纤维素酶高产菌的诱变选育及其酶学性质的研究
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摘要
纤维素类物质是自然界中含量最丰富、最廉价的可再生资源。据测算,地球表面每年因光合作用合成的干物质高达1500亿吨的干物质,其中纤维素、半纤维素的总量约为850亿吨,而纤维素的含量约占总干物质的6%,达220亿吨。可见,纤维素是自然界中取之不尽、用之不竭的可再生资源。纤维素酶是指能催化水解β-1.4葡萄糖苷键生成纤维二糖和葡萄糖的一类酶的总称,纤维素酶不是单一的酶,是由多个水解酶组成的一类复杂酶系。目前,在纤维素的分解转化的研究中,对纤维素的研究主要集中在纤维素水解转化的微生物的诱变、.筛选上,纤维素酶活性的提高。本论文以绿色木霉AS3.3711作为出发株,分别利用紫外线、快中子、γ射线、硫酸二乙酯和亚硝酸钠五种诱变因子,诱变选育纤维素酶高产的菌株,并且对其酶学性质进行了研究,结果如下：
1.采用五种不同的诱变方法对绿色木霉AS3.3711进行诱变选育,通过诱变致死率在70%～80%范围内作为筛选依据,试验结果如下：
(1)亚硝酸钠诱变：在菌悬液中分别加入醋酸缓冲液以及0.1mol/L, 0.05 mol/L, 0.03 mol/L, 0.02 mol/L等不同浓度的亚硝酸钠溶液,室温下分别保温5,10,15,20min,稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵复筛,得到亚硝酸钠诱变的最佳浓度为0.1mol/L,诱变时间为20min,突变株的CMC酶活是出发菌株酶活的1.74倍。
(2)硫酸二乙酯诱变处理：在硫酸二乙酯与菌液的体积比为1%,0.5%,0.3%,0.2%,30℃条件下分别振荡处理20,30,40,60min。稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵复筛,得到硫酸二乙酯诱变的最佳体积比为1%,诱变时间为40min,突变株的CMC酶活为出发菌株的2.8倍。
(3)紫外线辐照诱变：分别在256nm和354nm处,垂直距离为20,25,30cm,辐照处理60,120,180,240,300,360,600s,稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵复筛,得出紫外线最佳诱变条件：紫外波长为354nm,垂直距离30cm,照射时间240s,突变株的CMC酶活为出发菌株的2.64倍。
(4)γ-射线诱变：以照射剂量率为0.4Gy/min,照射时间为2.5,3.75,5,6.25,10min,将处理后的菌液稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵进行复筛,酶活提高最多的为出发菌株的1.29倍。
(5)快中子辐照诱变：采用剂量率为0.002Gy/s,分别照射5,10,20,30,40min,即0.6,1.2,2.4,3.6,4.8Gy 5个剂量进行处理,稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵复筛,得出当辐照剂量为1.2Gy时,正突变率为14.3%,筛选和连续传代培养,获得了稳定性较好的正突变株Fn10-1。突变株Fn10-1的羧甲基纤维素酶活(CMC酶活)可达914.80 U/mL,比出发菌株提高了30倍,滤纸酶活(FPA酶活)为633.63 U/mL,比出发菌株提高了4倍。
2.采用不同饱和度硫酸铵溶液对粗酶液进行盐析沉淀,当硫酸铵饱和度为80%时,盐析沉淀获得的纤维素酶的酶活力为原酶活的97.8%,即894.6U/mL。
3.对突变株Fn10-1所产纤维素酶进行酶学性质研究,包括酶促反应的最适pH值、pH稳定性、最适温度、热稳定性、激活剂和抑制剂。结果如下：纤维素酶的最适pH值为7.6,在pH值5.6-7.6之间有较好的稳定性,剩余酶活为原酶活的91.%；最适温度为70℃,并且在50℃以下,保温400min,剩余酶活为保温前的92%; Mg2+、Co2+、Na+、Mn2+、Fe2+、Al3+对该酶具有明显的激活作用,而Ba2+、Ag2+、Cu2+、K+对该酶具有明显的抑制作用。
Cellulose is rich in nature as the content of renewable resources and cheap. According to estimates, The cellulose material in synthetic substances of photosynthesis accounted for about 6%,that can reach 200 million tons on the earth's surface every year.At the moment,the create capacity of dry matter is reach as high as 15 million tons every year in the world. The amount of cellulose and hemicellulose about 850 million tons.So The cellulose is inexhaustible in nature. In the present for the transformation of the decomposition of cellulose research, mainly involves the development of cellulose microorganism resources, cellulose enzyme activity enhancement and optimization of reaction conditions. Cellulase is a kind of very highly active biological catalyst, it is the hydrolysis of cellulolytic enzymes generated a group of glucose non-functional. It consists of three components enzyme synergy:(])endo-1,4-β-D-glucanases (EQC1orCMC), (2)exo-1,4-β-D-glucanases(CBH or Cx),(3) 1,4-β-D-glucosidases (BG).
1. This study is using a hot issue in allusion to cellulose. To Trichoderma viride as the research object, Using physical and chemical respectively method for high-yielding cellulose enzyme activity mutagenesis breeding the degradation bacterium. Optimize enzyme production conditions, and study for enzymology character. Use five different mutation methods ,which is including fast neutron, ultraviolet rays, y-rays, diethyl sulfate, sodium nitrite, to Trichoderma viride AS3.3711 for mutagenesis breeding. Usually, after the mutation processing, the lethality and the radiation dosage has a linear positive correlation within the scope of certain. Through the test results:
(1) Sodium nitrite mutagenesis:During the fungus levitation liquid join acetic acid buffer fluid,and different concentrations of sodium nitrite solution with 0.1mol/L,0.05 mol/L,0.03 mol/L,0.02 mol/L. Respectively at room temperature heat preservation for 5,10,15,20min. The optimal concentration was 0.1mol/L and the optimal time is 20 min. The enzyme activity of CMC of mutations increased to 1.74 times.
(2) Diethyl sulfate mutagensis:The volume ratio of diethyl sulfate and mycete was 1%,0.5%,0.3%,0.2%. In 30℃conditions oscillation processing for 20,30,40,60min. The optimal volume ratio was 1% and the optimal time is 40 min. The enzyme activity of CMC of mutations increased to 2.8 times.
(3) Ultraviolet rays mutagensis:In 256nm and 354nm place respectively, the vertical distance to 20,25,30 cm, with irradiation treatment 60,120,180,240,300,360,600 s, The optimal wavelength was 354 nm, the optimal vertical distance was 30 cm and the optimal time was 240s. The enzyme activity of CMC of mutations increased to 2.64 times.
(4)γ-rays mutagensis:Use the Radiation dose rate 0.4 Gy/min and the irradiation time with 2.5,3.75,5,6.25, 10min, The enzyme activity of CMC of mutations only increased to 1.29 times.
(5) Fast neutron mutagensis:Use the Radiation dose rate 0.002 Gy/s and the irradiation time with 5,10,20, 30,40min, it meant 0.6,1.2,2.4,3.6,4.8Gy, The enzyme activity of carboxymethyl cellulose of Fn10-1 can reach 914.80 U/mL,that increased to 30 times than the primitive strain.
Use of Trichoderma viride AS3.3711 for fast neutron irradiation processing, to the hydrolysis circle of mutant strain on the congo red medium, whose diameter of the ratio of the colony, and cellulose enzyme as the basis, to obtain high mutation strain. Through the experiments found that low doses of fast neutron irradiation can greatly improve cellulose enzyme activity. The mutation rate is 14.3% when the irradiation dose for 1.2 Gy. We got the stability good of mutation Fn10-1 by means of screening and genetic stability experiment. The enzyme activity of carboxymethyl cellulose (enzyme activity of CMC) of Fn 10-1 can reach 914.80 U/mL,that increased to 30 times than the primitive strain. The enzyme activity of filter paper (enzyme activity of FPA) of Fn10-1 can reach 633.63 U/mL, that increased to 4 times than the primitive strain.
2. Through the ammonium sulfate grading precipitation method, the paper obtained the cellulose enzyme of Fn10-1 and measured this cellulose enzyme characteriztion. The enzymology character experiment including optimal pH value, stability, optimal temperature, thermal stability, activator and inhibitor. The results showed that determined 30% and 80% two saturation for grading precipitation gradient. First, joined solid ammonium sulphate to 30%, static overnighted, got the supernate after the centrifugation. And then joined solid ammonium sulphate to 80% again, static overnighted, got the precipitate after the centrifugation.
3.The precipitate was dissolved by phosphate buffer, namely for coarse enzyme liquid, to the enzymology character experiment subsequent. After the enzymology character experiment we got the results showed that its optimal pH value was 7.6, between 5.6 to 7.6, it had higer stability. Its optimal temperature was 70℃, And it had high stability when the temperrature was under 50℃. For this enzyme, Mg2+、Co2+、Na+、Mn2+、Fe2+ and Al3+ had obvious activation function, well, Ba2+、Ag2+、Cu2+ and K+ had inhibitory action on it.
1.采用五种不同的诱变方法对绿色木霉AS3.3711进行诱变选育,通过诱变致死率在70%～80%范围内作为筛选依据,试验结果如下：
(1)亚硝酸钠诱变：在菌悬液中分别加入醋酸缓冲液以及0.1mol/L, 0.05 mol/L, 0.03 mol/L, 0.02 mol/L等不同浓度的亚硝酸钠溶液,室温下分别保温5,10,15,20min,稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵复筛,得到亚硝酸钠诱变的最佳浓度为0.1mol/L,诱变时间为20min,突变株的CMC酶活是出发菌株酶活的1.74倍。
(2)硫酸二乙酯诱变处理：在硫酸二乙酯与菌液的体积比为1%,0.5%,0.3%,0.2%,30℃条件下分别振荡处理20,30,40,60min。稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵复筛,得到硫酸二乙酯诱变的最佳体积比为1%,诱变时间为40min,突变株的CMC酶活为出发菌株的2.8倍。
(3)紫外线辐照诱变：分别在256nm和354nm处,垂直距离为20,25,30cm,辐照处理60,120,180,240,300,360,600s,稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵复筛,得出紫外线最佳诱变条件：紫外波长为354nm,垂直距离30cm,照射时间240s,突变株的CMC酶活为出发菌株的2.64倍。
(4)γ-射线诱变：以照射剂量率为0.4Gy/min,照射时间为2.5,3.75,5,6.25,10min,将处理后的菌液稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵进行复筛,酶活提高最多的为出发菌株的1.29倍。
(5)快中子辐照诱变：采用剂量率为0.002Gy/s,分别照射5,10,20,30,40min,即0.6,1.2,2.4,3.6,4.8Gy 5个剂量进行处理,稀释涂布在刚果红筛选培养基上初筛,再以摇瓶发酵复筛,得出当辐照剂量为1.2Gy时,正突变率为14.3%,筛选和连续传代培养,获得了稳定性较好的正突变株Fn10-1。突变株Fn10-1的羧甲基纤维素酶活(CMC酶活)可达914.80 U/mL,比出发菌株提高了30倍,滤纸酶活(FPA酶活)为633.63 U/mL,比出发菌株提高了4倍。
2.采用不同饱和度硫酸铵溶液对粗酶液进行盐析沉淀,当硫酸铵饱和度为80%时,盐析沉淀获得的纤维素酶的酶活力为原酶活的97.8%,即894.6U/mL。
3.对突变株Fn10-1所产纤维素酶进行酶学性质研究,包括酶促反应的最适pH值、pH稳定性、最适温度、热稳定性、激活剂和抑制剂。结果如下：纤维素酶的最适pH值为7.6,在pH值5.6-7.6之间有较好的稳定性,剩余酶活为原酶活的91.%；最适温度为70℃,并且在50℃以下,保温400min,剩余酶活为保温前的92%; Mg2+、Co2+、Na+、Mn2+、Fe2+、Al3+对该酶具有明显的激活作用,而Ba2+、Ag2+、Cu2+、K+对该酶具有明显的抑制作用。
Cellulose is rich in nature as the content of renewable resources and cheap. According to estimates, The cellulose material in synthetic substances of photosynthesis accounted for about 6%,that can reach 200 million tons on the earth's surface every year.At the moment,the create capacity of dry matter is reach as high as 15 million tons every year in the world. The amount of cellulose and hemicellulose about 850 million tons.So The cellulose is inexhaustible in nature. In the present for the transformation of the decomposition of cellulose research, mainly involves the development of cellulose microorganism resources, cellulose enzyme activity enhancement and optimization of reaction conditions. Cellulase is a kind of very highly active biological catalyst, it is the hydrolysis of cellulolytic enzymes generated a group of glucose non-functional. It consists of three components enzyme synergy:(])endo-1,4-β-D-glucanases (EQC1orCMC), (2)exo-1,4-β-D-glucanases(CBH or Cx),(3) 1,4-β-D-glucosidases (BG).
1. This study is using a hot issue in allusion to cellulose. To Trichoderma viride as the research object, Using physical and chemical respectively method for high-yielding cellulose enzyme activity mutagenesis breeding the degradation bacterium. Optimize enzyme production conditions, and study for enzymology character. Use five different mutation methods ,which is including fast neutron, ultraviolet rays, y-rays, diethyl sulfate, sodium nitrite, to Trichoderma viride AS3.3711 for mutagenesis breeding. Usually, after the mutation processing, the lethality and the radiation dosage has a linear positive correlation within the scope of certain. Through the test results:
(1) Sodium nitrite mutagenesis:During the fungus levitation liquid join acetic acid buffer fluid,and different concentrations of sodium nitrite solution with 0.1mol/L,0.05 mol/L,0.03 mol/L,0.02 mol/L. Respectively at room temperature heat preservation for 5,10,15,20min. The optimal concentration was 0.1mol/L and the optimal time is 20 min. The enzyme activity of CMC of mutations increased to 1.74 times.
(2) Diethyl sulfate mutagensis:The volume ratio of diethyl sulfate and mycete was 1%,0.5%,0.3%,0.2%. In 30℃conditions oscillation processing for 20,30,40,60min. The optimal volume ratio was 1% and the optimal time is 40 min. The enzyme activity of CMC of mutations increased to 2.8 times.
(3) Ultraviolet rays mutagensis:In 256nm and 354nm place respectively, the vertical distance to 20,25,30 cm, with irradiation treatment 60,120,180,240,300,360,600 s, The optimal wavelength was 354 nm, the optimal vertical distance was 30 cm and the optimal time was 240s. The enzyme activity of CMC of mutations increased to 2.64 times.
(4)γ-rays mutagensis:Use the Radiation dose rate 0.4 Gy/min and the irradiation time with 2.5,3.75,5,6.25, 10min, The enzyme activity of CMC of mutations only increased to 1.29 times.
(5) Fast neutron mutagensis:Use the Radiation dose rate 0.002 Gy/s and the irradiation time with 5,10,20, 30,40min, it meant 0.6,1.2,2.4,3.6,4.8Gy, The enzyme activity of carboxymethyl cellulose of Fn10-1 can reach 914.80 U/mL,that increased to 30 times than the primitive strain.
Use of Trichoderma viride AS3.3711 for fast neutron irradiation processing, to the hydrolysis circle of mutant strain on the congo red medium, whose diameter of the ratio of the colony, and cellulose enzyme as the basis, to obtain high mutation strain. Through the experiments found that low doses of fast neutron irradiation can greatly improve cellulose enzyme activity. The mutation rate is 14.3% when the irradiation dose for 1.2 Gy. We got the stability good of mutation Fn10-1 by means of screening and genetic stability experiment. The enzyme activity of carboxymethyl cellulose (enzyme activity of CMC) of Fn 10-1 can reach 914.80 U/mL,that increased to 30 times than the primitive strain. The enzyme activity of filter paper (enzyme activity of FPA) of Fn10-1 can reach 633.63 U/mL, that increased to 4 times than the primitive strain.
2. Through the ammonium sulfate grading precipitation method, the paper obtained the cellulose enzyme of Fn10-1 and measured this cellulose enzyme characteriztion. The enzymology character experiment including optimal pH value, stability, optimal temperature, thermal stability, activator and inhibitor. The results showed that determined 30% and 80% two saturation for grading precipitation gradient. First, joined solid ammonium sulphate to 30%, static overnighted, got the supernate after the centrifugation. And then joined solid ammonium sulphate to 80% again, static overnighted, got the precipitate after the centrifugation.
3.The precipitate was dissolved by phosphate buffer, namely for coarse enzyme liquid, to the enzymology character experiment subsequent. After the enzymology character experiment we got the results showed that its optimal pH value was 7.6, between 5.6 to 7.6, it had higer stability. Its optimal temperature was 70℃, And it had high stability when the temperrature was under 50℃. For this enzyme, Mg2+、Co2+、Na+、Mn2+、Fe2+ and Al3+ had obvious activation function, well, Ba2+、Ag2+、Cu2+ and K+ had inhibitory action on it.
引文
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