普鲁兰酶产生菌的诱变选育及其发酵工艺的优化
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摘要
为了提高普鲁兰酶产酶酶活,以GX-6为出发菌株,采用低能离子束修饰技术对其进行诱变,并通过响应面法优化其发酵培养基。结果表明,最佳离子束诱变参数为:注入能量10 keV、诱变剂量1×1015ions·cm~(-2)、诱变时间38 s,此条件下菌株正突变率比负突变率高,利用离子束诱变技术反复诱变,最终获得一株普鲁兰酶酶活较高且遗传性稳定的突变菌株GX-6-2,其酶活为2. 13 U·mL~(-1),较出发菌株酶活(0. 65 U·mL~(-1))提高了2. 28倍。由Plackett-Burmen试验分析得到影响普鲁兰酶酶活的3个显著因素分别是玉米淀粉、麦芽糖和吐温-80。通过响应面试验得到最佳发酵培养参数为:玉米淀粉56. 5g·L~(-1)、麦芽糖11. 5 g·L~(-1)、吐温-80 1. 0 mL·L~(-1)、黄豆饼粉25 g·L~(-1)、pH值7. 0、发酵温度37℃、接种量3%、发酵时间24 h、装液量50 mL、转速180 r·min~(-1),此条件下诱变菌株的酶活为2. 57 U·mL~(-1),较出发菌株酶活提高了2. 95倍。对突变菌株的发酵特性进行初步研究发现,发酵培养24 h时,突变菌株酶活达到2. 67 U·mL~(-1),较出发菌株提高了3. 11倍。本研究结果为利用低能离子束修饰技术诱变选育普鲁兰酶产生菌提供了一定的理论参考。
In order to improve the enzymatic activity of pullulanase,the original strain named GX-6 was mutated with low energy ion beam implantation,and the fermentation medium was optimized by response surface methodology. The results showed that the strain had a higher positive rate when the implantation energy was 10 keV and the dose of N+implantation was 1 × 1015 ions·cm~(-2) with implantation time of 38 s. A high-yield and high genetic stability mutant strain of GX6-2 was obtained under this mutation dose through repeated mutagenesis by using ion beam and the activity was2. 28 times higher,up to 2. 13 U·mL~(-1),than original strain( 0. 65 U·mL~(-1)). From the Plackett-Burmen test,three significant factors affecting the enzyme activity of pullulase were corn starch,maltose and Tween-80. The optimized fermentation parameters was shown as follows: corn starch 56. 5 g·L~(-1),maltose 11. 5 g·L~(-1),Tween-80 1. 0 mL·L~(-1),soybean meal 25 g·L~(-1),pH 7. 0,inoculation volume of 3% fermentation temperature at 37℃,24 h fermentation,broth content 50 mL,with a spreed of 180 r·min~(-1). The activity of the mutagenic strain was 2. 57 U·mL~(-1),which was 2. 95 times higher than original strain under these conditions. The fermentation characteristics of mutant strain was also investigated. The activity of the mutant strain was 2. 67 U·mL~(-1),which was 3. 11 times higher than original strain when the strain was cultured for 24 h. The results of this study provide some theoretical references for the mutagenesis and breeding of pullulanase-producing strain by low energy ion beam implantation.
In order to improve the enzymatic activity of pullulanase,the original strain named GX-6 was mutated with low energy ion beam implantation,and the fermentation medium was optimized by response surface methodology. The results showed that the strain had a higher positive rate when the implantation energy was 10 keV and the dose of N+implantation was 1 × 1015 ions·cm~(-2) with implantation time of 38 s. A high-yield and high genetic stability mutant strain of GX6-2 was obtained under this mutation dose through repeated mutagenesis by using ion beam and the activity was2. 28 times higher,up to 2. 13 U·mL~(-1),than original strain( 0. 65 U·mL~(-1)). From the Plackett-Burmen test,three significant factors affecting the enzyme activity of pullulase were corn starch,maltose and Tween-80. The optimized fermentation parameters was shown as follows: corn starch 56. 5 g·L~(-1),maltose 11. 5 g·L~(-1),Tween-80 1. 0 mL·L~(-1),soybean meal 25 g·L~(-1),pH 7. 0,inoculation volume of 3% fermentation temperature at 37℃,24 h fermentation,broth content 50 mL,with a spreed of 180 r·min~(-1). The activity of the mutagenic strain was 2. 57 U·mL~(-1),which was 2. 95 times higher than original strain under these conditions. The fermentation characteristics of mutant strain was also investigated. The activity of the mutant strain was 2. 67 U·mL~(-1),which was 3. 11 times higher than original strain when the strain was cultured for 24 h. The results of this study provide some theoretical references for the mutagenesis and breeding of pullulanase-producing strain by low energy ion beam implantation.
引文
[1]邓传良,贾彦彦,任映雪,高武军,张涛,李鹏飞,卢龙斗.离子注入诱变莲花突变体分子机理的初步研究[J].遗传,2011,33(1):81-87
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[3]顾鹏飞,吴刚,胡永红,王春晓,丁志雯,杨文革.低能N+注入选育凝结芽孢杆菌高效生防菌株[J].河南农业科学,2016,45(5):87-90
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[7]李欢琴,王文磊,王昭凯,林祥志.低能离子束生物技术的应用[J].氨基酸和生物资源,2016,38(2):1-6
[8] He Q,Li N,Chen X,Ye Q,Bai J,Xiong J,Ying H. Mutation breeding of nuclease p1 production in Penicillium Citrinum,by lowenergy ion beam implantation[J]. Korean Journal of Chemical Engineering,2011,28(2):544-549
[9]肖随龙.普鲁兰酶基因工程研究进展[J].中国农业科技导报,2015,17(4):85-91
[10] Duan X,Chen J,Wu J. Improving the thermostability and catalytic efficiency of Bacillus deramificans pullulanase by site-directed mutagenesis[J]. Applied and Environmental Microbiology,2013,79(13):4072-4077
[11]常帆,薛文娇,马赛箭,安超,贾凤安.一株关中热泉液口普鲁兰酶产生菌的筛选鉴定及部分酶学性质研究[J].中国食品添加剂,2015(4):100-105
[12]孙会忠,王小东,朱金峰,李广良,孙明辉,侯小改.产普鲁兰酶菌株ZDJ35的分离鉴定及产酶特性[J].基因组学与应用生物学,2015,34(3):555-559
[13]王新.普鲁兰酶产生菌的选育及产酶特性研究[D].福州:福州大学,2014
[14]谢能中,王青艳,米慧芝,朱绮霞,秦艳,曹薇,朱婧,陆雁,黄日波.产普鲁兰酶克雷伯氏菌的分离鉴定及酶学性质研究[J].广西科学,2013,20(1):35-39
[15] Li S C,Liu H X,Gu S B,Wang M,Zhu Z Y,Wang D D.Screening of lipid high producing mutant from Rhodotorula Glutinis by low ion implantation and study on lipid extration technique[J].Advanced Materials Research,2012,343-344:1172-1181
[16] Maran J M,Manikandan S,Thirugnanasambandham K,Vigna N C,Dinesh R. Box-Behnken design based statistical modeling for ultrasound-assisted extraction of corn silk polysaccharide[J].Carbohydrate Polymers,2013,92(1):604
[17]刘志森,杨国庆.实验设计和响应面方法在油田开发方案设计中的应用[J].石油化工应用,2012,31(10):23-25
[18]臧文霞.利用微生物发酵麻疯果油粕生产生物有机肥的研究[D].海口:海南师范大学,2013
[19]王诗然,赵世光,薛正莲,钱森和,魏明. Plackett-Burman设计在漆酶发酵培养基主要影响因子筛选中的应用[J].安徽工程大学学报,2010,25(2):14-17
[20]陈华妮,潘洁萍,黄小丹,李振中,钱力.响应曲面法优化野生酸荔枝核中抗乙肝成分的提取工艺[J].中国实验方剂学杂志,2014,20(18):41-44
[21]章栋梁,钟蔚,张充,吕凤霞,别小妹,陆兆新.响应面法优化重组枯草芽孢杆菌产脂肪氧合酶条件[J].核农学报,2012,26(2):324-329
[22]金淼,周逸,徐亦及,唐健波,杨文鸽,张进杰.响应面法优化秘鲁鱿鱼(Dosidicus gigas)肌肉盐溶蛋白的提取和凝胶形成条件[J].核农学报,2013,27(7):1003-1011
[23]周杨,张慧慧,郑建仙.响应面法优化抗麻糬老化改良剂的试验研究[J].中国食品添加剂,2013(5):142-149
[24]周念波,李轶群,王海波,杨双喜.原生质体紫外诱变选育普鲁兰酶高产菌株[J].安徽农业科学,2011,39(15):9115-9117
[25]秦艳飞,薛正莲,苏燕南,王栋.紫外-微波复合诱变原生质体选育那西肽高产菌株[J].食品与发酵科技,2011,47(3):39-42
[26]郭宏文,邹东恢,杜国军,孙亚范.普鲁兰酶产生菌的诱变育种[J].食品研究与开发,2010,31(2):167-169
[27] Xu T T,Bai Z Z,Wang L J,He B F. Breeding of D(-)-lactic acid high producing strain by low-energy ion implantation and preliminary analysis of related metabolism[J]. Applied Biochemistry&Biotechnology,2010,160(2):314-321
[28] Nie G,Yang X,Liu H,Wang L,Gong G,Jin W,Zheng Z. N+ion beam implantation of tannase-producing Aspergillus niger, and optimization of its process parameters under submerged fermentation[J]. Annals of Microbiology,2013,63(1):279-287
[29]甄杰,胡政,李树芳,徐建勇,宋诙.一个新型耐热普鲁兰酶的结构与功能[J].生物工程学报,2014,30(1):119-128
[30]严伟,聂尧,徐岩.长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶在大肠杆菌中的活性表达与分泌调控[J].微生物学报,2013,53(2):145-153
[31]朱梦.普鲁兰酶产生菌的筛选及其酶学性质的研究[D].海口:海南大学,2010
[32]程景伟,景建洲,杨雪鹏,李书涛,樊攀,袁海晓,史文婷.普鲁兰酶产生菌的筛选鉴定及其发酵条件优化与酶学性质研究[J].广东化工,2012,39(6):37-39
[2] Hui L, Ma X, Shao L, Shen J, Song X. Enhancement of sophorolipid production of wickerhamiella domercqiae, var.sophorolipid,CGMCC 1576 by low-energy ion beam implantation[J]. Applied Biochemistry&Biotechnology,2012,167(3):510
[3]顾鹏飞,吴刚,胡永红,王春晓,丁志雯,杨文革.低能N+注入选育凝结芽孢杆菌高效生防菌株[J].河南农业科学,2016,45(5):87-90
[4]张建华,王乃彦,张丰收,王广甫.离子注入诱变微生物应用与研究进展[J].北京师范大学学报(自然科学版),2011,47(3):262-267
[5]于永昂.低能离子注入番茄生物效应的初步研究[D].新乡:河南师范大学,2012
[6]杜艳.碳离子束辐照拟南芥突变体筛选及诱变效应研究[D].北京:中国科学院大学,2015
[7]李欢琴,王文磊,王昭凯,林祥志.低能离子束生物技术的应用[J].氨基酸和生物资源,2016,38(2):1-6
[8] He Q,Li N,Chen X,Ye Q,Bai J,Xiong J,Ying H. Mutation breeding of nuclease p1 production in Penicillium Citrinum,by lowenergy ion beam implantation[J]. Korean Journal of Chemical Engineering,2011,28(2):544-549
[9]肖随龙.普鲁兰酶基因工程研究进展[J].中国农业科技导报,2015,17(4):85-91
[10] Duan X,Chen J,Wu J. Improving the thermostability and catalytic efficiency of Bacillus deramificans pullulanase by site-directed mutagenesis[J]. Applied and Environmental Microbiology,2013,79(13):4072-4077
[11]常帆,薛文娇,马赛箭,安超,贾凤安.一株关中热泉液口普鲁兰酶产生菌的筛选鉴定及部分酶学性质研究[J].中国食品添加剂,2015(4):100-105
[12]孙会忠,王小东,朱金峰,李广良,孙明辉,侯小改.产普鲁兰酶菌株ZDJ35的分离鉴定及产酶特性[J].基因组学与应用生物学,2015,34(3):555-559
[13]王新.普鲁兰酶产生菌的选育及产酶特性研究[D].福州:福州大学,2014
[14]谢能中,王青艳,米慧芝,朱绮霞,秦艳,曹薇,朱婧,陆雁,黄日波.产普鲁兰酶克雷伯氏菌的分离鉴定及酶学性质研究[J].广西科学,2013,20(1):35-39
[15] Li S C,Liu H X,Gu S B,Wang M,Zhu Z Y,Wang D D.Screening of lipid high producing mutant from Rhodotorula Glutinis by low ion implantation and study on lipid extration technique[J].Advanced Materials Research,2012,343-344:1172-1181
[16] Maran J M,Manikandan S,Thirugnanasambandham K,Vigna N C,Dinesh R. Box-Behnken design based statistical modeling for ultrasound-assisted extraction of corn silk polysaccharide[J].Carbohydrate Polymers,2013,92(1):604
[17]刘志森,杨国庆.实验设计和响应面方法在油田开发方案设计中的应用[J].石油化工应用,2012,31(10):23-25
[18]臧文霞.利用微生物发酵麻疯果油粕生产生物有机肥的研究[D].海口:海南师范大学,2013
[19]王诗然,赵世光,薛正莲,钱森和,魏明. Plackett-Burman设计在漆酶发酵培养基主要影响因子筛选中的应用[J].安徽工程大学学报,2010,25(2):14-17
[20]陈华妮,潘洁萍,黄小丹,李振中,钱力.响应曲面法优化野生酸荔枝核中抗乙肝成分的提取工艺[J].中国实验方剂学杂志,2014,20(18):41-44
[21]章栋梁,钟蔚,张充,吕凤霞,别小妹,陆兆新.响应面法优化重组枯草芽孢杆菌产脂肪氧合酶条件[J].核农学报,2012,26(2):324-329
[22]金淼,周逸,徐亦及,唐健波,杨文鸽,张进杰.响应面法优化秘鲁鱿鱼(Dosidicus gigas)肌肉盐溶蛋白的提取和凝胶形成条件[J].核农学报,2013,27(7):1003-1011
[23]周杨,张慧慧,郑建仙.响应面法优化抗麻糬老化改良剂的试验研究[J].中国食品添加剂,2013(5):142-149
[24]周念波,李轶群,王海波,杨双喜.原生质体紫外诱变选育普鲁兰酶高产菌株[J].安徽农业科学,2011,39(15):9115-9117
[25]秦艳飞,薛正莲,苏燕南,王栋.紫外-微波复合诱变原生质体选育那西肽高产菌株[J].食品与发酵科技,2011,47(3):39-42
[26]郭宏文,邹东恢,杜国军,孙亚范.普鲁兰酶产生菌的诱变育种[J].食品研究与开发,2010,31(2):167-169
[27] Xu T T,Bai Z Z,Wang L J,He B F. Breeding of D(-)-lactic acid high producing strain by low-energy ion implantation and preliminary analysis of related metabolism[J]. Applied Biochemistry&Biotechnology,2010,160(2):314-321
[28] Nie G,Yang X,Liu H,Wang L,Gong G,Jin W,Zheng Z. N+ion beam implantation of tannase-producing Aspergillus niger, and optimization of its process parameters under submerged fermentation[J]. Annals of Microbiology,2013,63(1):279-287
[29]甄杰,胡政,李树芳,徐建勇,宋诙.一个新型耐热普鲁兰酶的结构与功能[J].生物工程学报,2014,30(1):119-128
[30]严伟,聂尧,徐岩.长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶在大肠杆菌中的活性表达与分泌调控[J].微生物学报,2013,53(2):145-153
[31]朱梦.普鲁兰酶产生菌的筛选及其酶学性质的研究[D].海口:海南大学,2010
[32]程景伟,景建洲,杨雪鹏,李书涛,樊攀,袁海晓,史文婷.普鲁兰酶产生菌的筛选鉴定及其发酵条件优化与酶学性质研究[J].广东化工,2012,39(6):37-39
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