纺织用碱性耐热过氧化氢酶的微生物法生产及保存稳定性的研究
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摘要
过氧化氢酶,简称CAT,存在于所有好氧微生物和动、植物细胞内。其生理功能为作为特殊的过氧化物酶调节机体内H_2O_2的水平。各种来源的过氧化氢酶有着广泛的用途,如用于临床诊断和食品消毒中残余H_2O_2的分解等。最近,过氧化氢酶开始用于织物漂白工艺中替代水洗和化学试剂还原法去除H_2O_2。同从动植物组织中提取相比,微生物法合成过氧化氢酶具有许多优点。
本论文以一株嗜热子囊菌Thermoascus aurantiacus WSH 03-01为出发菌株,首先考察了诱导剂对过氧化氢酶合成的影响,并研究了接种量和搅拌转速对7 L罐中过氧化氢酶分批发酵的影响,确定了合适的接种量和搅拌转速;通过测定发酵过程中乙醇的消耗曲线确定了影响过氧化氢酶合成的关键因素,在发酵过程中流加乙醇实现了过氧化氢酶的高产,并将7 L罐实验结果放大到30 L的规模;此外还研究了稳定剂对Thermoascus aurantiacusWSH 03-01所产过氧化氢酶粗酶保存效果的影响,并考察了该酶去除H_2O_2的效果。主要研究内容如下:
(1)在发酵0 h添加88 mmol/L的H_2O_2,发酵终了过氧化氢酶酶活比对照提高了26.0%。如果在发酵的24 h或48 h分别添加1μmol/L的甲萘醌,发酵终了过氧化氢酶酶活比对照分别提高了11.0%和23.1%;而若在发酵的36 h和48 h各添加1μmol/L的甲萘醌,过氧化氢酶酶活可提高32.5%。在发酵0 h添加88 mmol/L的H_2O_2,同时在48 h添加1μmol/L的甲萘醌,过氧化氢酶酶活达到1725 U/mL,比对照(1074 U/mL)提高了60.6%。表明T.aurantiacus WSH 03-01合成过氧化氢酶是可以被诱导的。
(2)在7 L罐中考察了接种量和搅拌转速对过氧化氢酶分批发酵的影响,确定了合适的接种量为7%,最佳搅拌转速为200 rpm,过低或过高的搅拌转速均不利于过氧化氢酶的合成。在该条件下发酵96 h过氧化氢酶酶活最高为1851 U/mL。
(3)通过测定发酵过程中乙醇的消耗曲线确定了乙醇的流加速率为0.14 mL/h·L,在发酵过程中流加乙醇实现了过氧化氢酶的高产,发酵终了过氧化氢酶酶活为2453 U/mL。将7 L小罐实验结果放大到30 L的规模,发酵118 h时酶活达到最大值2287 U/mL。采用两阶段控制搅拌转速的方式较好地解决了后期溶氧偏低的问题,115 h细胞干重达到最大值9.56 g/L,发酵120 h酶活最高达到3652 U/mL。
(4)向发酵超滤液中加入稳定剂氯化钠20 mmol/L或甘油90g/L或明胶0.5g/L,在40℃保存60 d其酶活保留率均在90%以上。
(5) T. aurantiacus WSH03-01合成的过氧化氢酶在pH5.5-8.0,温度15℃-40℃时对H2O2都有很好的去除效果,15 min后100 ppmH_2O_2都能全部去除。
Catalase is widely distributed in nature and found in all aerobic microorganisms, plant andanimal cells. Physiologically it acts as a regulator of H2O2 levels in organelles and as a specificperoxidase. Catalases from various sources are utilized in numerous industrial applications, suchas diseases diagnosis and food sterilization. Recently, catalase is used in removal of hydrogenperoxide of bleaching wastewater as a substitute for washing with huge amount of wateror usingchemical reducing agents. Compared with extract from organelles, biosynthesis of catalase bymicroorganisms has much more advantages.
In this paper, the strain of Thermoascus aurantiacus WSH 03-01 was studied as a producerof catalase. The effect of revulsant on catalse fermentation was investigated in the beginning.Proper inoculum size and agitation speed were found by experiment in a 7 L fermentor. Thekey factor for producing catalse was determined by detecting ethanol's consuming processduring the catalse fermentation. Based on the result, catalse was high synthesized with themethod of adding ethanol continuously into the bioreactor. Thus, the scale-up of catalseproduction from 7 L fermentor to 30 L fermentor was also achieved. Furthermore, the effects ofvarious stabilizers on storage stabilities of catalase were observed, and the removal efficiency ofhydrogen peroxide by the obtained catalase was verified initially. The main content of thisdissertation is as follows:
(1) The catalase activity increased to 26.0% when 88 mmol/L hydrogen peroxide was addedat 0 h. When 1 μmol/L menadione was added at 24 h or 48 h, the catalse activity increased 11.0%and 23.1%, respectively, compared with that of control. The catalase activity increased 32.5%when menadione was added at 36h and 48h. According to the result of menadione additionoptimization, the catalse activity reached 1725 U/mL when 88 mmol/L hydrogen peroxide wasadded at 0 h and 1 μmol/L menadione was added at 48 h,which was 60.6% higher than that ofcontrol (1074 U/mL). It indicates that catalse can be induced by reactive oxygen speciesgenerated by menadione and hydrogen peroxide.
(2) The effects of inoculum size and agitation speed on catalse fermentation were optimizedin a 7 L fermentor. The proper conditions for catalse fermentation were: inoculum size 7%,agitation speed 200 rpm. The catalase activity reached 1851 U/mL in a 7 L fermentor at 96hunder the optimized conditions.
(3) The speed of adding ethanol was confirmed as 0.14 mL/(h·L) by determine ethanol'sconsuming curve in the course of catalse fermentation. The catalse activity reached 2453 U/mLwhen ethanol was added continuously into the fermentor. The result showed that the catalseactivity reached 2287 U/mL in a 30 L fermentor. A two-stage agitation speed control mode was
also developed to solve the low dissolved oxygen at the late phase of the fermentation. By thisway, the dry cell weight reached 9.56 g/L and the catalase activity reached 3652 U/mL.(4) The result of reserving condition study showed that the reservation efficiency of thecatalase activity exceeded 90% even it was conserved 60 days at 40℃ when 20 mmol/Lsodium chloride or 90g/L glycerol or 0.5g/L glutin was added into the concentrated catalase byultrafiltration.(5) The removal efficiency of hydrogen peroxide by the catalase obtained in this study washigher under the following conditions: pH range as 5.5~8.0, temperature between 15℃~40℃.Hydrogen peroxide at 100 ppm was removed completely by the enzyme after 15 minutesreaction.
本论文以一株嗜热子囊菌Thermoascus aurantiacus WSH 03-01为出发菌株,首先考察了诱导剂对过氧化氢酶合成的影响,并研究了接种量和搅拌转速对7 L罐中过氧化氢酶分批发酵的影响,确定了合适的接种量和搅拌转速;通过测定发酵过程中乙醇的消耗曲线确定了影响过氧化氢酶合成的关键因素,在发酵过程中流加乙醇实现了过氧化氢酶的高产,并将7 L罐实验结果放大到30 L的规模;此外还研究了稳定剂对Thermoascus aurantiacusWSH 03-01所产过氧化氢酶粗酶保存效果的影响,并考察了该酶去除H_2O_2的效果。主要研究内容如下:
(1)在发酵0 h添加88 mmol/L的H_2O_2,发酵终了过氧化氢酶酶活比对照提高了26.0%。如果在发酵的24 h或48 h分别添加1μmol/L的甲萘醌,发酵终了过氧化氢酶酶活比对照分别提高了11.0%和23.1%;而若在发酵的36 h和48 h各添加1μmol/L的甲萘醌,过氧化氢酶酶活可提高32.5%。在发酵0 h添加88 mmol/L的H_2O_2,同时在48 h添加1μmol/L的甲萘醌,过氧化氢酶酶活达到1725 U/mL,比对照(1074 U/mL)提高了60.6%。表明T.aurantiacus WSH 03-01合成过氧化氢酶是可以被诱导的。
(2)在7 L罐中考察了接种量和搅拌转速对过氧化氢酶分批发酵的影响,确定了合适的接种量为7%,最佳搅拌转速为200 rpm,过低或过高的搅拌转速均不利于过氧化氢酶的合成。在该条件下发酵96 h过氧化氢酶酶活最高为1851 U/mL。
(3)通过测定发酵过程中乙醇的消耗曲线确定了乙醇的流加速率为0.14 mL/h·L,在发酵过程中流加乙醇实现了过氧化氢酶的高产,发酵终了过氧化氢酶酶活为2453 U/mL。将7 L小罐实验结果放大到30 L的规模,发酵118 h时酶活达到最大值2287 U/mL。采用两阶段控制搅拌转速的方式较好地解决了后期溶氧偏低的问题,115 h细胞干重达到最大值9.56 g/L,发酵120 h酶活最高达到3652 U/mL。
(4)向发酵超滤液中加入稳定剂氯化钠20 mmol/L或甘油90g/L或明胶0.5g/L,在40℃保存60 d其酶活保留率均在90%以上。
(5) T. aurantiacus WSH03-01合成的过氧化氢酶在pH5.5-8.0,温度15℃-40℃时对H2O2都有很好的去除效果,15 min后100 ppmH_2O_2都能全部去除。
Catalase is widely distributed in nature and found in all aerobic microorganisms, plant andanimal cells. Physiologically it acts as a regulator of H2O2 levels in organelles and as a specificperoxidase. Catalases from various sources are utilized in numerous industrial applications, suchas diseases diagnosis and food sterilization. Recently, catalase is used in removal of hydrogenperoxide of bleaching wastewater as a substitute for washing with huge amount of wateror usingchemical reducing agents. Compared with extract from organelles, biosynthesis of catalase bymicroorganisms has much more advantages.
In this paper, the strain of Thermoascus aurantiacus WSH 03-01 was studied as a producerof catalase. The effect of revulsant on catalse fermentation was investigated in the beginning.Proper inoculum size and agitation speed were found by experiment in a 7 L fermentor. Thekey factor for producing catalse was determined by detecting ethanol's consuming processduring the catalse fermentation. Based on the result, catalse was high synthesized with themethod of adding ethanol continuously into the bioreactor. Thus, the scale-up of catalseproduction from 7 L fermentor to 30 L fermentor was also achieved. Furthermore, the effects ofvarious stabilizers on storage stabilities of catalase were observed, and the removal efficiency ofhydrogen peroxide by the obtained catalase was verified initially. The main content of thisdissertation is as follows:
(1) The catalase activity increased to 26.0% when 88 mmol/L hydrogen peroxide was addedat 0 h. When 1 μmol/L menadione was added at 24 h or 48 h, the catalse activity increased 11.0%and 23.1%, respectively, compared with that of control. The catalase activity increased 32.5%when menadione was added at 36h and 48h. According to the result of menadione additionoptimization, the catalse activity reached 1725 U/mL when 88 mmol/L hydrogen peroxide wasadded at 0 h and 1 μmol/L menadione was added at 48 h,which was 60.6% higher than that ofcontrol (1074 U/mL). It indicates that catalse can be induced by reactive oxygen speciesgenerated by menadione and hydrogen peroxide.
(2) The effects of inoculum size and agitation speed on catalse fermentation were optimizedin a 7 L fermentor. The proper conditions for catalse fermentation were: inoculum size 7%,agitation speed 200 rpm. The catalase activity reached 1851 U/mL in a 7 L fermentor at 96hunder the optimized conditions.
(3) The speed of adding ethanol was confirmed as 0.14 mL/(h·L) by determine ethanol'sconsuming curve in the course of catalse fermentation. The catalse activity reached 2453 U/mLwhen ethanol was added continuously into the fermentor. The result showed that the catalseactivity reached 2287 U/mL in a 30 L fermentor. A two-stage agitation speed control mode was
also developed to solve the low dissolved oxygen at the late phase of the fermentation. By thisway, the dry cell weight reached 9.56 g/L and the catalase activity reached 3652 U/mL.(4) The result of reserving condition study showed that the reservation efficiency of thecatalase activity exceeded 90% even it was conserved 60 days at 40℃ when 20 mmol/Lsodium chloride or 90g/L glycerol or 0.5g/L glutin was added into the concentrated catalase byultrafiltration.(5) The removal efficiency of hydrogen peroxide by the catalase obtained in this study washigher under the following conditions: pH range as 5.5~8.0, temperature between 15℃~40℃.Hydrogen peroxide at 100 ppm was removed completely by the enzyme after 15 minutesreaction.
引文
[1] 怀斯曼.酶应用手册.上海科学技术出版社,1986.
[2] 方允中, 李文杰. 自由基与酶. 科学出版社,1989.
[3] Herbert D,Pinsent.Production and utilization of catalase using Saccharomyces cerevisiae. Biochem,1948,43:193-203.
[4] Sumner J B, Dounce A L.Purification and characterization of a thermostable catalase from culture broth of Thermoascus aurantiacus. J Biol Chem, 1937, 121:417-424.
[5] Goth L.Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture.Clin Chim Acta.1991, 196: 143.
[6] 冯国基.现代诊断与治疗. 北京: 人民卫生出版社, 1994.
[7] 莫简主. 医学自由基生物学导论.北京: 人民卫生出版社, 1989.
[8] Genencor.PCT Int.Appl.WO 931-721, 1993.
[9] Novo-Nordisk.PCT Int.Appl. WO 9217571, 1992.
[10] 财团法人生物技术开发中心.Int.Cl.CN 1219588A,1999.
[11] 相尺考亮.酶应用手册.上海:上海科学技术出版社,1989.
[12] Sosa R.C,Masy D,Rouxhet P.G.Influence of surface properties of carbon black on the activity of adsorbed catalse.Carbon,1994,32(7):1369-1375.
[13] Marinka Gudelj,Gilbert Otto Fruhwirth,Andreas Paar. A catalase-peroxidase from a newly isolated thermoalkaliphilic Bacillus sp.with potential for the treatment of textile bleaching effluents.Extremophiles,2001,5:423～429.
[14] Laurent Le Pape,Emmanuel Perret,Isabelle Mechaud-Soret. Magnetization studies of the active and fluoride-inhibited derivatives of the reduced catalase of Lactobacillus plantarum:toward a general picture of the anion-inhibited and active forms of the reduced dimanganese catalases.J Biol Inorg-Chem,2002,7:445-450.
[15] Goncalves V M, Cezar de Cerqueira Leite L, Raw I, et al. Influence of surface properties of carbon black on the activity of adsorbed catalse. Biotechnol Appl Biochem,1999,29: 73-77.
[16] Maurizio P, Massimiliano F, Paola P, et al. Effect of stirrer speed and buffering agents on the production of glucose oxidase and catalase by Penicillium variabile(P16) in benchtop bioreactor. Enzyme and Microb Technol, 1995, 17:336-339.
[17] 陆挺,汪成富,戴英,等.过氧化氢酶发酵性能的研究.苏州大学学报,2001,17(3): 95-97.
[18] 周一,严自正,卢运玉,等.耐热过氧化氢酶产生菌的筛选和发酵条件的研究.微生物学报,1990,30(3): 223-227.
[19] Venkateshwaran G,Somashekar D,Prakash M H,et al.Production and utilization of catalase using Saccharomyces cerevisiae. Process Biochem,1999,34:187-191.
[20] Fiedurek J,Gromada A.Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture.J Appl Microbiol,2000,89: 85-89.
[21] Minagawa S,Tawara T,Terao I.Production of catalase.Jp 09-047285,1997.
[22] 王凡强,王正祥,邵蔚蓝,等.热稳定性过氧化氢酶工程菌株发酵条件的研究.食品与发酵工业,2002,28 (2):11-14.
[23] 黄文涛.酶应用手册.上海:上海科技出版社, 1989.
[24] Wang H X,Yukiko Y,Shinoyama H,et al.Purification and characterization of a thermostable catalase from culture broth of Thermoascus aurantiacus.J Ferment Bioeng,1998,85(2):169-173.
[25] Liu J Z,Huang Y Y,Liu J,et al.Effect of metal ions simultaneous production of glucose oxidase and catalase by Aspergillus niger. Lett in Appl Micro,2001,32: 16-19.
[26] 方芳.纺织用碱性耐热过氧化氢酶的微生物法生产及其应用.江南大学硕士论文,2004,3.
[27] Nishikawa Y, Kawata Y,Nagai J.Effect of triton X-10 on catalase production by Aspergillus terreus IFO 6123 [J]. J Fermentat Bioeng,1993,76:235-236.
[28] Bergmeger H U,Bergmeyer J,Grabl M,et al.Methods of Enzymatic Analysis.3rd ed, Vol3, Weinheim: Verlag Chemie Press, 1983,257-259.
[29] 许申鸿,杭瑚,李运平.超氧化物歧化酶邻苯三酚测活法的研究及改进.化学通报,2001,8:516-519.
[30] 王微青.淀粉科学手册.中国轻工业出版社,1990.
[31] 魏述众.生物化学.北京:中国轻工出版社,1996.
[32] Thor H,Smith MT,Hartzell P,et al.The metabolism of menadione (2-methyl-1,4-naphthaquinone) by isolated hepatocytes .J Biol Chem, 1982, 275:12419-12425.
[33] Michaela Kreiner,Linda M.Harvey,Brian Mcneil.Oxidative stress response of a recombinant Aspergillus niger to exogenous menadione and H2O2 addition . Enzyme Microb Technol,2002,30:346-353.
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[2] 方允中, 李文杰. 自由基与酶. 科学出版社,1989.
[3] Herbert D,Pinsent.Production and utilization of catalase using Saccharomyces cerevisiae. Biochem,1948,43:193-203.
[4] Sumner J B, Dounce A L.Purification and characterization of a thermostable catalase from culture broth of Thermoascus aurantiacus. J Biol Chem, 1937, 121:417-424.
[5] Goth L.Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture.Clin Chim Acta.1991, 196: 143.
[6] 冯国基.现代诊断与治疗. 北京: 人民卫生出版社, 1994.
[7] 莫简主. 医学自由基生物学导论.北京: 人民卫生出版社, 1989.
[8] Genencor.PCT Int.Appl.WO 931-721, 1993.
[9] Novo-Nordisk.PCT Int.Appl. WO 9217571, 1992.
[10] 财团法人生物技术开发中心.Int.Cl.CN 1219588A,1999.
[11] 相尺考亮.酶应用手册.上海:上海科学技术出版社,1989.
[12] Sosa R.C,Masy D,Rouxhet P.G.Influence of surface properties of carbon black on the activity of adsorbed catalse.Carbon,1994,32(7):1369-1375.
[13] Marinka Gudelj,Gilbert Otto Fruhwirth,Andreas Paar. A catalase-peroxidase from a newly isolated thermoalkaliphilic Bacillus sp.with potential for the treatment of textile bleaching effluents.Extremophiles,2001,5:423～429.
[14] Laurent Le Pape,Emmanuel Perret,Isabelle Mechaud-Soret. Magnetization studies of the active and fluoride-inhibited derivatives of the reduced catalase of Lactobacillus plantarum:toward a general picture of the anion-inhibited and active forms of the reduced dimanganese catalases.J Biol Inorg-Chem,2002,7:445-450.
[15] Goncalves V M, Cezar de Cerqueira Leite L, Raw I, et al. Influence of surface properties of carbon black on the activity of adsorbed catalse. Biotechnol Appl Biochem,1999,29: 73-77.
[16] Maurizio P, Massimiliano F, Paola P, et al. Effect of stirrer speed and buffering agents on the production of glucose oxidase and catalase by Penicillium variabile(P16) in benchtop bioreactor. Enzyme and Microb Technol, 1995, 17:336-339.
[17] 陆挺,汪成富,戴英,等.过氧化氢酶发酵性能的研究.苏州大学学报,2001,17(3): 95-97.
[18] 周一,严自正,卢运玉,等.耐热过氧化氢酶产生菌的筛选和发酵条件的研究.微生物学报,1990,30(3): 223-227.
[19] Venkateshwaran G,Somashekar D,Prakash M H,et al.Production and utilization of catalase using Saccharomyces cerevisiae. Process Biochem,1999,34:187-191.
[20] Fiedurek J,Gromada A.Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture.J Appl Microbiol,2000,89: 85-89.
[21] Minagawa S,Tawara T,Terao I.Production of catalase.Jp 09-047285,1997.
[22] 王凡强,王正祥,邵蔚蓝,等.热稳定性过氧化氢酶工程菌株发酵条件的研究.食品与发酵工业,2002,28 (2):11-14.
[23] 黄文涛.酶应用手册.上海:上海科技出版社, 1989.
[24] Wang H X,Yukiko Y,Shinoyama H,et al.Purification and characterization of a thermostable catalase from culture broth of Thermoascus aurantiacus.J Ferment Bioeng,1998,85(2):169-173.
[25] Liu J Z,Huang Y Y,Liu J,et al.Effect of metal ions simultaneous production of glucose oxidase and catalase by Aspergillus niger. Lett in Appl Micro,2001,32: 16-19.
[26] 方芳.纺织用碱性耐热过氧化氢酶的微生物法生产及其应用.江南大学硕士论文,2004,3.
[27] Nishikawa Y, Kawata Y,Nagai J.Effect of triton X-10 on catalase production by Aspergillus terreus IFO 6123 [J]. J Fermentat Bioeng,1993,76:235-236.
[28] Bergmeger H U,Bergmeyer J,Grabl M,et al.Methods of Enzymatic Analysis.3rd ed, Vol3, Weinheim: Verlag Chemie Press, 1983,257-259.
[29] 许申鸿,杭瑚,李运平.超氧化物歧化酶邻苯三酚测活法的研究及改进.化学通报,2001,8:516-519.
[30] 王微青.淀粉科学手册.中国轻工业出版社,1990.
[31] 魏述众.生物化学.北京:中国轻工出版社,1996.
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