双酶耦合催化法合成特定聚合度β-1,3-葡寡糖研究
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摘要
采用蔗糖磷酸化酶和昆布二糖磷酸化酶双酶耦合催化工艺合成β-1,3-葡寡糖。以蔗糖为底物,通过蔗糖磷酸化酶磷酸解蔗糖生成α-D-葡萄糖-1-磷酸(α-D-Glucose-1-phosphate,G1P),再以来自纤细眼虫(Euglena gracilis Z)中的昆布二糖磷酸化酶为β-1,3-葡寡糖合成酶实现G1P所带的葡萄糖单元往引物葡萄糖上的添加,通过控制催化反应时间得到不同聚合度的寡糖。在该策略下,反应0.5 h时耦合催化合成寡糖聚合度在4~6之间;反应4 h时,有7~11糖生成;4 h之后反应趋于平衡,并无更高聚合度的寡糖生成,聚合度稳定在4~11之间。对合成的寡糖进行电喷雾串联质谱(electrospray ionization tandem mass spectrometry,ESI-MS/MS)和氢核磁共振(hydrogen nuclear magnetic resonance,~1H-NMR)分析,可证明产物为β-1,3-葡寡糖。对耦合催化条件进行优化,发现在100 mmol/L的磷酸缓冲液(pH 6.5)、100 mmol/L的蔗糖、50 mmol/L的葡萄糖、5 mmol/L的MgCl_2、0.1 U/mL昆布二糖磷酸化酶、0.2 U/mL蔗糖磷酸化酶,40℃条件下反应4 h,葡萄糖的转化率可达39.4%。
With unique physiological function,β-1,3-glucooligosaccharides has been widely used as food additives and health care products. It is hard to achieve β-1,3-glucooligosaccharides with low production cost and definite degree of polymerization. Previous studies showed that sucrose phosphorylase and laminaribiose phosphorylase could be used as coupled catalysts for the production of β-1,3-glucooligosaccharides. Sucrose phosphorylase purchased from SIGMA-ALDRICH can phosphorolysis sucrose into glucose-1-phosphate and laminaribiose phosphorylase extracted from Euglena gracilis Z can realize the affixion of α-D-glucose-1-phosphate onto primer glucose,thus β-1,3-glucooligosaccharides is produced. We found that after reaction for 0. 5 h,the degree of polymerization was 4 to 6. When the reaction time reached 4 h,oligosaccharides with degree of polymerization 7 to 11 occurred. After that,the reaction system remained stable and no oligosaccharides with higher degree of polymerization were produced. To achieve better conversion rate of glucose,we optimized the reaction condition and found that the conversion rate could reached 39. 4% after reaction with 100 mmol/L phosphate buffer( pH 6. 5),100 mmol/L sucrose,50 mmol/L glucose,5 mmol/L MgCl_2,0. 1 U/mL laminaribiose phosphorylase and 0. 2 U/mL sucrose phosphorylase at 40 ℃ for 4 h. ESIMS/MS and ~1 H-NMR results of the reaction products showed that obtained products was β-1,3-glucooligosaccharides.
With unique physiological function,β-1,3-glucooligosaccharides has been widely used as food additives and health care products. It is hard to achieve β-1,3-glucooligosaccharides with low production cost and definite degree of polymerization. Previous studies showed that sucrose phosphorylase and laminaribiose phosphorylase could be used as coupled catalysts for the production of β-1,3-glucooligosaccharides. Sucrose phosphorylase purchased from SIGMA-ALDRICH can phosphorolysis sucrose into glucose-1-phosphate and laminaribiose phosphorylase extracted from Euglena gracilis Z can realize the affixion of α-D-glucose-1-phosphate onto primer glucose,thus β-1,3-glucooligosaccharides is produced. We found that after reaction for 0. 5 h,the degree of polymerization was 4 to 6. When the reaction time reached 4 h,oligosaccharides with degree of polymerization 7 to 11 occurred. After that,the reaction system remained stable and no oligosaccharides with higher degree of polymerization were produced. To achieve better conversion rate of glucose,we optimized the reaction condition and found that the conversion rate could reached 39. 4% after reaction with 100 mmol/L phosphate buffer( pH 6. 5),100 mmol/L sucrose,50 mmol/L glucose,5 mmol/L MgCl_2,0. 1 U/mL laminaribiose phosphorylase and 0. 2 U/mL sucrose phosphorylase at 40 ℃ for 4 h. ESIMS/MS and ~1 H-NMR results of the reaction products showed that obtained products was β-1,3-glucooligosaccharides.
引文
[1]陆光兴.热凝胶葡寡糖的高效定向水解制备及其生物活性研究[D].无锡:江南大学,2015.
[2]张洪涛.微生物β-1,3-葡聚糖的强化合成及最小功能单元挖掘[D].无锡:江南大学,2011.
[3]SHINYA T,MNARD R,KOZONE I,et al.Novelβ‐1,3-,1,6-oligoglucan elicitor from Alternaria alternata 102for defense responses in tobacco[J].Febs Journal,2006,273(11):2 421-2 431.
[4]KAWASHIMA S,HIROSE K,IWATA A,et al.β-glucan curdlan induces IL-10-producing CD4+T cells and inhibits allergic airway inflammation[J].Journal of Immunology,2012,189(12):5 713-5 721.
[5]ZHU Feng-mei,DU Bin,XU Bao-jun.A critical review on production and industrial applications of beta-glucans[J].Food Hydrocolloids,2016(52):275-288.
[6]侯庆华,宋文东.昆布寡糖对2型糖尿病大鼠睾丸的保护作用[J].中药新药与临床药理,2011,22(2):160-164.
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[8]DESCROIX K,VETVI CKA V,LAURENT I,et al.New oligo-β-(1,3)-glucan derivatives as immunostimulating agents[J].Bioorganic&Medicinal Chemistry,2010,18(1):348-357.
[9]MARECHAL L R.Beta-1,3-oligoglucan:orthophosphate glucosyltransferases from Euglena gracilis.II.Comparative studies between laminaribiose-and beta-1,3-oligoglucan phosphorylase[J].Biochim Biophys Acta,1967,146(2):431-442.
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[11]侯顾伟,马江锋,隋姗姗,等.蔗糖磷酸化酶制备及应用的研究进展[J].中国酿造,2010(6):17-20.
[12]VANDAMME E J,VAN L J,LAPORTE D.Dynamics and regulation of sucrose phosphorylase formation in Leuconostoc mesenteroides fermentations[J].Biotechnology and Bioengineering,1987,29(1):8-15.
[13]SILVERSTEIN R,VOET J,REED D,et al.Purification and mechanism of action of sucrose phosphorylase[J].The Journal of Biological Chemistry,1967,242(6):1 338-1 346.
[14]RUSSELL R R,MUKASA H,SHIMAMURA A,et al.Streptococcus mutans gtf A gene specifies sucrose phosphorylase[J].Infection and Immunity,1988,56(10):2 763-2 765.
[15]KULLIN B,ABRATT V R,REID S J.A functional analysis of the Bifidobacterium longum csc A and scr P genes in sucrose utilization[J].Applied Microbiology and Biotechnology,2006,72(5):975-981.
[16]VAN DEN BROEK L A M,VAN BOXTEL E L,KIEVIT R P,et al.Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083[J].Applied Microbiology And Biotechnology,2004,65(2):219-227.
[17]KITAOKA M,SASAKI T,TANIGUCHI H.Conversion of sucrose into laminaribiose using sucrose phosphorylase,xylose isomerase and laminaribiose phosphorylase[J].Journal of the Japanese Society of Starch Science,1993,40(3):311-314.
[18]YAMAMOTO Y,KAWASHIMA D,HASHIZUME A,et al.Purification and characterization of 1,3-β-D-glucan phosphorylase from Ochromonas danica[J].Bioscience Biotechnology&Biochemistry,2013,77(9):1 949-1 954.
[19]SAHEKI S,TAKEDA A,SHIMAZU T.Assay of inorganic phosphate in the mild p H range,suitable for measurement of glycogen phosphorylase activity[J].Analytical Biochemistry,1985,148(2):277-281.
[20]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1-2):248-254.
[21]KITAOKA M,SASAKI T,TANIGUCHI H.Synthesis of laminarioligosaccharides using crude extract of euglenagracilis z-cells[J].Agricultural and Biological Chemistry,1991,55(5):1 431-1 432.
[22]李恬.蔗糖为底物双酶法合成直链糊精的研究[D].无锡:江南大学,2013.
[23]NIHIRA T,SAITO Y,KITAOKA M,et al.Characterization of a laminaribiose phosphorylase from Acholeplasma laidlawii PG-8A and production of 1,3-β-d-glucosyl disaccharides[J].Carbohydrate Research,2012(361):49-54.
[24]饶名祯,顾晨荣,李云霞,等.MALDI-TOF MS技术在食品微生物领域的应用研究[J].上海师范大学学报(自然科学版),2015,44(6):681-686.
[25]PALMA A S,LIU Yan,ZHANG Hong-tao,et al.Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry[J].Molecular&Cellular Proteomics,2015,14(4):974-988.
[26]ARIFKHODZHAEV A O.Galactans and galactan-containing polysaccharides of higher plants[J].Chemistry of Natural Compounds,2000,36(3):229-244.
[27]李潇.褐藻酸寡糖的分离分析及其抗氧化活性的研究[D].西安:西北大学,2011.
[2]张洪涛.微生物β-1,3-葡聚糖的强化合成及最小功能单元挖掘[D].无锡:江南大学,2011.
[3]SHINYA T,MNARD R,KOZONE I,et al.Novelβ‐1,3-,1,6-oligoglucan elicitor from Alternaria alternata 102for defense responses in tobacco[J].Febs Journal,2006,273(11):2 421-2 431.
[4]KAWASHIMA S,HIROSE K,IWATA A,et al.β-glucan curdlan induces IL-10-producing CD4+T cells and inhibits allergic airway inflammation[J].Journal of Immunology,2012,189(12):5 713-5 721.
[5]ZHU Feng-mei,DU Bin,XU Bao-jun.A critical review on production and industrial applications of beta-glucans[J].Food Hydrocolloids,2016(52):275-288.
[6]侯庆华,宋文东.昆布寡糖对2型糖尿病大鼠睾丸的保护作用[J].中药新药与临床药理,2011,22(2):160-164.
[7]HIDA T H,ISHIBASHI K,MIURA N N,et al.Cytokine induction by a linear 1,3-glucan,curdlan-oligo,in mouse leukocytes in vitro[J].Inflammation Research,2009,58(1):9-14.
[8]DESCROIX K,VETVI CKA V,LAURENT I,et al.New oligo-β-(1,3)-glucan derivatives as immunostimulating agents[J].Bioorganic&Medicinal Chemistry,2010,18(1):348-357.
[9]MARECHAL L R.Beta-1,3-oligoglucan:orthophosphate glucosyltransferases from Euglena gracilis.II.Comparative studies between laminaribiose-and beta-1,3-oligoglucan phosphorylase[J].Biochim Biophys Acta,1967,146(2):431-442.
[10]KITAOKA M,SASAKI T,TANIGUCHI H.Purification and properties of laminaribiose phosphorylase(EC 2.4.1.31)from Euglena gracilis Z[J].Archives of Biochemistry and Biophysics,1993,304(2):508-514.
[11]侯顾伟,马江锋,隋姗姗,等.蔗糖磷酸化酶制备及应用的研究进展[J].中国酿造,2010(6):17-20.
[12]VANDAMME E J,VAN L J,LAPORTE D.Dynamics and regulation of sucrose phosphorylase formation in Leuconostoc mesenteroides fermentations[J].Biotechnology and Bioengineering,1987,29(1):8-15.
[13]SILVERSTEIN R,VOET J,REED D,et al.Purification and mechanism of action of sucrose phosphorylase[J].The Journal of Biological Chemistry,1967,242(6):1 338-1 346.
[14]RUSSELL R R,MUKASA H,SHIMAMURA A,et al.Streptococcus mutans gtf A gene specifies sucrose phosphorylase[J].Infection and Immunity,1988,56(10):2 763-2 765.
[15]KULLIN B,ABRATT V R,REID S J.A functional analysis of the Bifidobacterium longum csc A and scr P genes in sucrose utilization[J].Applied Microbiology and Biotechnology,2006,72(5):975-981.
[16]VAN DEN BROEK L A M,VAN BOXTEL E L,KIEVIT R P,et al.Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083[J].Applied Microbiology And Biotechnology,2004,65(2):219-227.
[17]KITAOKA M,SASAKI T,TANIGUCHI H.Conversion of sucrose into laminaribiose using sucrose phosphorylase,xylose isomerase and laminaribiose phosphorylase[J].Journal of the Japanese Society of Starch Science,1993,40(3):311-314.
[18]YAMAMOTO Y,KAWASHIMA D,HASHIZUME A,et al.Purification and characterization of 1,3-β-D-glucan phosphorylase from Ochromonas danica[J].Bioscience Biotechnology&Biochemistry,2013,77(9):1 949-1 954.
[19]SAHEKI S,TAKEDA A,SHIMAZU T.Assay of inorganic phosphate in the mild p H range,suitable for measurement of glycogen phosphorylase activity[J].Analytical Biochemistry,1985,148(2):277-281.
[20]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1-2):248-254.
[21]KITAOKA M,SASAKI T,TANIGUCHI H.Synthesis of laminarioligosaccharides using crude extract of euglenagracilis z-cells[J].Agricultural and Biological Chemistry,1991,55(5):1 431-1 432.
[22]李恬.蔗糖为底物双酶法合成直链糊精的研究[D].无锡:江南大学,2013.
[23]NIHIRA T,SAITO Y,KITAOKA M,et al.Characterization of a laminaribiose phosphorylase from Acholeplasma laidlawii PG-8A and production of 1,3-β-d-glucosyl disaccharides[J].Carbohydrate Research,2012(361):49-54.
[24]饶名祯,顾晨荣,李云霞,等.MALDI-TOF MS技术在食品微生物领域的应用研究[J].上海师范大学学报(自然科学版),2015,44(6):681-686.
[25]PALMA A S,LIU Yan,ZHANG Hong-tao,et al.Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry[J].Molecular&Cellular Proteomics,2015,14(4):974-988.
[26]ARIFKHODZHAEV A O.Galactans and galactan-containing polysaccharides of higher plants[J].Chemistry of Natural Compounds,2000,36(3):229-244.
[27]李潇.褐藻酸寡糖的分离分析及其抗氧化活性的研究[D].西安:西北大学,2011.