匍枝根霉纤维素酶高产菌株的诱变选育及发酵优化
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摘要
为提高纤维素酶产量,以匍枝根霉TP-02为出发菌株,通过诱变选育纤维素酶高产突变株,并研究碳源、氮源以及氨基酸等物质对菌株产酶能力的影响.经紫外和甲基磺酸乙酯复合诱变后筛选得到一株高产菌株TZ-03,其滤纸酶活(FPA)为4.96IU/mL,较TP-02提高了37.8%.优化后最佳发酵培养基为:微晶纤维素20g/L,鱼粉蛋白胨10g/L,麸皮浸出汁2.5%,CaCl_22g/L,MgSO_4·7H_2O 4g/L,KH_2PO_43g/L,谷氨酰胺1g/L,PEG-4000 0.25g/L,Tween 80 200μL/L,微量元素液1mL/L.此条件下TZ-03的FPA酶活高达11.58IU/mL.通过对发酵过程中溶氧、pH和补料条件的控制,进行10L发酵罐放大实验,最终使该突变株的FPA酶活在84h达到峰值21.32IU/mL,与摇瓶发酵相比提高了89.8%.β-葡萄糖苷酶(BG)、内切酶(EG)和外切酶(CBH)的酶活峰值分别为41.0IU/mL、26.76IU/mL和16.94IU/mL.通过诱变及发酵优化成功提高了纤维素酶产量,为纤维素酶的工业化应用奠定了一定的基础.
To improve the yield of cellulase,the parent strain RhizopusstoloniferTP-02was mutagenized to obtain a high-yielding mutant.The effect of carbon sources,nitrogen sources and amino acid addition on the cellulase production was studied.Through the UV mutagenesis combined with ethyl methanesulfonate(EMS)mutagenesis,a high-yielding strain TZ-03was obtained.The filter paper activity(FPA)of the mutant was 4.96IU/mL(37.8%higher than that of TP-02).The optimal media were confirmed as follows:Avicel 20g/L,peptone 10g/L,wheat bran extract 2.5%,CaCl_22g/L,MgSO_4·7H_2O 4g/L,KH_2PO_43g/L,glutamine 1g/L,Tween 80 200μL/L,PEG-4000 0.25g/L,trace element solution1mL/L.In the above condition,the FPA of TZ-03peaked at 11.58IU/mL.The amplification experiment was carried out in 10Lfermentor with the control of dissolved oxygen,pH and feeding conditions in the fermentation process.The results showed that the maximum of FPA was 21.32IU/mL peaked at 84h(89.8%higher than that in the shake flask culture conditions).The highestβ-glycosidase(BG)activity,endoglucanase(EG)activity and cellobiohydrolase(CBH)activity were 41.06IU/mL,26.76IU/mL and16.94IU/mL,respectively.In this study,the yield of cellulase from Rhizopus Stolonifer TZ-03was effectively increased by the breeding of high-yielding mutant and optimization of fermentation conditions,laying a foundation for the industrial production and application of cellulase.
To improve the yield of cellulase,the parent strain RhizopusstoloniferTP-02was mutagenized to obtain a high-yielding mutant.The effect of carbon sources,nitrogen sources and amino acid addition on the cellulase production was studied.Through the UV mutagenesis combined with ethyl methanesulfonate(EMS)mutagenesis,a high-yielding strain TZ-03was obtained.The filter paper activity(FPA)of the mutant was 4.96IU/mL(37.8%higher than that of TP-02).The optimal media were confirmed as follows:Avicel 20g/L,peptone 10g/L,wheat bran extract 2.5%,CaCl_22g/L,MgSO_4·7H_2O 4g/L,KH_2PO_43g/L,glutamine 1g/L,Tween 80 200μL/L,PEG-4000 0.25g/L,trace element solution1mL/L.In the above condition,the FPA of TZ-03peaked at 11.58IU/mL.The amplification experiment was carried out in 10Lfermentor with the control of dissolved oxygen,pH and feeding conditions in the fermentation process.The results showed that the maximum of FPA was 21.32IU/mL peaked at 84h(89.8%higher than that in the shake flask culture conditions).The highestβ-glycosidase(BG)activity,endoglucanase(EG)activity and cellobiohydrolase(CBH)activity were 41.06IU/mL,26.76IU/mL and16.94IU/mL,respectively.In this study,the yield of cellulase from Rhizopus Stolonifer TZ-03was effectively increased by the breeding of high-yielding mutant and optimization of fermentation conditions,laying a foundation for the industrial production and application of cellulase.
引文
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[12]刘政,杨绍斌,宋小美,等.氨基酸类营养对微生物生长及活性的影响[J].黑龙江农业科学,2010,5:13-15.
[13]汪天虹,王春卉,高培基.纤维素酶纤维素吸附区的结构与功能[J].生物工程进展,2000,20(2):37-40.
[14]张丹.纤维素酶的研究进展及其展望[J].青海畜牧兽医杂志,2017,47(3):49-52.
[15]J G SHEWALE.Beta-Glucosidase:its role in cellulase synthesis and hydrolysis of cellulose[J].International Journal of Biochemistry,1982,14(6):435-443.
[16]J WEBER,F A AGBLEVOR.Microbubble fermentation of Trichoderma reesei for cellulase production[J].Process Biochemistry,2005,40(2):669-676.
[17]T JUHSZ,Z SZENGYEL,N SZIJRT,et al.Effect of pH on cellulase production of trichoderma reesei RUT C30[J].Applied Biochemistry&Biotechnology,2004,113(1-3):201-211.
[18]周若飞,汤斌,李松,等.匍枝根霉淀粉发酵产纤维素酶的条件研究[J].食品与发酵工业,2016(6):93-97.
[2]黄娟,包振伟.纤维素酶的应用研究进展[J].产业与科技论坛,2016(16):38-39.
[3]孙尚琛,南怀燕,王永刚,等.一株高效降解纤维素菌株的筛选及其产酶能力研究[J].中国微生态学杂志,2017(10):1 131-1 135.
[4] X Y ZHANG,L H ZI,X M GE,et al.Development of Trichoderma reesei mutants by combined mutagenesis and induction of cellulase by low-cost corn starch hydrolysate[J].Process Biochemistry,2017(54):96-101.
[5]张飞.利用人工锌指蛋白技术提高里氏木霉Rut-C30纤维素酶产量[D].大连:大连理工大学,2016.
[6]汤斌,许钟源,李松,等.匍枝根霉纤维素酶发酵条件优化及分批发酵动力学模型的构建[J].食品与发酵工业,2014,40(1):85-90.
[7] S SHAHBAZI,K ISPAREH,M KARIMI,et al.Gamma and UV radiation induced mutagenesis in trichoderma reesei to enhance cellulases enzyme activity[J].International Journal of Farming and Allied Sciences,2014,3(5):543-554.
[8]张秋卓,蔡伟民.纤维素酶高产菌株的复合交替诱变选育[J].工业微生物,2008,38(6):32-37.
[9]汤斌,潘海波,张庆庆,等.基于响应面法优化匍枝根霉TP-02液态发酵纤维素酶条件[J].工业微生物,2010,40(2):30-34.
[10]许志,曾柏全,宋睿.高产纤维素酶菌株筛选及诱变选育[J].中国农学通报,2011,27(24):108-111.
[11]张克旭,陈宁,张蓓.代谢控制发酵[M].北京:中国轻工业出版社,2005.
[12]刘政,杨绍斌,宋小美,等.氨基酸类营养对微生物生长及活性的影响[J].黑龙江农业科学,2010,5:13-15.
[13]汪天虹,王春卉,高培基.纤维素酶纤维素吸附区的结构与功能[J].生物工程进展,2000,20(2):37-40.
[14]张丹.纤维素酶的研究进展及其展望[J].青海畜牧兽医杂志,2017,47(3):49-52.
[15]J G SHEWALE.Beta-Glucosidase:its role in cellulase synthesis and hydrolysis of cellulose[J].International Journal of Biochemistry,1982,14(6):435-443.
[16]J WEBER,F A AGBLEVOR.Microbubble fermentation of Trichoderma reesei for cellulase production[J].Process Biochemistry,2005,40(2):669-676.
[17]T JUHSZ,Z SZENGYEL,N SZIJRT,et al.Effect of pH on cellulase production of trichoderma reesei RUT C30[J].Applied Biochemistry&Biotechnology,2004,113(1-3):201-211.
[18]周若飞,汤斌,李松,等.匍枝根霉淀粉发酵产纤维素酶的条件研究[J].食品与发酵工业,2016(6):93-97.