以甘油为底物产二羟丙酮（DHA）微生物的选育及甘油脱氢酶酶学性质的研究
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摘要
二羟丙酮(DHA)是一种重要的化工原料,在精细化工、制药、食品等方面具有广泛的应用前景。本研究从自然界筛选出21株能够以甘油为唯一碳源产二羟丙酮的菌株,经初步发酵测定发酵液中DHA含量,其中菌株6-8 DHA产量最高达6.2 g/L。对其进行常规生理生化鉴定实验,并结合16S rDNA基因分析,比对结果表明,菌株6-8与Acinetobacter sp. B8-22相似性最高,达99.7%,在细菌分类学上属于假单胞菌目莫拉菌科不动杆菌属,将其命名为Acinetobacter sp.6-8。在研究Acinetobacter sp. 6-8在LB培养基中的生长曲线的基础上,考察了培养时间与培养温度对Acinetobacter sp. 6-8发酵DHA的影响,最终确定37℃时发酵72 h即可获得较高的DHA产量。在摇瓶水平上,分别考察了碳源和氮源对Acinetobacter sp. 6-8发酵产DHA的影响,并对碳氮源综合水平进行了正交实验优化,最终确定采用甘油10.0%、山梨醇2.0%、酵母膏0.5%、玉米浆0.2%作为发酵培养基的碳氮源浓度,以此条件发酵,DHA产量为7.21 g/L,比未优化时DHA产量提高了9.8%;
建立了简单且准确测定二羟丙酮(DHA)的高效液相色谱(HPLC)方法,以Alltima C18(5μm,250×4.6 mm)为分离柱,5%甲醇水溶液(0.05%H3PO4调pH至3.0)为流动相,用紫外检测器在203 nm处检测DHA。结果测得DHA标准样品在203nm的保留时间为6.2 min,并测得DHA的线性范围为0.1～10.0 g/L,用HPLC法测得以甘油为底物发酵产DHA发酵液中DHA含量与显色法测得的DHA含量一致。
以克雷伯氏菌基因组DNA为模板,扩增得到编码甘油脱氢酶(GDH)基因dhaD,将其克隆到大肠杆菌表达载体pET-28a(+)上,在E.coli BL21(DE3)中诱导表达,利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni2+柱纯化具有表达活性的甘油脱氢酶(GDH),纯化后比酶活达到156 U/mg,纯化倍数达4.6倍,回收率为67.4%。并初步研究了该酶的酶学性质:酶反应的最适pH为11.0,在pH7.0～12.0范围内稳定;酶反应的最适温度为30℃,稳定范围为25℃～45℃;Mg2+、Cu2+、Zn2+、K+对酶促反应有一定的激活作用;酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49μmol/(mL·min),说明了此酶对甘油的亲和性较高。本实验采用了生物全细胞转化的方法,将重组大肠杆菌培养至对数生长后期,经IPTG诱导收集菌体,将其加入转化液中,利用全细胞转化法只需12 h甘油转化为DHA达1.4 g/L。为利用基因工程菌的工业化应用打下了良好的基础。
Dihydroxyacetone is a kind of important chemical materials, which has been widely applied in chemistry, pharmacy, food and so on. More than 20 strains capable of producing dihydroxyacetone from glycerol were isolated from 4 different natural environment samples by using two detection methods. The strain 6-8 which could grow on medium containing glycerol as sole carbon source had a higher converting capability. Under a better culture, the highest DHA production of the strain 6-8 reached 6.2 g/L. In addition to general morphological and biochemical characteristics, the strain 6-8 was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of the strain 6-8 had similarity of 99.7% with Acinetobacter sp. B8-22 suggesting that the strain 6-8 is one of subspecies of Acinetobacter sp. The experiment was used to optimiaze parameters of the fermentation condition of Acinetobacter sp. 6-8. The maximal yield of DHA (7.21 g/L) could be obtained when the concentrations of glycerol, mannitol, yeast extract, and corn steep liquor were set at 10.0%, 2.0%, 0.5%, and 0.2%. In this condition the production of DHA was enhanced by 9.8% than that of the prior to optimization.
A method to determine dihydroxyacetone (DHA) in fermentation broth was developed by high performance liquid chromatography (HPLC). DHA was separated on an Alltima C18( 5μm,250×4.6 mm ). The mobile phase was 0.5% methanol solution (pH adjusted to 3.0 with H3PO4), the flow-rate was 1.0 mL/min and the detective wavelength was 203 nm. The detection limits of DHA was 0.1 g/L～10.0 g/L. 6.2 g/L DHA in the fermentation broth was detected by HPLC method, which was in agreement with the result by spectrophotometric method. The method in this paper was simple, rapid and applicable for DHA determination in the fermentation broth. The method was applicable for DHA determination in the fermentation process.
The dhaD gene encoding glycerol dehydrogenase (GDH) from Klebsiella sp. was amplified and inserted into expression vector pET-28a(+), the plasmid pET-28a-dhaD was constructed and introduced into Escherichia coli BL21 (DE3).SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21. Then GDH was purified by Ni-NTA affinity chromatography, the result showed a single band about 39 kDa on SDS-PAGE gel, and the specified activity was about 156 U/mg, the special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%. The optimum reaction pH was 11.0, and the GDH activity have little change when incubated in the buffer of pH 7.0~11.0. The optimum reactive temperature was 30℃,and the GDH was more stable on the temperature of 25℃~45℃. The Km value was 0.54 mmol/L and Vmax was 0.49μmol/(mL·min) in the glycerol. The production of dihydroxyacetone from glycerol was 1.4 g/L by whole cell catalysed conversion of recombinant E.coli BL21.
建立了简单且准确测定二羟丙酮(DHA)的高效液相色谱(HPLC)方法,以Alltima C18(5μm,250×4.6 mm)为分离柱,5%甲醇水溶液(0.05%H3PO4调pH至3.0)为流动相,用紫外检测器在203 nm处检测DHA。结果测得DHA标准样品在203nm的保留时间为6.2 min,并测得DHA的线性范围为0.1～10.0 g/L,用HPLC法测得以甘油为底物发酵产DHA发酵液中DHA含量与显色法测得的DHA含量一致。
以克雷伯氏菌基因组DNA为模板,扩增得到编码甘油脱氢酶(GDH)基因dhaD,将其克隆到大肠杆菌表达载体pET-28a(+)上,在E.coli BL21(DE3)中诱导表达,利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni2+柱纯化具有表达活性的甘油脱氢酶(GDH),纯化后比酶活达到156 U/mg,纯化倍数达4.6倍,回收率为67.4%。并初步研究了该酶的酶学性质:酶反应的最适pH为11.0,在pH7.0～12.0范围内稳定;酶反应的最适温度为30℃,稳定范围为25℃～45℃;Mg2+、Cu2+、Zn2+、K+对酶促反应有一定的激活作用;酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49μmol/(mL·min),说明了此酶对甘油的亲和性较高。本实验采用了生物全细胞转化的方法,将重组大肠杆菌培养至对数生长后期,经IPTG诱导收集菌体,将其加入转化液中,利用全细胞转化法只需12 h甘油转化为DHA达1.4 g/L。为利用基因工程菌的工业化应用打下了良好的基础。
Dihydroxyacetone is a kind of important chemical materials, which has been widely applied in chemistry, pharmacy, food and so on. More than 20 strains capable of producing dihydroxyacetone from glycerol were isolated from 4 different natural environment samples by using two detection methods. The strain 6-8 which could grow on medium containing glycerol as sole carbon source had a higher converting capability. Under a better culture, the highest DHA production of the strain 6-8 reached 6.2 g/L. In addition to general morphological and biochemical characteristics, the strain 6-8 was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of the strain 6-8 had similarity of 99.7% with Acinetobacter sp. B8-22 suggesting that the strain 6-8 is one of subspecies of Acinetobacter sp. The experiment was used to optimiaze parameters of the fermentation condition of Acinetobacter sp. 6-8. The maximal yield of DHA (7.21 g/L) could be obtained when the concentrations of glycerol, mannitol, yeast extract, and corn steep liquor were set at 10.0%, 2.0%, 0.5%, and 0.2%. In this condition the production of DHA was enhanced by 9.8% than that of the prior to optimization.
A method to determine dihydroxyacetone (DHA) in fermentation broth was developed by high performance liquid chromatography (HPLC). DHA was separated on an Alltima C18( 5μm,250×4.6 mm ). The mobile phase was 0.5% methanol solution (pH adjusted to 3.0 with H3PO4), the flow-rate was 1.0 mL/min and the detective wavelength was 203 nm. The detection limits of DHA was 0.1 g/L～10.0 g/L. 6.2 g/L DHA in the fermentation broth was detected by HPLC method, which was in agreement with the result by spectrophotometric method. The method in this paper was simple, rapid and applicable for DHA determination in the fermentation broth. The method was applicable for DHA determination in the fermentation process.
The dhaD gene encoding glycerol dehydrogenase (GDH) from Klebsiella sp. was amplified and inserted into expression vector pET-28a(+), the plasmid pET-28a-dhaD was constructed and introduced into Escherichia coli BL21 (DE3).SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21. Then GDH was purified by Ni-NTA affinity chromatography, the result showed a single band about 39 kDa on SDS-PAGE gel, and the specified activity was about 156 U/mg, the special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%. The optimum reaction pH was 11.0, and the GDH activity have little change when incubated in the buffer of pH 7.0~11.0. The optimum reactive temperature was 30℃,and the GDH was more stable on the temperature of 25℃~45℃. The Km value was 0.54 mmol/L and Vmax was 0.49μmol/(mL·min) in the glycerol. The production of dihydroxyacetone from glycerol was 1.4 g/L by whole cell catalysed conversion of recombinant E.coli BL21.
引文
1.张育川.二羟丙酮(DHA)[J].专用化学品, 2003, 22: 21
2. Rosaria C, Giovanni P, Cristina D P, et al. One-pot electroactalytic oxidation of glycerol to DHA [J]. Tetrahedron letters, 2006. 22(3):6993-6995
3. Flickineger M C, Perlman D. Application of oxygen-enriches aeration in the conversion of glycerol to dihydroxyacetone by Gluconobacter nelanogenus IFO3293 [J]. Applied and Environmental Microbiology, 1997, 33(3): 706-712
4. Green S R, Whalen E A, Molokie E. Dihydroxyacetone: production and uses [J]. Journal of Biochemical and Microbiological Technology and Engineering, 1961, 3(4): 351-355
5.左华,赵宝祥. 1,3-二羟基丙酮衍生物的合成[J].有机化学, 2004, 4, 331-333
6. Claus Peter, Demirel Seval, Lucas Martin, et al. Method for the selective production of dihydeoxyacetone from glycerin, and method for the production of a metal catalyst used for the selective oxidation of glycerin[P]. European Patent.WO2007033807. 2007-03-29
7. Charney William. Process for the production of dihydroxyacetone[P]. American Patent US4076589. 1978-02-28
8.张霞.微生物转化法生产二羟基丙酮[D]: [硕士学位论文].杭州:浙江工业大学, 2001
9. Claret C, Salmon J M, Romieu C, et al. Physiology of Gluconobacter oxydans during dihydroxyacetone production from glycerol [J]. Appl Microbiol Biotechnol, 1994, 41: 359-365
10. Yamada S, Nabe K, Izuo N, et al. Enzymatic Production of dihydroxyacetone by Acetobacter suboxydans ATCC621 [J]. J Ferment Technol, 1979, 57(3): 221-226
11.郑裕国,张霞,沈寅初,等.微生物转化甘油生产1,3-二羟基丙酮的菌株筛选[J].浙江工业大学学报, 2001, 29(2):124-127
12. Bories A, Claret C, Soucaille P. Production of dihydroxyacetone by cultivation of Acetobater suboxydans [J]. J Ferment Technol, 1980, 58(3):221-226
13. Bories A, Claret C, Soucaille P. Kinetic study and optimization of the production of dihydroxyacetone from glycerol using Gluconobacter oxydans[J]. Process Biochemistry, 1991, 26: 243-248
14. Batzing B L, Claus G W. Biphasic growth of Actobater suboxydans on a glycerol limiting medium [J]. Journal of Bacteriology, 1971, 108(1): 592-595
15. Underkofler L A, Ellis I F. The production of dihydroxyacetone by the action of the action of Acetobacter xylinum [J]. American Chemistry Society, 1937, 59: 301-302
16. Yamada S, Nabe K, Izuo N, et al. Fermentativ production of dihydroxyacetone by Acetobacter suboxydans ATCC621 [J]. J Ferment Technol, 1979, 57(3): 221-226
17. Nabe K, Izuo N, Yamada S. Conversion of glycerol to dihydroxyacetone by immobilized whole cells of Acetobacter xylinum [J]. Applied and Environmental Microbiology, 1979, 38(6): 1056-1060
18.赵金红,陈守义,李军涛.二羟基丙酮的生产方法[P].中国专利, 200510033823.5. 2005-10-26
19. Lin E, Levin A, Magasanik B. The effect of aerobic metabolism on the inducible glycerol dehydrogenase of Aerobacter aerogenes [J], J Biol Chem, 1960, 235(3): 1824-1828
20. Tang C T, Ruch F E, Lin E C C, Purification and properies of a NAD+-linked dehydrogenase that serves an Escherichia coli mutant for glycerol catabolism [J]. J. Bacteriol, 1979. 140 (1): 182-187
21.张小梅,诸葛健.含甘油脱水酶激活因子编码基因的产1,3-PD新型重组菌的构建[J].中国生物工程学报, 2007, 23(5): 842-845
22.张勇,郑裕国. 1,3-二羟基丙酮生物技术法生产及其在生物体内的代谢[J].中国现代应用药学杂志, 2005, 22(7): 622-624
23.张智宏,孙小娟.二羟基丙酮的气相色谱分析[J].分析测试学报, 1999, 18: 56-63
24. Wang L L, Qian J, Hu Zh C. Determination of dihydroxyacetone and glycerol in fermentation broth by pyrolytic methylation/gas chromatography [J]. Analytica Chimica Acta, 2006, 557,262-266
25. Claret C, Bories A. Soucaille P. Glycerol inhibition of growth and dihydroxyacetone production by Gluconobater oxydans [J]. Cerrent Microbiology, 1992, 25:149-155
26.陈宏文.生物法合成1,3-丙二醇的过程工程研究[D]: [硕士学位论文].天津:天津大学化工学院, 2005
27.诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社, 1994, 223-225
28. Lambert M, Neish A C. Rapid method for estimation of glycerol in fermentation solutions [J]. Can J Res, 1956, 28: 83-89
29.陈立仁,蒋生祥,刘霞等.高效液相色谱基础与实践[M].第一版.北京:科学出版社, 2001, 114-120
30.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社, 2001, 188-192
31. Joseph S, David W R. Molecular Cloning. 3rd. New York: Cold Spring Harbor Laboratory Press, 2001, 106-454
32.冯屏,周家春,徐玉佩.膜生物反应器连续发酵法制取二羟基丙酮的研究[J].食品与发酵工业, 2003, 12(29): 40-43
33.褚以文.微生物培养基优化方法及其OPTI优化软件[J].国外医药抗生素册, 1999, 20(2): 58- 61
34.吴有炜.试验设计与数据处理[M].苏州:苏州大学出版社, 2002
35. Varuzhan H A. A novel method of production of theanine by immobilized pseudomonas nitroreducens cells [J]. Biosci Biotech Biochem, 1993, 57(3): 481-483
36. Tang J C T, Forage R G, Lin E C C, Derepression of an NAD+-linked dehydrogenase that serves an Escherichia coli mutant for growth on glycerol [J]. J. Bacteriology, 1982, 152(3), 1001-1007
37. Bradford M M. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Anal Biochem, 1976, 72: 248-254
38.李育阳主编.基因表达技术[M].北京:科学出版社, 2001
39. Leichus B, Blanchard J. Isotopic Analysis of the reaction catalyzed by glycerol dehydrogense [J], Biochem, 1994, 33(48): 14642-14647
40. Veronica T, Winfried B, Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase [J]. J Bacteriology, 1994, 176(6): 1796-1800
41. Hekmat D, Bauer R, Neff V. Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans [J]. Process Biochemistry, 2006, 42(1) ,71-76
42.陈宏文,吴雅红,吴振华,等.克雷伯杆菌甘油脱氢酶的分离纯化及性质[J].无锡轻工大学学报, 2005, 24(1): 1-5
2. Rosaria C, Giovanni P, Cristina D P, et al. One-pot electroactalytic oxidation of glycerol to DHA [J]. Tetrahedron letters, 2006. 22(3):6993-6995
3. Flickineger M C, Perlman D. Application of oxygen-enriches aeration in the conversion of glycerol to dihydroxyacetone by Gluconobacter nelanogenus IFO3293 [J]. Applied and Environmental Microbiology, 1997, 33(3): 706-712
4. Green S R, Whalen E A, Molokie E. Dihydroxyacetone: production and uses [J]. Journal of Biochemical and Microbiological Technology and Engineering, 1961, 3(4): 351-355
5.左华,赵宝祥. 1,3-二羟基丙酮衍生物的合成[J].有机化学, 2004, 4, 331-333
6. Claus Peter, Demirel Seval, Lucas Martin, et al. Method for the selective production of dihydeoxyacetone from glycerin, and method for the production of a metal catalyst used for the selective oxidation of glycerin[P]. European Patent.WO2007033807. 2007-03-29
7. Charney William. Process for the production of dihydroxyacetone[P]. American Patent US4076589. 1978-02-28
8.张霞.微生物转化法生产二羟基丙酮[D]: [硕士学位论文].杭州:浙江工业大学, 2001
9. Claret C, Salmon J M, Romieu C, et al. Physiology of Gluconobacter oxydans during dihydroxyacetone production from glycerol [J]. Appl Microbiol Biotechnol, 1994, 41: 359-365
10. Yamada S, Nabe K, Izuo N, et al. Enzymatic Production of dihydroxyacetone by Acetobacter suboxydans ATCC621 [J]. J Ferment Technol, 1979, 57(3): 221-226
11.郑裕国,张霞,沈寅初,等.微生物转化甘油生产1,3-二羟基丙酮的菌株筛选[J].浙江工业大学学报, 2001, 29(2):124-127
12. Bories A, Claret C, Soucaille P. Production of dihydroxyacetone by cultivation of Acetobater suboxydans [J]. J Ferment Technol, 1980, 58(3):221-226
13. Bories A, Claret C, Soucaille P. Kinetic study and optimization of the production of dihydroxyacetone from glycerol using Gluconobacter oxydans[J]. Process Biochemistry, 1991, 26: 243-248
14. Batzing B L, Claus G W. Biphasic growth of Actobater suboxydans on a glycerol limiting medium [J]. Journal of Bacteriology, 1971, 108(1): 592-595
15. Underkofler L A, Ellis I F. The production of dihydroxyacetone by the action of the action of Acetobacter xylinum [J]. American Chemistry Society, 1937, 59: 301-302
16. Yamada S, Nabe K, Izuo N, et al. Fermentativ production of dihydroxyacetone by Acetobacter suboxydans ATCC621 [J]. J Ferment Technol, 1979, 57(3): 221-226
17. Nabe K, Izuo N, Yamada S. Conversion of glycerol to dihydroxyacetone by immobilized whole cells of Acetobacter xylinum [J]. Applied and Environmental Microbiology, 1979, 38(6): 1056-1060
18.赵金红,陈守义,李军涛.二羟基丙酮的生产方法[P].中国专利, 200510033823.5. 2005-10-26
19. Lin E, Levin A, Magasanik B. The effect of aerobic metabolism on the inducible glycerol dehydrogenase of Aerobacter aerogenes [J], J Biol Chem, 1960, 235(3): 1824-1828
20. Tang C T, Ruch F E, Lin E C C, Purification and properies of a NAD+-linked dehydrogenase that serves an Escherichia coli mutant for glycerol catabolism [J]. J. Bacteriol, 1979. 140 (1): 182-187
21.张小梅,诸葛健.含甘油脱水酶激活因子编码基因的产1,3-PD新型重组菌的构建[J].中国生物工程学报, 2007, 23(5): 842-845
22.张勇,郑裕国. 1,3-二羟基丙酮生物技术法生产及其在生物体内的代谢[J].中国现代应用药学杂志, 2005, 22(7): 622-624
23.张智宏,孙小娟.二羟基丙酮的气相色谱分析[J].分析测试学报, 1999, 18: 56-63
24. Wang L L, Qian J, Hu Zh C. Determination of dihydroxyacetone and glycerol in fermentation broth by pyrolytic methylation/gas chromatography [J]. Analytica Chimica Acta, 2006, 557,262-266
25. Claret C, Bories A. Soucaille P. Glycerol inhibition of growth and dihydroxyacetone production by Gluconobater oxydans [J]. Cerrent Microbiology, 1992, 25:149-155
26.陈宏文.生物法合成1,3-丙二醇的过程工程研究[D]: [硕士学位论文].天津:天津大学化工学院, 2005
27.诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社, 1994, 223-225
28. Lambert M, Neish A C. Rapid method for estimation of glycerol in fermentation solutions [J]. Can J Res, 1956, 28: 83-89
29.陈立仁,蒋生祥,刘霞等.高效液相色谱基础与实践[M].第一版.北京:科学出版社, 2001, 114-120
30.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社, 2001, 188-192
31. Joseph S, David W R. Molecular Cloning. 3rd. New York: Cold Spring Harbor Laboratory Press, 2001, 106-454
32.冯屏,周家春,徐玉佩.膜生物反应器连续发酵法制取二羟基丙酮的研究[J].食品与发酵工业, 2003, 12(29): 40-43
33.褚以文.微生物培养基优化方法及其OPTI优化软件[J].国外医药抗生素册, 1999, 20(2): 58- 61
34.吴有炜.试验设计与数据处理[M].苏州:苏州大学出版社, 2002
35. Varuzhan H A. A novel method of production of theanine by immobilized pseudomonas nitroreducens cells [J]. Biosci Biotech Biochem, 1993, 57(3): 481-483
36. Tang J C T, Forage R G, Lin E C C, Derepression of an NAD+-linked dehydrogenase that serves an Escherichia coli mutant for growth on glycerol [J]. J. Bacteriology, 1982, 152(3), 1001-1007
37. Bradford M M. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Anal Biochem, 1976, 72: 248-254
38.李育阳主编.基因表达技术[M].北京:科学出版社, 2001
39. Leichus B, Blanchard J. Isotopic Analysis of the reaction catalyzed by glycerol dehydrogense [J], Biochem, 1994, 33(48): 14642-14647
40. Veronica T, Winfried B, Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase [J]. J Bacteriology, 1994, 176(6): 1796-1800
41. Hekmat D, Bauer R, Neff V. Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans [J]. Process Biochemistry, 2006, 42(1) ,71-76
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