海藻酸钠包埋法固定硝基还原假单胞菌的研究
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摘要
目的优化海藻酸钠和CaCl_2对硝基还原假单胞菌SP.001细胞的固定化条件。方法以海藻酸钠为载体,通过包埋法固定化谷氨酰胺酶,通过单因素实验和正交实验对固定化细胞制备条件进行优化。结果单因素实验中,当海藻酸钠和CaCl_2的浓度分别为4%和3%、酶液量与海藻酸钠的体积比为1:2、固定化时间为3 h时,包埋效果最好,测得固定化细胞回收率较高,可达80.5%。通过正交实验得出最佳组合为:酶液量与海藻酸钠体积比1:3,固定化时间3 h,海藻酸钠浓度4%,CaCl_2浓度3%,固定化细胞回收率达到85.78%。结论本研究优化了海藻酸钠包埋法固定硝基还原假单胞菌的条件,并对固定化细胞的性质进行探讨,为硝基还原假单胞菌所产的谷氨酰胺酶的固定化和应用提供参考。
Objective To optimize the immobilization conditions of sodium alginate and CaCl_2 on nitro-reduced Pseudomonas SP.001 cells. Methods Using sodium alginate as carrier, glutaminase was immobilized by embedding method, and the preparation conditions of immobilized cells were optimized by single factor experiment and orthogonal experiment. Results In the single factor experiment, when the concentration of sodium alginate and CaCl_2 were 4% and 3%, the volume ratio of enzyme liquid volume to sodium alginate was 1:2, and the immobilization time was 3 h, the embedding effect was the best, and the recovery rate of immobilized cells was high,reaching 80.5%. Through orthogonal test, the best combination was obtained as follows: the volume ratio of enzyme liquid volume to sodium alginate was 1:3, the immobilization time was 3 h, the concentration of sodium alginate was 4%, the concentration of CaCl_2 was 3%, and the recovery rate of immobilized cells reached 85.78%. Conclusion This study optimizes the conditions for the immobilization of nitroreductive pseudomonas by sodium alginate, and discusses the properties of the immobilized cells, in order to provide reference for the immobilization and application of glutaminase produced by nitroreductive Pseudomonas.
Objective To optimize the immobilization conditions of sodium alginate and CaCl_2 on nitro-reduced Pseudomonas SP.001 cells. Methods Using sodium alginate as carrier, glutaminase was immobilized by embedding method, and the preparation conditions of immobilized cells were optimized by single factor experiment and orthogonal experiment. Results In the single factor experiment, when the concentration of sodium alginate and CaCl_2 were 4% and 3%, the volume ratio of enzyme liquid volume to sodium alginate was 1:2, and the immobilization time was 3 h, the embedding effect was the best, and the recovery rate of immobilized cells was high,reaching 80.5%. Through orthogonal test, the best combination was obtained as follows: the volume ratio of enzyme liquid volume to sodium alginate was 1:3, the immobilization time was 3 h, the concentration of sodium alginate was 4%, the concentration of CaCl_2 was 3%, and the recovery rate of immobilized cells reached 85.78%. Conclusion This study optimizes the conditions for the immobilization of nitroreductive pseudomonas by sodium alginate, and discusses the properties of the immobilized cells, in order to provide reference for the immobilization and application of glutaminase produced by nitroreductive Pseudomonas.
引文
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[18]张斌,孙倩,任延鹏,等.海藻酸钠固定化β-葡萄糖醛酸苷酶的研究[J].中国食品添加剂,2011,(1):110-115.Zhang B,Sun Q,Ren YP,et al.Study on the immobilization ofβ-glucuronidase by sodium alginate[J].China Food Add,2011,(1):110-115.
[19]管悦.固定化微生物转化药用植物黄芩的研究[D].哈尔滨:东北林业大学,2014.Guan Y.Study on transformation of medicinal plant scutellaria baicalensis georgi with immobilized microorganisms[D].Harbin:Northeast Forestry University,2014.
[20]魏杰,石钟辉,候潇,等.谷氨酰胺合成酶产生菌的固定化研究[J].微生物学杂志,2007(03):73-76.Wei J,Shi ZH,Hou X,et al.Immobilization of Glutamine Synthase producing strain[J].J Microbiol,2007,(3):73-76.
[21]朱磊.产琼胶酶降解菌的筛选及酶的固定化研究[D].大连:大连工业大学,2016.Zhu L.Screening of agarase-degrading bacteria and immobilization of agarase[D].Dalian:Dalian Polytechnic University,2016.
[2]Matsuura S,Chiba M,Tomon E,et al.Synthesis of amino acid using a flow-type microreactor containing enzyme-mesoporous silica micros Phere composites[J].Rsc Adv,2014,4:9021-9030.
[3]Liu B,Li P,Zhang CL.Theanine synthesized by immobilizing Pseudomonas nitroreducens LY in nanofibrous membranes[J].Process Biochem,2010,45:1330-1333.
[4]Itoh T,Hoshikawa Y,Matsuura S,et al.Production of L-theanine using glutaminase encapsulated in carbon-coated mesoporous silica with high phstability[J].Biochem Eng J,2012,68:207-214.
[5]Lamboley L,Lacroix C,Champagne CP.Continuous mixed strain mesophilic lactic starter production in supplemented whey permeate medium using immobilized cell technology[J].Biotechnol Bioeng,2008,56(5):502-516.
[6]蒋宇红,黄霞,俞毓馨.几种固定化细胞载体的比较[J].环境科学,1993,(2):11-15,92.Jiang YH,Huang X,Yu YX.Comparison of several immobilized cell carriers[J].Environ Sci,1993,(2):11-15,92.
[7]Chen Y,Liu Q,Zhou T,et al.Ethanol production by repeated batch and continuous fermentations by Saccharomyces cerevisiae immobilized in a fibrous bed bioreactor[J].J Microbiol Biotech,2013,23(4):511-517.
[8]Tachiki T,Yamada T,Mizuno K.Gamma-glutamyl transfer reactions by glutaminase from Pseudomonas nitroreducens IFO12694 and their application for the syntheses of theanine and gamma-glutamylmethylamide[J].Biosci Biotechnol Bioch,1998,62:1279-1283.
[9]Binod P,Sindhu R,Madhavan A,et al.Recent developments in L-glutaminase production and applications an overview[J].Bioresource Technol,2017,245:1766-1774.
[10]Wanmeng M,Tao Z,Bo J.An overview of biological production of L-theanine[J].Biotechnol Adv,2015,33(3-4):335-342.
[11]Chiyakuraishi,Katsutoshiyamazaki,Yasuyukisusa,et al.Its utilization in the food industry[J].Food Revint,2001,17(2):221-246.
[12]Shuai YY,Zhang T,Jiang B,et al.Development ofefficient enzymatic production of theanine byγ-glutamyl-transpeptidase from a newly isolated strain of Bacillus subti-lis SK11.004[J].J Sci Food Agric,2010,90(15):2563-2567.
[13]杨成,张涛,江波,等.硝基还原假单胞菌谷氨酰胺酶的分离纯化及酶学性质[J].食品与发酵工业,2012,38(12):16-21.Yang C,Zhang T,Jiang B,et al.Purification and characterization of glutaminase from Pseudomonas nitroreducens[J].Food Ferment Ind,2012,38(12):16-21.
[14]贾晓鹤,陈莉,赵宁伟,等.生物转化法应用重组谷氨酰转肽酶合成L-茶氨酸[J].食品工业科技,2008,29(2):166-169.Jia XH,Chen L,Zhao NW,et al.Synthesis of L-theanine by biotransformation using recombinant Glutamyltranspeptidase[J].Food Ind Sci Technol,2008,29(2):166-169.
[15]王春晖.茶氨酸生物合成研究[D].北京:中国农业科学院茶叶研究所,2005.Wang CH.Study on theanine biosynthesis[D].Beijing:Tea Research Institute,Chinese Academy of Agricultural Sciences,2005.
[16]酒卫敬,汪苹,李奥搏,等.海藻酸钠作为固定化细胞包埋剂的研究[J].科技创新导报,2011,(2):12-13.Jiu WJ,Wang P,Li AB,et al.Study on sodium alginate as immobilized cell embedding agent[J].Sci Technol Innov Herald,2011,(2):12-13.
[17]Liu B,Li P,Zhang CL.Theanine synthesized by immobilizing Pseudomonas nitroreducens LY in nanofibrous membranes[J].Process Biochem,2010,45:1330-1333.
[18]张斌,孙倩,任延鹏,等.海藻酸钠固定化β-葡萄糖醛酸苷酶的研究[J].中国食品添加剂,2011,(1):110-115.Zhang B,Sun Q,Ren YP,et al.Study on the immobilization ofβ-glucuronidase by sodium alginate[J].China Food Add,2011,(1):110-115.
[19]管悦.固定化微生物转化药用植物黄芩的研究[D].哈尔滨:东北林业大学,2014.Guan Y.Study on transformation of medicinal plant scutellaria baicalensis georgi with immobilized microorganisms[D].Harbin:Northeast Forestry University,2014.
[20]魏杰,石钟辉,候潇,等.谷氨酰胺合成酶产生菌的固定化研究[J].微生物学杂志,2007(03):73-76.Wei J,Shi ZH,Hou X,et al.Immobilization of Glutamine Synthase producing strain[J].J Microbiol,2007,(3):73-76.
[21]朱磊.产琼胶酶降解菌的筛选及酶的固定化研究[D].大连:大连工业大学,2016.Zhu L.Screening of agarase-degrading bacteria and immobilization of agarase[D].Dalian:Dalian Polytechnic University,2016.