盐单胞菌S62 β-半乳糖苷酶合成低聚半乳糖的研究
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摘要
以盐单胞菌S62 β-半乳糖苷酶为研究对象,探究其合成低聚半乳糖效率。为了提高低聚半乳糖产率,对反应条件进行了优化,并以反应温度、pH、加酶量、底物浓度为考察对象进行正交试验,得到最优反应条件:反应温度40℃,pH 7.0,加酶量50 U/mL,底物质量浓度300 g/L。反应6 h时可获得最大低聚半乳糖产率(41.91±0.27)%,乳糖消耗率为(82.47±0.38)%。反应4～8 h内低聚半乳糖产率都维持在40%以上,此时乳糖消耗率均在80%以上,在提高乳糖利用率的同时实现了低聚半乳糖的高产,有利于降低生产成本,为低温S62 β-半乳糖苷酶工业化应用奠定了基础。
We used the Halomonas sp. S62 β-galactosidase to synthesize galactooligosaccharides from lactose,to explore galactooligosaccharides synthesis efficiency. To improve the yield of galactooligosaccharides,the reaction conditions were optimized,and the orthogonal test was carried out by the reaction temperature,p H,enzyme dosage and substrate concentration. The optimal reaction conditions were as follows: reaction temperature 40 ℃,p H 7. 0,enzyme dosage 50 U/m L,substrate concentration 300 g/L.The maximal yield of galactooligosaccharides was( 41. 91±0. 27) % at 6 h and the lactose consumption rate was( 82. 47±0. 38) %.Within 4-8 h,the yield of galactooligosaccharides was above 40% and the lactose consumption rate was over80%.The yield of galactooligosaccharides was maintained above 40% within 4-8 h of the reaction. The consumption rate of lactose was all above 80%. The utilization rate of lactose was increased while the high production of galactooligosaccharides was achieved,which was beneficial to the reduction of production cost,laying the foundation for the industrial application of low-temperature S62 β-galactosidase.
We used the Halomonas sp. S62 β-galactosidase to synthesize galactooligosaccharides from lactose,to explore galactooligosaccharides synthesis efficiency. To improve the yield of galactooligosaccharides,the reaction conditions were optimized,and the orthogonal test was carried out by the reaction temperature,p H,enzyme dosage and substrate concentration. The optimal reaction conditions were as follows: reaction temperature 40 ℃,p H 7. 0,enzyme dosage 50 U/m L,substrate concentration 300 g/L.The maximal yield of galactooligosaccharides was( 41. 91±0. 27) % at 6 h and the lactose consumption rate was( 82. 47±0. 38) %.Within 4-8 h,the yield of galactooligosaccharides was above 40% and the lactose consumption rate was over80%.The yield of galactooligosaccharides was maintained above 40% within 4-8 h of the reaction. The consumption rate of lactose was all above 80%. The utilization rate of lactose was increased while the high production of galactooligosaccharides was achieved,which was beneficial to the reduction of production cost,laying the foundation for the industrial application of low-temperature S62 β-galactosidase.
引文
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[24] 邢肖肖,齐崴,王梦凡,等. β-半乳糖苷酶酶学性质及其在低聚半乳糖合成中的应用[J].生物加工过程,2015,13(2):30-34.
[25] RODRIGUEZ-COLINAS B,DE ABREU M A,FERNANDEZ-ARROJO L,et al.Production of galacto-oligosaccharides by the β-galactosidase from Kluyveromyces lactis:comparative analysis of permeabilized cells versus soluble enzyme[J].J Agric Food Chem,2011,59(19):10477-10484.
[26] URRUTIA P,RODRIGUEZCOLINAS B,FERNANDEZARROJO L,et al.Detailed analysis of galactooligosaccharides synthesis with β-galactosidase from Aspergillus oryzae[J].J Agric Food Chem,2013,61(5):1081.
[27] YANAHIRA S,KOBAYASHI T,SUGURI T,et al.Formation of oligosaccharides from lactose by Bacillus circulans beta-galactosidase[J].Biosci Biotech Biochem,1995,59(6):1021-1026.
[28] VERA C,GUERRERO C,ILLANES A.Determination of the transgalactosylation activity of Aspergillus oryzae β-galactosidase:effect of pH,temperature,and galactose and glucose concentrations.[J].Carbohydr Res,2011,346(6):745-752.
[29] FAN Y,HUA X,ZHANG Y,et al.Cloning,expression and structural stability of a cold-adapted β-galactosidase from Rahnella sp.R3[J].Protein Expr Purif,2015,115:158-164.
[30] COKER J A,SHERIDAN P P,LOVELANDCURTZE J,et al.Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate[J].J Bacteriol,2003,185(18):5473-5482.
[31] GEIGER B,NGUYEN H M,WENIG S,et al.From by-product to valuable components:efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus[J].Biochem Eng J,2016,116:45-53.
[32] VASILJEVIC T,JELEN P.Oligosaccharide production and proteolysis during lactose hydrolysis using crude cellular extracts from lactic acid bacteria[J].Dairy Sci Technol,2003,83(6):453-467.
[2] NAGY Z,KISS T,SZENTIRMAI A,et al.β-Galactosidase of Penicillium chrysogenum:production,purification,and characterization of the enzyme[J].Protein Expr Purif,2001,21(1):24-29.
[3] FLOOD M T,KONDO M.Toxicity evaluation of a β-galactosidase preparation produced by Penicillium multicolor[J].Regul Toxicol Pharmacol,2004,40(3):281-292.
[4] HAIDER T,HUSAIN Q.Preparation of lactose-free milk by using salt-fractionated almond (Amygdalus communis) β-galactosidase[J].J Sci Food Agric,2010,87(7):1278-1283.
[5] TORRES,D P M,GONCALVES M D P F,TEIXEIRA J A,et al.Galacto-oligosaccharides:production,properties,applications,and significance as prebiotics[J].Compr Rev Food Sci Food Safety,2010,9(5):438-454.
[6] JURADO E,CAMACHO F,LUZóN G,et al.A new kinetic model proposed for enzymatic hydrolysis of lactose by a β-galactosidase from Kluyveromyces fragilis[J].Enzyme Microb Technol,2002,31(3):300-309.
[7] LEE D H,KANG S G,SUH S G,et al.Purification and characterization of a beta-galactosidase from peach (Prunus persica)[J].Mol Cells,2003,15(1):68-74.
[8] KLEWICKI R.Formation of gal-sorbitol during lactose hydrolysis with β-galactosidase[J].Food Chem,2007,100(3):1196-1201.
[9] VAN LEEUWEN S S,KUIPERS B J,DIJKHUIZEN L,et al.Comparative structural characterization of 7 commercial galacto-oligosaccharide (GOS) products[J].Carbohydr Res,2016,425:48-58.
[10] MUSSATTO S I,MANCILHA I M.Non-digestible oligosaccharides:a review[J].Carbohydr Polym,2007,68(3):587-597.
[11] ABURTO C,GUERRERO C,VERA C,et al.Simultaneous synthesis and purification (SSP) of galacto-oligosaccharides in batch operation[J].LWT - Food Sci Technol,2016,72:81-89.
[12] TZORTZIS G,VULEVIC J.Galacto-oligosaccharide prebiotics[M].New York:Springer,2009.
[13] KONAR E,SARKAR S,SINGHAL R S.ChemInform abstract:galactooligosaccharides:chemistry,production,properties,market status and applications[J].Cheminform,2012,https://doi.org/10.1002/chin.201204253.
[14] OOZEER R,VAN L K,LUDWIG T,et al.Intestinal microbiology in early life:specific prebiotics can have similar functionalities as human-milk oligosaccharides[J].Am J Clin Nutr,2013,98(2):561S-571S.
[15] GOURBEYRE P,DENERY S,BODINIER M.Probiotics,prebiotics,and synbiotics:impact on the gut immune system and allergic reactions[J].J Leukocyte Biol,2011,89(5):685-695.
[16] MONTEAGUDOMERA A,ARTHUR J C,JOBIN C,et al.High purity galacto-oligosaccharides enhance specific Bifidobacterium species and their metabolic activity in the mouse gut microbiome[J].Benef Microbes,2016,7(2):247-264.
[17] ARSLANOGLU S,MOROO G,SCHMITT J,et al.Early dietary intervention with a mixture of prebiotic oligosaccharides reduces the incidence of allergic manifestations and infections during the first two years of life[J].J Nutr,2008,138(6):1091-1095.
[18] HOFFEN E V,RUITER B,FABER J,et al.A specific mixture of short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides induces a beneficial immunoglobulin profile in infants at high risk for allergy[J].Allergy,2009,64(3):484-487.
[19] VULEVIC J,DRAKOULARAKOU A,YAQOOB P,et al.Modulation of the fecal microflora profile and immune function by a novel trans-galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers[J].Am J Clin Nutr,2008,88(5):1438-1446.
[20] TREMARLI V,B?CKHED F.Functional interactions between the gut microbiota and host metabolism[J].Nature,2012,71(7415):242-249.
[21] SHADID R,HAARMAN M J,THEIS W,et al.Effects of galactooligosaccharide and long-chain fructooligosaccharide supplementation during pregnancy on maternal and neonatal microbiota and immunity:a randomized,double-blind,placebo-controlled study[J].Am J Clin Nutr,2007,86(5):1426-1437.
[22] WANG G X,GAO Y,HU B,et al.A novel cold-adapted β-galactosidase isolated from Halomonas sp.S62:gene cloning,purification and enzymatic characterization[J].World J Microb Biotechnol,2013,29(8):1473-1480.
[23] 王欣,吴斌,何冰芳. β-半乳糖苷酶的筛选、克隆表达、酶学性质及其酶法合成低聚半乳糖[J].生物加工过程,2015,13(6):30-35.
[24] 邢肖肖,齐崴,王梦凡,等. β-半乳糖苷酶酶学性质及其在低聚半乳糖合成中的应用[J].生物加工过程,2015,13(2):30-34.
[25] RODRIGUEZ-COLINAS B,DE ABREU M A,FERNANDEZ-ARROJO L,et al.Production of galacto-oligosaccharides by the β-galactosidase from Kluyveromyces lactis:comparative analysis of permeabilized cells versus soluble enzyme[J].J Agric Food Chem,2011,59(19):10477-10484.
[26] URRUTIA P,RODRIGUEZCOLINAS B,FERNANDEZARROJO L,et al.Detailed analysis of galactooligosaccharides synthesis with β-galactosidase from Aspergillus oryzae[J].J Agric Food Chem,2013,61(5):1081.
[27] YANAHIRA S,KOBAYASHI T,SUGURI T,et al.Formation of oligosaccharides from lactose by Bacillus circulans beta-galactosidase[J].Biosci Biotech Biochem,1995,59(6):1021-1026.
[28] VERA C,GUERRERO C,ILLANES A.Determination of the transgalactosylation activity of Aspergillus oryzae β-galactosidase:effect of pH,temperature,and galactose and glucose concentrations.[J].Carbohydr Res,2011,346(6):745-752.
[29] FAN Y,HUA X,ZHANG Y,et al.Cloning,expression and structural stability of a cold-adapted β-galactosidase from Rahnella sp.R3[J].Protein Expr Purif,2015,115:158-164.
[30] COKER J A,SHERIDAN P P,LOVELANDCURTZE J,et al.Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate[J].J Bacteriol,2003,185(18):5473-5482.
[31] GEIGER B,NGUYEN H M,WENIG S,et al.From by-product to valuable components:efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus[J].Biochem Eng J,2016,116:45-53.
[32] VASILJEVIC T,JELEN P.Oligosaccharide production and proteolysis during lactose hydrolysis using crude cellular extracts from lactic acid bacteria[J].Dairy Sci Technol,2003,83(6):453-467.