基因重组蛋清溶菌酶的分离纯化
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摘要
以毕赤氏基因重组酵母发酵液为研究对象,经过离心分离、超滤、膜分离、阳离子交换树脂吸附后,得到的纯化液,真空干燥得到酶活为20000U·mg~(-1)的蛋清溶菌酶粉末。研究结果表明:发酵液在5000r/min、25℃下离心15min,得到离心液溶菌酶活性为360.1U·mL~(-1),菌体细胞的去除率达99.99%;超滤最佳操作条件:压力为0.15MPa,pH为6.5;一级超滤得到的溶菌酶活性为455.4U·mL~(-1),收率为90.6%,纯化程度提高了23.7%;二级超滤得到的溶菌酶活性为648U·mL~(-1),收率为61.2%,纯化程度提高了42.3%; D152阳离子交换树脂对料液进行离子交换层析,得溶菌酶的酶活性为770.5U·mL~(-1),收率达90%,纯化程度提高了18.9%。
After centrifugation,ultrafiltration,membrane separation and cation exchange resin adsorption,the purified solution obtained from Pichia pastoris yeast fermentation broth with Pichia pastoris gene was dried in vacuum to obtain the lysozyme powder with enzyme activity of 20000 U mg~(-1). The results showed that the fermentation broth was centrifuged at 5000 r/min and 25℃ for 15 min,and the lysozyme activity of the centrifuge was 360.1 U · mL~(-1). The removal rate of the cells was 99.99%. The optimal operating conditions for ultrafiltration were:pressure was 0.15 MPa and pH value was 6.5;lysozyme activity obtained by first-stage ultrafiltration was 455.4 U · mL~(-1),the yield was 90.6%,and the purification degree increased by 23.7%;lysozyme activity obtained by second-stage ultrafiltration was 648 U · mL~(-1),yield was 61.2%,and the purification degree increased by 42.3%;D152 cation exchange resin for ion exchange chromatography,the enzyme activity of lysozyme was 770.5 U · mL~(-1),the yield was 90%,and the purification degree increased by 18.9%.
After centrifugation,ultrafiltration,membrane separation and cation exchange resin adsorption,the purified solution obtained from Pichia pastoris yeast fermentation broth with Pichia pastoris gene was dried in vacuum to obtain the lysozyme powder with enzyme activity of 20000 U mg~(-1). The results showed that the fermentation broth was centrifuged at 5000 r/min and 25℃ for 15 min,and the lysozyme activity of the centrifuge was 360.1 U · mL~(-1). The removal rate of the cells was 99.99%. The optimal operating conditions for ultrafiltration were:pressure was 0.15 MPa and pH value was 6.5;lysozyme activity obtained by first-stage ultrafiltration was 455.4 U · mL~(-1),the yield was 90.6%,and the purification degree increased by 23.7%;lysozyme activity obtained by second-stage ultrafiltration was 648 U · mL~(-1),yield was 61.2%,and the purification degree increased by 42.3%;D152 cation exchange resin for ion exchange chromatography,the enzyme activity of lysozyme was 770.5 U · mL~(-1),the yield was 90%,and the purification degree increased by 18.9%.
引文
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[2]Sharon N,Seifter S.A transglycosylation reaction catalyzed by lysozyme[J].Joumal of Biological Chemistry,1964,239(7):2398-2399.
[3]Callewaert L,Michiels C W.Lysozymes in the animal kingdom[J].Jourmal of Biosciences,2010,35(1):127-160.
[4]Blake C C F,Koenig D F,Mair G A,etal.Structure ofhen egg white lysozyme:A three-dimensional fourier synthesis at2?resolution[J].Nature,1965,206(4986):757-761.
[5]Johnson L N,Pillips D C.Structure of some crystalline lysozymeinhibitor complexes determined by x-ray analysis at 6?Resolution[J].Nature,1965,206(4986):761-763.
[6]Xue Q G,Itoh N,Schey K L,et al,A new lysozyme from the eastern oyster(Crassostrea virginica)indicates adaptive evolution of i-type lysozyme[J].Cellular&Molecular Life Sciences,2007,64(1):82-95.
[7]博冰,季秀玲,俞汇颖,等.鸡蛋清中溶菌酶的分离提取[J].浙江农业学报,2013,25(6):1364-1367.
[8]侯启瑞,王金玉,谢凯舟,等.测定鸡蛋蛋清中溶菌酶含量和活力标准方法的建立[J].中国畜牧杂志,2010,46(3):49-52.
[9]孙玮遥,王向东,林剑.外源蛋白在毕赤酵母中的高效表达策略[J].中国酿造,2016,35(9):11-15.
[10]王镜岩,朱长庚,徐长发,等.生物化学[M].高等教育出版社,2002.
[11]姚如华,等.微生物工程工艺原理[M].华南理工大学出版社,2005.
[12]孙彦.生物分离工程[M].化学工业出版社,2013.
[13]Lauri H J Lajunen,Gregory R.Choppin.Analytical chemistry of the lanthanides part 1.atomic absorption and plasma atomic emission spectroscopic methods[J].Reviews in Analytical Chemistry,1989,9(3).
[14]沈萍,陈向东.微生物学实验[M].第三版.北京:高等教育出版社.
[15]陈琛.超滤技术在IgY分离中的应用[J].精细与专用化学品,2010,18(3):21-24.
[16]Lu Wang,Tanya Khan,Kaustubha Mohanty,Raja Ghosh.Cascade ultrafiltration bioreactor-separator system for continuous production of F(ab’)2 fragment from immunoglobulin G[J].Journal of Membrane Science,2010,351(1):96-103.
[17]罗磊,樊金铃,孙红姣.预处理猪血清IgG料液的超滤浓缩研究[J].食品科學,2008,29(6):159-163.
[18]刘闪闪.用于牛血清蛋白液分离的聚醚砜超滤膜的抗污染研究[D].宁波大学,2011.
[19]孟凡红.重组人血清白蛋白在汉逊酵母中的表达与纯化[D].大连理工大学,2006.
[20]亓晅,吕阳成,陈扬,等.牛血清白蛋白为分离剂超滤拆分色氨酸的机理和模型(II)[J].化工学报,2007,58(1):119-124.
[21]杜利成,杨葵华.提高从鸡蛋清中提取溶菌酶产率的研究[J].四川食品与发酵,2007(6):27-31.