高产纤维分解酶黑曲霉诱变选育与发酵条件优化
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摘要
本试验旨在研究高产纤维分解酶黑曲霉诱变选育与发酵条件优化。通过对黑曲霉X1诱变选育(紫外、硫酸二乙酯、紫外-硫酸二乙酯复合诱变),获得产纤维分解酶能力强的突变菌黑曲霉,并对其产酶发酵条件和水解秸秆能力进行研究。结果表明,黑曲霉X1经紫外、硫酸二乙酯及紫外-硫酸二乙酯复合诱变,筛选出1株产酶能力高的突变菌黑曲霉X1U4-1;与原菌相比,突变菌滤纸酶活性提高了66.7%,β-葡萄糖苷酶活性提高了22.3%,木聚糖酶活性提高了3.8%,还原糖含量(12 h)提高了6.8%。黑曲霉X1U4-1最适发酵产酶条件为发酵时间72 h,玉米芯∶麸皮=3∶7,接种量1.2 mL,硫酸铵浓度4%;在此条件下,滤纸酶活性达到0.77 U/g,纤维素酶活性达到93.80 U/g,木聚糖酶活性达到9 383.18 U/g。本研究表明,采用紫外-硫酸二乙酯复合诱变可有效改善黑曲霉产酶性能和水解秸秆能力。
This experiment was conducted to study the mutagenesis breeding and optimal fermentation condition optimization of high yield fiber decomposition enzyme of Aspergillus niger. High cellulose decomposition enzyme of mutant strain was obtained through the mutagenesis breeding (ultraviolet,diethyl sulfate and ultraviolet-diethyl sulfate compound mutagenesis) of Aspergillus niger X1,and the fermentation conditions of enzyme production and the hydrolysis ability of strawwere studied. The results showed that a mutant strain of Aspergillus niger X1 U4-1 was selected through ultraviolet,diethyl sulfate and ultraviolet-diethyl sulfate compound mutagenesis of the original strain of Aspergillus niger X1; compared with the original strain,the filter paper enzyme activity increased by 66. 7%,the β-glucosidase activity increased by 22. 3%,the xylanase activity increased by 3.8% and the reducing sugar content (12 h) increased by 6.8% in mutant strain. The optimization of fermentation condition of Aspergillus niger X1 U4-1 was that fermentation time was 84 h, corn kernel ∶ bran = 3:7,inoculation quantity was 1.2 mL,ammonium sulphate concentration was 4%; in this condition,the filter paper enzyme activity reached 0.77 U/g,cellulase activity reached 93.80 U/g,xylanase activity reached 9 383.18 U/g. This study shows that the mutagenesis of ultraviolet-diethyl sulfuric acid is effective in mutagenesis breeding of Aspergillus niger,thus to improve enzyme producing ability and strawhydrolysis capacity.
This experiment was conducted to study the mutagenesis breeding and optimal fermentation condition optimization of high yield fiber decomposition enzyme of Aspergillus niger. High cellulose decomposition enzyme of mutant strain was obtained through the mutagenesis breeding (ultraviolet,diethyl sulfate and ultraviolet-diethyl sulfate compound mutagenesis) of Aspergillus niger X1,and the fermentation conditions of enzyme production and the hydrolysis ability of strawwere studied. The results showed that a mutant strain of Aspergillus niger X1 U4-1 was selected through ultraviolet,diethyl sulfate and ultraviolet-diethyl sulfate compound mutagenesis of the original strain of Aspergillus niger X1; compared with the original strain,the filter paper enzyme activity increased by 66. 7%,the β-glucosidase activity increased by 22. 3%,the xylanase activity increased by 3.8% and the reducing sugar content (12 h) increased by 6.8% in mutant strain. The optimization of fermentation condition of Aspergillus niger X1 U4-1 was that fermentation time was 84 h, corn kernel ∶ bran = 3:7,inoculation quantity was 1.2 mL,ammonium sulphate concentration was 4%; in this condition,the filter paper enzyme activity reached 0.77 U/g,cellulase activity reached 93.80 U/g,xylanase activity reached 9 383.18 U/g. This study shows that the mutagenesis of ultraviolet-diethyl sulfuric acid is effective in mutagenesis breeding of Aspergillus niger,thus to improve enzyme producing ability and strawhydrolysis capacity.
引文
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[11]陈英连.玉米秸秆蛋白发酵饲料中木质纤维素酶高产菌株的选育及应用[D].硕士学位论文.青岛:中国海洋大学,2014:1-92.
[12]刘明启,关荣发,陈文伟.黑曲霉固体发酵产木聚糖酶的响应面优化设计及其酶学性质的研究[J].农业生物技术学报,2010,18(1):52-60.
[13]黄丹梅,宋育阳,刘延琳,等.产果胶酶黑曲霉的UV诱变及基于葡萄皮渣的固态发酵[J].食品工业科技,2017,38(17):125-129,162.
[14]张谦,贾佳,林智,等.产脂肪酶黑曲霉摇瓶发酵条件优化研究[J].生物技术通报,2015,31(12):227-233.
[15]吴升山,倪辉,肖安风,等.黑曲霉DB056产柚苷酶发酵条件初步优化[J].微生物学通报,2010,37(9):1305-1311.
[2]SHRIVASTAVA B,NANDAL P,SHARMA A,et al.Solid state bioconversion of w heat straw into digestible and nutritive ruminant feed by G anoderma sp.rckk02[J].Bioresource Technology,2012,107:347-351.
[3]陈仕伟.黑曲霉、绿色木霉的诱变选育及其在秸秆腐熟剂中的应用初探[D].硕士学位论文.武汉:华中农业大学,2013:1-59.
[4]王春丽,武改红,陈畅,等.黑曲霉原生质体诱变选育β-葡萄糖苷酶高产菌株[J].生物工程学报,2009,25(12):1921-1926.
[5]陈娜,付传颖,张玉新,等.黑曲霉高产β-葡萄糖苷酶菌株的诱变、筛选及发酵条件优化[J].基因组学与应用生物学,2016,35(4):901-906.
[6]JAMAL P,TIJANI R I D,ALAM M Z,et al.Effect of operational parameters on solid state fermentation of cassava peel to an enriched animal feed[J].Journal of Applied Sciences,2012,12(11):1166-1170.
[7]颜玉兰.产木聚糖酶木霉菌株固态发酵条件研究[D].硕士学位论文.南昌:江西农业大学,2012:1-38.
[8]刘春芬.纤维素酶高产菌株的选育及酶的分离纯化研究[D].硕士学位论文.重庆:西南农业大学,2005:1-53.
[9]许志.高产纤维素酶菌株选育、分子鉴定及发酵工艺研究[D].硕士学位论文.长沙:中南林业科技大学,2012:1-64.
[10]董桓.纤维素酶和半纤维素酶高产菌株的筛选[D].硕士学位论文.保定:河北大学,2014:1-63.
[11]陈英连.玉米秸秆蛋白发酵饲料中木质纤维素酶高产菌株的选育及应用[D].硕士学位论文.青岛:中国海洋大学,2014:1-92.
[12]刘明启,关荣发,陈文伟.黑曲霉固体发酵产木聚糖酶的响应面优化设计及其酶学性质的研究[J].农业生物技术学报,2010,18(1):52-60.
[13]黄丹梅,宋育阳,刘延琳,等.产果胶酶黑曲霉的UV诱变及基于葡萄皮渣的固态发酵[J].食品工业科技,2017,38(17):125-129,162.
[14]张谦,贾佳,林智,等.产脂肪酶黑曲霉摇瓶发酵条件优化研究[J].生物技术通报,2015,31(12):227-233.
[15]吴升山,倪辉,肖安风,等.黑曲霉DB056产柚苷酶发酵条件初步优化[J].微生物学通报,2010,37(9):1305-1311.
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