根癌农杆菌D-阿洛酮糖-3-差向异构酶基因克隆、结构预测及原核表达
|
摘要
D-阿洛酮糖-3-差向异构酶能够催化D-果糖转化为D-阿洛酮糖。为实现D-阿洛酮糖-3-差向异构酶的异源表达,设计引物,克隆并分离其序列,通过生物信息学软件分析D-阿洛酮糖-3-差向异构酶DNA和蛋白质的结构特点。结果表明,该基因开放阅读框870 bp,编码289个氨基酸;蛋白质二级结构中α-螺旋占38. 41%,β-折叠占47. 06%,无规则卷曲占14. 53%;该蛋白为亲水性蛋白,不含信号肽,无跨膜区,定位于细胞膜;构建原核表达载体并导入E. coli BL21宿主中,表达的D-阿洛酮糖-3-差向异构酶分子质量约为33 k Da,1 mmol/L IPTG诱导E. coli BL21重组菌28 h后,目的蛋白表达量和酶活分别为0. 32 g/L和3. 8 U/m L。根癌农杆菌D-阿洛酮糖-3-差向异构酶基因能够在大肠杆菌中实现表达。
D-Psicose can be converted from D-fructose catalyzed by D-psicose-3-epimerase. To realize the heterologous expression of D-psicose-3-epimerase( DPEase),the primers were designed to clone and isolate the gene. The DNA sequences and structural characteristics of DPEase were analyzed by bioinformatics. The results showed the gene possessed 870 bp open reading frame and encoded 289 amino acids.Alpha helix,beta folding,and random coil respectively accounted for 38. 41%,47. 06%,and 14. 53%in the secondary structure of the protein. The protein located at the cell membrane belonged to a hydrophilic protein without the signal peptide and transmembrane region. The prokaryotic expression vector was constructed and transferred into the E. coli BL21. The molecular weight of DPEase was appropriately 33 k Da. After an induction of 1 mmol/L IPTG for 28 h,the protein amount and enzymatic activity of the recombinant E. coli BL21 were 0. 32 g/L and 3. 8 U/m L. The DPEase gene of A. tumefaciens could be expressed in the engineered E. coli.
D-Psicose can be converted from D-fructose catalyzed by D-psicose-3-epimerase. To realize the heterologous expression of D-psicose-3-epimerase( DPEase),the primers were designed to clone and isolate the gene. The DNA sequences and structural characteristics of DPEase were analyzed by bioinformatics. The results showed the gene possessed 870 bp open reading frame and encoded 289 amino acids.Alpha helix,beta folding,and random coil respectively accounted for 38. 41%,47. 06%,and 14. 53%in the secondary structure of the protein. The protein located at the cell membrane belonged to a hydrophilic protein without the signal peptide and transmembrane region. The prokaryotic expression vector was constructed and transferred into the E. coli BL21. The molecular weight of DPEase was appropriately 33 k Da. After an induction of 1 mmol/L IPTG for 28 h,the protein amount and enzymatic activity of the recombinant E. coli BL21 were 0. 32 g/L and 3. 8 U/m L. The DPEase gene of A. tumefaciens could be expressed in the engineered E. coli.
引文
[1] CHUNG M Y,OH D K,LEE K W. Hypoglycemic health benefits of D-psicose[J]. Journal of Agricultural and Food Chemistry,2012,60(4):863-869.
[2] IIDA T,HAYASHI N,YOSHIKAWA Y,et al. Failure of D-psicose absorbed in the small intestine to metabolize into energy and its low large intestinal fermentability in humans[J]. Metabolism Clinical and Experimental,2010,59(2):206-214.
[3] HAYASHI N,IIDA T,YAMADA T,et al. Study on the postprandial blood glucose suppression effect of D-psicose in borderline diabetes and the safety of long-term ingestion by normal human subjects[J]. Bioscience Biotechnology and Biochemistry,2010,74(3):510-519.
[4] MURAO K,YU X,CAO W M,et al. D-Psicose inhibits the expression of MCP-1 induced by high-glucose stimulation in HUVECs[J]. Life Sciences,2007,81(7):592-599.
[5] CHUNG Y M,HYUN L J,YOUL K D,et al. Dietary D-psicose reduced visceral fat mass in high-fat diet-induced obese rats[J]. Journal of Food Science,2012,77(2):53-58.
[6] IIDA T,KISHIMOTO Y,YOSHIKAWA Y,et al. Acute D-psicose administration decreases the glycemic responses to an oral maltodextrin tolerance test in normal adults[J]. Journal of Nutritional Science and Vitaminology,2008. 54(6):511-514.
[7] TAKATA M K,YAMAGUCHI F,NAKANOSE K,et al.Neuroprotective effect of D-psicose on 6-hydroxydopamine-induced apoptosis in rat pheochromocytoma(PC12)cells[J]. Journal of Bioscience and Bioengineering,2005,100(5):511-516.
[8]王成福,方春雷,杜瑞峰,等.一种阿洛酮糖的制备方法及其应用:104447888A[P]. 2015-03-25.
[9] ZHU Y,MEN Y,BAI W,et al. Overexpression of D-psicose 3-epimerase from Ruminococcus sp. in Escherichia coli and its potential application in D-psicose production[J]. Biotechnology Letters,2012,34(10):1901-1906.
[10] HE W,MUW,JIANG B,et al. Food-grade expression of D-psicose 3-epimerase with tandem repeat genes in Bacillus subtilis[J]. Journal of Agricultural and Food Chemistry,2016,64(28):5701-5707.
[11] LI Z,LI Y,DUAN S,et al. Bioconversion of D-glucose to D-psicose with immobilized D-xylose isomerase and D-psicose 3-epimerase on Saccharomyces cerevisiae spores[J]. Journal of Industrial Microbiology and Biotechnology,2015,42(8):1117-1128.
[12] MU W M,CHU F,XING Q C,et al. Cloning,expression,and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10[J]. Journal of Agricultural and Food Chemistry,2011,59(14):7785-7792.
[13] CHAN H C,ZHU Y,HU Y M,et al. Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars[J]. Protein and Cell,2012,3(2):123-131.
[14] KIM H J,LIM B C,YEOM S J,et al. Roles of Ile66and Ala107 of D-psicose 3-epimerase from Agrobacterium tumefaciens in binding O6 of its substrate,D-fructose[J]. Biotechnology Letters,2010,32(1):113-118.
[15] KIM H J,HYUN E,KIM Y S,et al. Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose[J]. Applied and Environmental Microbiology,2006,72(2):981-985.
[16] ZHANG L T,JIANG B,MU W M,et al. Bioproduction of D-psicose using permeabilized cells of newly isolated Rhodobacter sphaeroides SK011[J]. Frontiers of Chemical Engineering in China,2009,3(4):393-398.
[17] ZHANG W L,ZHANG T,JIANG B,et al. Biochemical characterization of a D-psicose 3-epimerase from Treponema primitia ZAS-1 and its application on enzymatic production of D-psicose[J]. Journal of Science of Food and Agriculture,2016,96(1):49-56.
[18] JIA M,MU W M,CHU F F,et al. A D-psicose 3-epimerase with neutral p H optimum from Clostridium bolteae for D-psicose production:cloning,expression,purification,and characterization[J]. Applied Microbiology and Biotechnology,2014,98(2):717-725.
[19] HAN Y,HAN H,KIM A H,et al. D-allulose supplementation normalized the body weight and fat-pad mass in diet-induced obese mice via the regulation of lipid metabolism under isocaloric fed condition[J]. Molecular Nutrition and Food Research,2016,60(7):1695-1706.
[20] PARK C S,PARK C S,SHIN K C,et al. Production of D-psicose from D-fructose by whole recombinant cells with high-level expression of D-psicose 3-epimerase from Agrobacterium tumefaciens[J]. Journal of Bioscience and Bioengineering,2016,121(2):186-190.
[21] KIM H J,YEOM S J,KIM K,et al. Mutational analysis of the active site residues of a D-psicose 3-epimerase from Agrobacterium tumefaciens[J]. Biotechnology Letters,2010,32(2):261-268.
[2] IIDA T,HAYASHI N,YOSHIKAWA Y,et al. Failure of D-psicose absorbed in the small intestine to metabolize into energy and its low large intestinal fermentability in humans[J]. Metabolism Clinical and Experimental,2010,59(2):206-214.
[3] HAYASHI N,IIDA T,YAMADA T,et al. Study on the postprandial blood glucose suppression effect of D-psicose in borderline diabetes and the safety of long-term ingestion by normal human subjects[J]. Bioscience Biotechnology and Biochemistry,2010,74(3):510-519.
[4] MURAO K,YU X,CAO W M,et al. D-Psicose inhibits the expression of MCP-1 induced by high-glucose stimulation in HUVECs[J]. Life Sciences,2007,81(7):592-599.
[5] CHUNG Y M,HYUN L J,YOUL K D,et al. Dietary D-psicose reduced visceral fat mass in high-fat diet-induced obese rats[J]. Journal of Food Science,2012,77(2):53-58.
[6] IIDA T,KISHIMOTO Y,YOSHIKAWA Y,et al. Acute D-psicose administration decreases the glycemic responses to an oral maltodextrin tolerance test in normal adults[J]. Journal of Nutritional Science and Vitaminology,2008. 54(6):511-514.
[7] TAKATA M K,YAMAGUCHI F,NAKANOSE K,et al.Neuroprotective effect of D-psicose on 6-hydroxydopamine-induced apoptosis in rat pheochromocytoma(PC12)cells[J]. Journal of Bioscience and Bioengineering,2005,100(5):511-516.
[8]王成福,方春雷,杜瑞峰,等.一种阿洛酮糖的制备方法及其应用:104447888A[P]. 2015-03-25.
[9] ZHU Y,MEN Y,BAI W,et al. Overexpression of D-psicose 3-epimerase from Ruminococcus sp. in Escherichia coli and its potential application in D-psicose production[J]. Biotechnology Letters,2012,34(10):1901-1906.
[10] HE W,MUW,JIANG B,et al. Food-grade expression of D-psicose 3-epimerase with tandem repeat genes in Bacillus subtilis[J]. Journal of Agricultural and Food Chemistry,2016,64(28):5701-5707.
[11] LI Z,LI Y,DUAN S,et al. Bioconversion of D-glucose to D-psicose with immobilized D-xylose isomerase and D-psicose 3-epimerase on Saccharomyces cerevisiae spores[J]. Journal of Industrial Microbiology and Biotechnology,2015,42(8):1117-1128.
[12] MU W M,CHU F,XING Q C,et al. Cloning,expression,and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10[J]. Journal of Agricultural and Food Chemistry,2011,59(14):7785-7792.
[13] CHAN H C,ZHU Y,HU Y M,et al. Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars[J]. Protein and Cell,2012,3(2):123-131.
[14] KIM H J,LIM B C,YEOM S J,et al. Roles of Ile66and Ala107 of D-psicose 3-epimerase from Agrobacterium tumefaciens in binding O6 of its substrate,D-fructose[J]. Biotechnology Letters,2010,32(1):113-118.
[15] KIM H J,HYUN E,KIM Y S,et al. Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose[J]. Applied and Environmental Microbiology,2006,72(2):981-985.
[16] ZHANG L T,JIANG B,MU W M,et al. Bioproduction of D-psicose using permeabilized cells of newly isolated Rhodobacter sphaeroides SK011[J]. Frontiers of Chemical Engineering in China,2009,3(4):393-398.
[17] ZHANG W L,ZHANG T,JIANG B,et al. Biochemical characterization of a D-psicose 3-epimerase from Treponema primitia ZAS-1 and its application on enzymatic production of D-psicose[J]. Journal of Science of Food and Agriculture,2016,96(1):49-56.
[18] JIA M,MU W M,CHU F F,et al. A D-psicose 3-epimerase with neutral p H optimum from Clostridium bolteae for D-psicose production:cloning,expression,purification,and characterization[J]. Applied Microbiology and Biotechnology,2014,98(2):717-725.
[19] HAN Y,HAN H,KIM A H,et al. D-allulose supplementation normalized the body weight and fat-pad mass in diet-induced obese mice via the regulation of lipid metabolism under isocaloric fed condition[J]. Molecular Nutrition and Food Research,2016,60(7):1695-1706.
[20] PARK C S,PARK C S,SHIN K C,et al. Production of D-psicose from D-fructose by whole recombinant cells with high-level expression of D-psicose 3-epimerase from Agrobacterium tumefaciens[J]. Journal of Bioscience and Bioengineering,2016,121(2):186-190.
[21] KIM H J,YEOM S J,KIM K,et al. Mutational analysis of the active site residues of a D-psicose 3-epimerase from Agrobacterium tumefaciens[J]. Biotechnology Letters,2010,32(2):261-268.