产生菌的抗阻遏选育及其产酶条件优化
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摘要
纤维素酶是一种多组分复合酶系,一般由内切-β-1,4-葡聚糖苷酶、外切-β-1,4-葡聚糖苷酶和β-1,4-葡萄糖苷酶三种组分构成。纤维素酶自被发现以来就受到众多学者的青睐,主要是纤维素酶具有非常广泛的应用领域,如食品工业、纺织工业、畜牧业、能源等,但生产成本高限制了纤维素酶的工业化进程。提高纤维素酶产生菌的产酶能力和优化生产菌的产酶条件是提高酶产量、降低酶成本的重要途径。
本文从自然界筛选产酶能力较高的野生菌,通过紫外、微波诱变结合阻遏剂筛选对其进行抗阻遏选育,获得抗阻遏效能和发酵产酶能力提高的变异菌株,利用响应面法对变异菌株的发酵条件进行优化,探讨了纤维素酶的部分酶学性质。本文的主要工作内容和研究结果如下：
(1)从沂蒙山区的灌木枯枝、腐土中采样,用羧甲基纤维素刚果红培养基初筛,然后通过摇瓶发酵测定各菌株的酶活进行复筛,筛选出一株CMCA和FPA相对较高的菌株,通过菌落形态、菌丝和孢子显微形态观察,依据《真菌鉴定手册》和《普通真菌学》等书籍的参考,将其初步鉴定为绿色木霉(T. viride Persex Fx NS90)。
(2)分别考察了不同浓度阻遏剂甘油、纤维二糖、葡萄糖对绿色木霉生长和产酶的影响,结果表明：对菌体生长,在4%浓度范围内,甘油、葡萄糖、纤维二糖的浓度越高,菌体量也越大。对发酵产酶,当甘油在0.4%时能促进酶的生产,而在2%时阻遏酶的分泌；纤维二糖对酶的发酵没有呈现明显的影响；葡萄糖除0.4%外,其他浓度均对酶的生产有阻遏作用。经紫外照射90s,微波辐射60s,选育到的突变株相对于原始菌株在相同浓度的阻遏剂下其CMCA和FPA分别提高了41%和44.95%。突变株在生长过程中,菌落由黄色逐渐变为绿色,呈现黄色孢子。
(3)采用响应面分析法优化绿色木霉变异菌株UW-Ⅲ的发酵产酶条件。根据模型预测和实验验证,得到绿色木霉变异菌株UW-Ⅲ产酶的响应面模型优化发酵条件为微晶纤维素、麸皮和发酵时间分别为6.1 g/L、8.5 g/L和96 h,且CMC酶活达362.523 U/mL
(4)研究了绿色木霉纤维素酶的部分酶学性质,包括最适温度、最适pH、温度稳定性、pH稳定性以及甘油、葡萄糖、纤维二糖的影响等,结果显示：绿色木霉纤维素酶的最适反应温度是50℃,最适pH是5.0左右,酶具有相对较好的温度稳定性和pH稳定性；纤维二糖在低添加量下对绿色木霉纤维素酶活具有促进作用,葡萄糖对纤维素酶的抑制作用明显。
Cellulase is a multi-component enzyme complex, which is generally composed of endo-β-1,4-glucanase, exo-(3-1,4-glucanase andβ-1,4-glucosidase. Cellulose is interested by many scholars, mainly because it has a very wide range of applications, such as food industry, textile industry, animal husbandry, energy and so on. The industrialization process of cellulase is limited by its high production costs. The important ways to raise enzyme yield and reduce enzyme costs are increasing of cellulase producing capability and optimizing of fermentation conditions.
In this study, a higher yield cellulase-producting wild strain was screened. Then through UV and microwave irradiation, combining with repressor agent screening, a cellulase-producing mutant was bred, which had higher ability of anti-repressor and enzyme production. Subsequently, the fermentation conditions of mutant were optimized by response surface methodology and parts of enzymatic properties of cellulase was discussed. The main contents and results of this thesis are as follows:
Samples were collected from dead branches and rotten soil in Yimeng. A strain of high CMCA and FPA was screened by carboxymethylcellulose congo red medium and fermentation culture. By observing colony morphology, microscopic morphology of mycelium and spores, and based on "Fungal Identification Manual", "ordinary Mycology", it was identified as Trichoderma viride (T. Viride Persex Fx NS90).
The effect of different concentrations of glycerol, glucose, cellobiose on Trichoderma viride growth and enzyme production was studied. The results show that, in the concentration range of 4%, the biomass of strain growing showed positive correlation to the concentrations of glycerol, glucose, cellobiose in the medium.When the concentration of glycerol in the medium was 0.4%, the production of enzyme was promoted, but the concentration was 2%, the production was repressed. Cellobiose did not show significant effects on the enzyme production.Except the 0.4% concentration, other concentrations of glucose all repress the enzyme production.After 90s UV radiation and 60s microwave treatment, compared to the original strain in the same concentration of repressor, the CMCA and FPA of the mutant produced had increased 41% and 44.95% respectively. In the growth process of the mutant strain, the color of mutant colony changed from yellow to green, and the spores presented yellow during the cells culture.
The fermentation conditions of mutant UW-Ⅲwas optimizated by response surface analysis. According to the model prediction and experimental verification, response surface model for enzyme production fermentation conditions of mutant UW-Ⅲwere optimized that microcrystalline cellulose, wheat bran and fermentation time were the value was 6.1 g/L,8.5 g/L and 96 h, and the CMCA was up to 362.523 U/mL.
Parts of the enzymatic properties, including optimum temperature, optimum pH, temperature stability, pH stability and the effect of glycerol, glucose, cellobiose addition was studied. The results showed that:the optimum temperature of cellulase was 50℃. The optimum pH was around 5.0. Cellulose has a relatively good temperature stability and pH stability. Under the low amount of addition, cellobiose has positive effects on cellulose. Glucose has strong inhibition to cellulase.
本文从自然界筛选产酶能力较高的野生菌,通过紫外、微波诱变结合阻遏剂筛选对其进行抗阻遏选育,获得抗阻遏效能和发酵产酶能力提高的变异菌株,利用响应面法对变异菌株的发酵条件进行优化,探讨了纤维素酶的部分酶学性质。本文的主要工作内容和研究结果如下：
(1)从沂蒙山区的灌木枯枝、腐土中采样,用羧甲基纤维素刚果红培养基初筛,然后通过摇瓶发酵测定各菌株的酶活进行复筛,筛选出一株CMCA和FPA相对较高的菌株,通过菌落形态、菌丝和孢子显微形态观察,依据《真菌鉴定手册》和《普通真菌学》等书籍的参考,将其初步鉴定为绿色木霉(T. viride Persex Fx NS90)。
(2)分别考察了不同浓度阻遏剂甘油、纤维二糖、葡萄糖对绿色木霉生长和产酶的影响,结果表明：对菌体生长,在4%浓度范围内,甘油、葡萄糖、纤维二糖的浓度越高,菌体量也越大。对发酵产酶,当甘油在0.4%时能促进酶的生产,而在2%时阻遏酶的分泌；纤维二糖对酶的发酵没有呈现明显的影响；葡萄糖除0.4%外,其他浓度均对酶的生产有阻遏作用。经紫外照射90s,微波辐射60s,选育到的突变株相对于原始菌株在相同浓度的阻遏剂下其CMCA和FPA分别提高了41%和44.95%。突变株在生长过程中,菌落由黄色逐渐变为绿色,呈现黄色孢子。
(3)采用响应面分析法优化绿色木霉变异菌株UW-Ⅲ的发酵产酶条件。根据模型预测和实验验证,得到绿色木霉变异菌株UW-Ⅲ产酶的响应面模型优化发酵条件为微晶纤维素、麸皮和发酵时间分别为6.1 g/L、8.5 g/L和96 h,且CMC酶活达362.523 U/mL
(4)研究了绿色木霉纤维素酶的部分酶学性质,包括最适温度、最适pH、温度稳定性、pH稳定性以及甘油、葡萄糖、纤维二糖的影响等,结果显示：绿色木霉纤维素酶的最适反应温度是50℃,最适pH是5.0左右,酶具有相对较好的温度稳定性和pH稳定性；纤维二糖在低添加量下对绿色木霉纤维素酶活具有促进作用,葡萄糖对纤维素酶的抑制作用明显。
Cellulase is a multi-component enzyme complex, which is generally composed of endo-β-1,4-glucanase, exo-(3-1,4-glucanase andβ-1,4-glucosidase. Cellulose is interested by many scholars, mainly because it has a very wide range of applications, such as food industry, textile industry, animal husbandry, energy and so on. The industrialization process of cellulase is limited by its high production costs. The important ways to raise enzyme yield and reduce enzyme costs are increasing of cellulase producing capability and optimizing of fermentation conditions.
In this study, a higher yield cellulase-producting wild strain was screened. Then through UV and microwave irradiation, combining with repressor agent screening, a cellulase-producing mutant was bred, which had higher ability of anti-repressor and enzyme production. Subsequently, the fermentation conditions of mutant were optimized by response surface methodology and parts of enzymatic properties of cellulase was discussed. The main contents and results of this thesis are as follows:
Samples were collected from dead branches and rotten soil in Yimeng. A strain of high CMCA and FPA was screened by carboxymethylcellulose congo red medium and fermentation culture. By observing colony morphology, microscopic morphology of mycelium and spores, and based on "Fungal Identification Manual", "ordinary Mycology", it was identified as Trichoderma viride (T. Viride Persex Fx NS90).
The effect of different concentrations of glycerol, glucose, cellobiose on Trichoderma viride growth and enzyme production was studied. The results show that, in the concentration range of 4%, the biomass of strain growing showed positive correlation to the concentrations of glycerol, glucose, cellobiose in the medium.When the concentration of glycerol in the medium was 0.4%, the production of enzyme was promoted, but the concentration was 2%, the production was repressed. Cellobiose did not show significant effects on the enzyme production.Except the 0.4% concentration, other concentrations of glucose all repress the enzyme production.After 90s UV radiation and 60s microwave treatment, compared to the original strain in the same concentration of repressor, the CMCA and FPA of the mutant produced had increased 41% and 44.95% respectively. In the growth process of the mutant strain, the color of mutant colony changed from yellow to green, and the spores presented yellow during the cells culture.
The fermentation conditions of mutant UW-Ⅲwas optimizated by response surface analysis. According to the model prediction and experimental verification, response surface model for enzyme production fermentation conditions of mutant UW-Ⅲwere optimized that microcrystalline cellulose, wheat bran and fermentation time were the value was 6.1 g/L,8.5 g/L and 96 h, and the CMCA was up to 362.523 U/mL.
Parts of the enzymatic properties, including optimum temperature, optimum pH, temperature stability, pH stability and the effect of glycerol, glucose, cellobiose addition was studied. The results showed that:the optimum temperature of cellulase was 50℃. The optimum pH was around 5.0. Cellulose has a relatively good temperature stability and pH stability. Under the low amount of addition, cellobiose has positive effects on cellulose. Glucose has strong inhibition to cellulase.
引文
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