纤维素酶产生菌Trichoderma viride L4C发酵产酶条件及诱变的研究

发布时间：2018-05-12 06:26

  本文选题：绿色木霉 + 液态发酵　； 参考：《甘肃农业大学》2015年硕士论文

【摘要】：本论文以实验室保存的产纤维素酶菌株绿色木霉(Trichoderma viride)L4C为试验材料,采用单因素试验和正交试验方法优化该菌株液体发酵产酶条件;采用紫外诱变、亚硝基胍诱变及复合诱变方法,经过纤维素水解圈的初筛及DNS法测酶活选育出产酶活力较高且遗传性稳定的菌株,结果如下:1、通过对菌株绿色木霉L4C液体产酶发酵条件进行单因素的优化试验,得到产酶培养的优化条件:稻草和纤维素粉复合物为最佳碳源,硫酸铵为最适氮源,接种量为10%,培养时间为4 d,培养温度为28℃,培养基初始pH为4.5。在单因素的基础上,通过多因素正交试验得到最佳产酶条件:接种量为15%,培养时间为5 d,培养温度为28℃,初始pH为5.0。在最佳产酶条件下,CMCase为921.56U/mL,FPA为745.02 U/mL,半纤维素酶活为556.71 U/mL,分别比优化前基础培养基产酶提高了56.7%,65.0%和66.8%。2、通过紫外线(UV)诱变试验,明确了紫外诱变的适宜条件:15 w紫外灯,距离30 cm,照射处理时间300 s,孢子悬浮液稀释度为10-6。此条件下的诱变效应为:致死率为85.0%。诱变后,获得一株高产纤维素酶菌株-绿色木霉Z15(Trichoderma viride.Z15)。摇瓶发酵后,CMCase为1196.21 U/mL,FPA为863.56U/mL,其产酶能力分别较出发菌株提高了1.30倍和1.16倍。传代试验证明,该菌株具有较好的遗传稳定性。3、通过亚硝基胍(NTG)诱变试验,明确了NTG诱变的适宜条件:NTG 0.6mg/mL,处理时间为50 min。该条件下的诱变效应为:孢子致死率为80.4%,正突变率8.01%。诱变后,获得一株高产纤维素酶菌株-绿色木霉N12(Trichoderma viride.N12)。摇瓶发酵后,CMCase为1410.02 U/mL,FPA为992.67U/mL,其产酶能力较出发菌株提高了18.5%和9.7%。此菌株具有较理想的遗传稳定性。4、通过复合诱变试验,确定了UV+NTG复合诱变的条件:NTG的最佳浓度为0.6 mg/mL,15 w紫外灯,距离30 cm,照射处理时间120 s。该条件下的诱变效应为:孢子致死率80.8%,正突变率8.76%。诱变后,获得一株高产纤维素酶菌株-绿色木霉ZN4(Trichoderma viride.ZN4)。摇瓶发酵后,CMCase为1678.78U/mL,FPA为1031.56 U/mL,其产酶能力较出发菌株提高了19.1%和12.3%。经传代验证,该菌株具有较理想的遗传稳定性和产酶潜力。5、利用出发菌株绿色木霉L4C和诱变菌株ZN4发酵处理秸杆3d、4d、5d后,结果表明:诱变菌株的效果优于出发菌株,干物质中粗蛋白的提高率在5 d时达81.69%,粗纤维的降解率在5 d时达39.98%。
[Abstract]:The optimum conditions for producing enzyme were studied by means of single factor test and orthogonal test . The optimum conditions were as follows : 1 . The optimum conditions for producing enzyme were obtained by means of orthogonal experiments . The optimum conditions for producing enzyme were as follows : 1 . The optimum conditions were as follows : 1 . Under optimum enzyme production conditions , CMCase was 921.56U / mL , and the initial pH was 5.0 . Under the condition of optimum enzyme production , the optimum conditions were as follows : 1 . The optimum conditions for producing enzyme were as follows : 1 . Under the optimum enzyme production condition , the optimum conditions were as follows : 1 . The optimum conditions for producing enzyme were as follows : The lethal rate was 85.0 % . After the mutation , a high yield cellulase strain , Trichoderma viride . Z15 was obtained . After shake flask fermentation , CMCase was 1196 . 21 U / mL , FPA was 863.56U / mL , and its enzyme - producing ability increased by 1.30 and 1.16 times respectively . After fermentation , CMCase was 1410 . 02 U / mL , FPA was 992.67U / mL . The yield of NTG was improved by 18.5 % and 9.7 % . The optimum concentration of NTG was 0.6 mg / mL , 15 w UV lamp , 30 cm distance and 120 s irradiation treatment time . The mutagenic effect of NTG was 80.8 % , positive mutation rate 8.76 % . After mutagenesis , a high yield cellulase strain , Trichoderma viride . ZN4 was obtained . After fermentation of shaking flask , CMCase was 1678.78U / mL , FPA was 1031 . 56 U / mL , and its enzyme - producing ability was improved by 19.1 % and 12.3 % . The results showed that the strain had better genetic stability and enzyme - producing potential .

【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816

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