米曲霉来源α-半乳糖苷酶基因gal A在毕赤酵母中的表达及其对豆浆中大豆寡糖的酶解效果
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摘要
本试验旨在研究米曲霉来源α-半乳糖苷酶基因gal A在毕赤酵母中的表达及其对豆浆中大豆寡糖的酶解效果。以NCBI数据库中米曲霉来源α-半乳糖苷酶基因gal A的mRNA序列(GenBank登录号:XP_001817311.1)为依据,利用PCR扩增得到gal A基因并根据毕赤酵母使用密码子的偏好性优化基因序列,构建野生型和优化型毕赤酵母工程菌株,摇瓶发酵120 h,测定酶学性质。设置25和45℃2个温度,每个温度下各设置0.6、1.2和2.4 U 3个加酶量,用发酵得到的α-半乳糖苷酶酶解10 mL豆浆中的大豆寡糖。结果显示:该α-半乳糖苷酶基因gal A全长1 605 bp,不含内含子,编码534个氨基酸,诱导120 h后优化型工程菌株的α-半乳糖苷酶活性为1.952 U/mL,比野生型工程菌株提高了285%。发酵得到的α-半乳糖苷酶的最适pH为4.33,最适温度为55℃;在pH 3.00～8.00间稳定性良好,在55℃条件下保持40 min后残余α-半乳糖苷酶相对活性为60%;该酶对大部分金属离子具有抗性,但其被MnSO4抑制;以对硝基苯基-β-D-吡喃半乳糖苷(p NPG)为底物时的酶动力学参数米氏常数(Km)为0.024 3 mol/L,最大反应速度(Vmax)为1.0×10-7mol/(L·s)。酶解试验结果显示,45℃下加酶量为2.4 U反应12 h后,大豆寡糖中棉籽糖的降解率为50.0%,水苏糖的降解率为31.9%。由此可见,本试验中生产的α-半乳糖苷酶对豆浆中的大豆寡糖有一定的降解作用。
The aim of this experiment was to express the α-galactosidase gene gal-A from Aspergillus oryzae in Pichia pastoris and study its enzymolysis effect on soybean oligosaccharides in soymilk.The specific primers were designed on the basis of mRNA sequence published in NCBI database(GenBank accession number:XP_001817311.1),and were used in polymerase chain reaction(PCR) to obtain the sequence of gal-A gene.Codon sequence was optimized according to the codon bias of Pichia pastoris.Wild type and optimized type Pichia pastoris engineered strains were constructed,fermented in shaking flask for 120 h and the enzyme properties were determined.Ten mL soymilk were treated with different concentrations(0.6,1.2 and 2.4 U) of α-galactosidase under different temperatures(25 and 45 ℃).The results showed that the full length of α-galactosidase gene gal-A was 1 605 bp without intron,coding 534 amino acids.After inducing 120 h,the α-galactosidase activity of optimized type engineered strain was 1.952 U/mL,which improved 285% compared with the wild type engineered strain.Optimal pH and temperature of this α-galactosidase was 4.33 and 55 ℃,respectively.The enzyme showed good pH stability at the range of pH 3.00 to 8.00.For temperature stability,after incubating 40 min in 55 ℃,the relative activity of residue α-galactosidase was 60%.The α-galactosidase showed resistance to most of metal ions detected,while was inhibited by M nSO4.For enzyme kinetic characteristics,the M ichaelis constant(Km) and the maximum reaction velocity(Vmax) of α-galactosidase using 4-nitrophenyl-β-D-galactoside(p NPG) as substrate were 0.024 3 mol/L and 1.0×10-7 mol/(L ·s),respectively.Enzymatic hydrolysis results showed that soybean oligosaccharides were degradated under 45 ℃ with 2.4 U α-galactosidase,after 12 h,the degradation rate of raffinose and stachyose were 50.0% and 31.9%,respectively.These results indicate that α-galactosidase obtained in this experiment can degrade soybean oligosaccharides in soymilk to some extent.
The aim of this experiment was to express the α-galactosidase gene gal-A from Aspergillus oryzae in Pichia pastoris and study its enzymolysis effect on soybean oligosaccharides in soymilk.The specific primers were designed on the basis of mRNA sequence published in NCBI database(GenBank accession number:XP_001817311.1),and were used in polymerase chain reaction(PCR) to obtain the sequence of gal-A gene.Codon sequence was optimized according to the codon bias of Pichia pastoris.Wild type and optimized type Pichia pastoris engineered strains were constructed,fermented in shaking flask for 120 h and the enzyme properties were determined.Ten mL soymilk were treated with different concentrations(0.6,1.2 and 2.4 U) of α-galactosidase under different temperatures(25 and 45 ℃).The results showed that the full length of α-galactosidase gene gal-A was 1 605 bp without intron,coding 534 amino acids.After inducing 120 h,the α-galactosidase activity of optimized type engineered strain was 1.952 U/mL,which improved 285% compared with the wild type engineered strain.Optimal pH and temperature of this α-galactosidase was 4.33 and 55 ℃,respectively.The enzyme showed good pH stability at the range of pH 3.00 to 8.00.For temperature stability,after incubating 40 min in 55 ℃,the relative activity of residue α-galactosidase was 60%.The α-galactosidase showed resistance to most of metal ions detected,while was inhibited by M nSO4.For enzyme kinetic characteristics,the M ichaelis constant(Km) and the maximum reaction velocity(Vmax) of α-galactosidase using 4-nitrophenyl-β-D-galactoside(p NPG) as substrate were 0.024 3 mol/L and 1.0×10-7 mol/(L ·s),respectively.Enzymatic hydrolysis results showed that soybean oligosaccharides were degradated under 45 ℃ with 2.4 U α-galactosidase,after 12 h,the degradation rate of raffinose and stachyose were 50.0% and 31.9%,respectively.These results indicate that α-galactosidase obtained in this experiment can degrade soybean oligosaccharides in soymilk to some extent.
引文
[1]CHEN L Y,VADLANI P V,MADL R L.High-efficiency removal of phytic acid in soy meal using twostage temperature-induced Aspergillus oryzae solidstate fermentation[J].Journal of the Science of Food and Agriculture,2014,94(1):113-118.
[2]EL-SHEMY H,ABDEL-RAHIM E,SHABAN O,et al.Comparison of nutritional and antinutritional factors in soybean and fababean seeds with or without cortex[J].Soil Science and Plant Nutrtion,2000,46(2):515-524.
[3]ZOU W,WANG M,YAO D,et al.Fluorescence and circular dichroism spectroscopy to understand the interactions between cyclodextrins andα-galactosidase from green coffee beans[J].Food Bioscience,2017,20:110-115.
[4]TSANIKLIDIS G,BENOVIAS A,DELIS C,et al.Acidic alpha galactosidase during the maturation and cold storage of cherry tomatoes[J].Acta Physiologiae Plantarum,2016,38(2):57.
[5]ZHA H G,FLOWERS V L,YANG M,et al.Acidicα-galactosidase is the most abundant nectarin in floral nectar of common tobacco(Nicotiana tabacum)[J].Annals of Botany,2012,109(4):735-745.
[6]AULITTO M,FUSCO S,FIORENTINO G,et al.Thermus thermophilus as source of thermozymes for biotechnological applications:homologous expression and biochemical characterization of anα-galactosidase[J].Microbial Cell Factories,2017,16:28.
[7]XU H B,QIN Y J,HUANG Z Q,et al.Characterization and site-directed mutagenesis of anα-galactosidase from the deep-sea bacterium Bacillus megaterium[J].Enzyme and Microbial Technology,2014,56:46-52.
[8]LEE A,CHOI K H,YOON D,et al.Characterization of a thermostable glycoside hydrolase family 36α-galactosidase from Caldicellulosiruptor bescii[J].Journal of Bioscience and Bioengineering,2017,124(3):289-295.
[9]GONG W,XU L,GU G F,et al.Efficient and regioselective synthesis of globotriose by a novelα-galactosidase from Bacteroides fragilis[J].Applied M icrobiology and Biotechnology,2016,100(15):6693-6702.
[10]GOLOTIN V A,BALABANOVA L A,NOSKOVA Y A,et al.Optimization of cold-adapted alpha-galactosidase expression in Escherichia coli[J].Protein Expression and Purification,2016,123:14-18.
[11]TEMUUJIN U,PARK J S,HONG S K.Molecular characterization of theα-galactosidase SCO0284 from Streptomyces coelicolor A3(2),a family 27 glycosyl hydrolase[J].Journal of M icrobiology and Biotechnology,2016,26(9):1650-1656.
[12]MARUTA A,YAMANE M,MATSUBARA M,et al.A novelα-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain[J].Enzyme and M icrobial Technology,2017,103:25-33.
[13]HU Y J,ZHU M J,TIAN G T,et al.Isolation of a protease-resistant and pH-stableα-galactosidase displaying hydrolytic efficacy toward raffinose family oligosaccharides from the button mushroom Agaricus bisporus[J].International Journal of Biological M acromolecules,2017,104:576-583.
[14]胡玉净.榆黄蘑、红平菇和双孢菇子实体中α-半乳糖苷酶理化性质的研究[D].博士学位论文.北京:中国农业大学,2016:39-59.
[15]莫海飞,陈炼红,黄林,等.响应面法优化里氏木霉固体发酵产α-半乳糖苷酶培养条件[J].中国畜牧兽医,2016,43(8):2038-2046.
[16]NELSON M P,BOUTIN M,TSE T E,et al.The lysosomal enzyme alpha-galactosidase A is deficient in Parkinson’s disease brain in association with the pathologic accumulation of alpha-synuclein[J].Neurobiology of Disease,2018,110:68-81.
[17]EYLER N,BIKO L,HOSE D,et al.Comprehensive and differential long-term characterization of the alpha-galactosidase A deficient mouse model of Fabry disease focusing on the sensory system and pain development[J].M olecular Pain,2016,12,doi:10.1177/1744806916646379.
[18]ALCALAY R N,WOLF P,LEVY O A,et al.Alpha galactosidase A activity in Parkinson’s disease[J].Neurobiology of Disease,2018,112:85-90.
[19]张波,王继强,张宝彤.α-半乳糖苷酶水解底物的特异性及其在保育猪日粮中的应用[J].中国饲料,2013(1):18-20.
[20]许尧兴,许少春,李艳丽,等.在玉米-豆粕型日粮中添加α-半乳糖苷酶对生长猪生产性能的影响[J].饲料工业,2008,29(4):9-11.
[21]WANG C L,LU W Q,LI D F,et al.Effects of alphagalactosidase supplementation to corn-soybean meal diets on nutrient utilization,performance,serum indices and organ weight in broilers[J].Asian-Australasian Journal of Animal Science,2005,18(12):1761-1768.
[22]缪志军,王向荣,蒋桂韬,等.复合非淀粉多糖酶制剂中补充α-半乳糖苷酶对番鸭生产性能的影响[J].中国饲料,2010(12):17-19.
[23]沈仁权,陈中孚,顾其敏,等.嗜热脂肪芽孢杆菌中一株对分解代谢物不敏感突变型的α-半乳糖苷酶的纯化及其性质[J].复旦学报(自然科学版),1981,20(2):177-185.
[24]SHANKAR S K,MULIMANI V H.α-galactosidase production by Aspergillus oryzae in solid-state fermentation[J].Bioresource Technology,2007,98(4):958-961.
[25]GAO X L,YIN Y Y,ZHOU C S.Purification,characterisation and salt-tolerance molecular mechanisms of aspartyl aminopeptidase from Aspergillus oryzae 3.042[J].Food Chemistry,2018,240:377-385.
[26]KE Y,HUANG W Q,LI J Z,et al.Enzymatic characteristics of a recombinant neutral proteaseⅠ(r NpⅠ)from Aspergillus oryzae expressed in Pichia pastoris[J].Journal of Agricultural and Food Chemistry,2012,60(49):12164-12169.
[27]LEE S K,HWANG J Y,CHOI S H,et al.Purification and characterization of Aspergillus oryzae LK-101salt-tolerant acid protease isolated from soybean paste[J].Food Science and Biotechnology,2010,19(2):327-334.
[28]ZAAK H,SASSI M,FERNANDEZ-LAFUENTE R.A newheterofunctional amino-vinyl sulfone support to immobilize enzymes:application to the stabilization ofβ-galactosidase from Aspergillus oryzae[J].Process Biochemistry,2018,64:200-205.
[29]REZESSY-SZABJ M,NGUYEN Q D,HOSCHKE A,et al.A novel thermostableα-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS395.62/b:purification and characterization[J].Biochimica et Biophysica Acta:General Subjects,2007,1770(1):55-62.
[30]CHANG S W,LEE G C,SHAW J F.Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase[J].Journal of Agricultural and Food Chemistry,2006,54(3):815-822.
[31]KATROLIA P,JIA H Y,YAN Q J,et al.Characterization of a protease-resistantα-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides[J].Bioresource Technology,2012,110:578-586.
[32]HUANG Y,ZHANG H,BEN P P,et al.Characterization of a novel GH36α-galactosidase from Bacillus megaterium and its application in degradation of raffinose family oligosaccharides[J].International Journal of Biological M acromolecules,2018,108:98-104.
[33]KAPNOOR S,MULIMANI V H.Production ofα-galactosidase by Aspergillus oryzae through solid-state fermentation and its application in soymilk galactooligosaccharide hydrolysis[J].Brazilian Archives of Biology and Technology,2010,53(1):211-218.
[34]VIANA S D F,GUIMARES V M,JOSI C,et al.Hydrolysis of oligosaccharides in soybean flour by soybeanα-galactosidase[J].Food Chemistry,2005,93(4):665-670.
[2]EL-SHEMY H,ABDEL-RAHIM E,SHABAN O,et al.Comparison of nutritional and antinutritional factors in soybean and fababean seeds with or without cortex[J].Soil Science and Plant Nutrtion,2000,46(2):515-524.
[3]ZOU W,WANG M,YAO D,et al.Fluorescence and circular dichroism spectroscopy to understand the interactions between cyclodextrins andα-galactosidase from green coffee beans[J].Food Bioscience,2017,20:110-115.
[4]TSANIKLIDIS G,BENOVIAS A,DELIS C,et al.Acidic alpha galactosidase during the maturation and cold storage of cherry tomatoes[J].Acta Physiologiae Plantarum,2016,38(2):57.
[5]ZHA H G,FLOWERS V L,YANG M,et al.Acidicα-galactosidase is the most abundant nectarin in floral nectar of common tobacco(Nicotiana tabacum)[J].Annals of Botany,2012,109(4):735-745.
[6]AULITTO M,FUSCO S,FIORENTINO G,et al.Thermus thermophilus as source of thermozymes for biotechnological applications:homologous expression and biochemical characterization of anα-galactosidase[J].Microbial Cell Factories,2017,16:28.
[7]XU H B,QIN Y J,HUANG Z Q,et al.Characterization and site-directed mutagenesis of anα-galactosidase from the deep-sea bacterium Bacillus megaterium[J].Enzyme and Microbial Technology,2014,56:46-52.
[8]LEE A,CHOI K H,YOON D,et al.Characterization of a thermostable glycoside hydrolase family 36α-galactosidase from Caldicellulosiruptor bescii[J].Journal of Bioscience and Bioengineering,2017,124(3):289-295.
[9]GONG W,XU L,GU G F,et al.Efficient and regioselective synthesis of globotriose by a novelα-galactosidase from Bacteroides fragilis[J].Applied M icrobiology and Biotechnology,2016,100(15):6693-6702.
[10]GOLOTIN V A,BALABANOVA L A,NOSKOVA Y A,et al.Optimization of cold-adapted alpha-galactosidase expression in Escherichia coli[J].Protein Expression and Purification,2016,123:14-18.
[11]TEMUUJIN U,PARK J S,HONG S K.Molecular characterization of theα-galactosidase SCO0284 from Streptomyces coelicolor A3(2),a family 27 glycosyl hydrolase[J].Journal of M icrobiology and Biotechnology,2016,26(9):1650-1656.
[12]MARUTA A,YAMANE M,MATSUBARA M,et al.A novelα-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain[J].Enzyme and M icrobial Technology,2017,103:25-33.
[13]HU Y J,ZHU M J,TIAN G T,et al.Isolation of a protease-resistant and pH-stableα-galactosidase displaying hydrolytic efficacy toward raffinose family oligosaccharides from the button mushroom Agaricus bisporus[J].International Journal of Biological M acromolecules,2017,104:576-583.
[14]胡玉净.榆黄蘑、红平菇和双孢菇子实体中α-半乳糖苷酶理化性质的研究[D].博士学位论文.北京:中国农业大学,2016:39-59.
[15]莫海飞,陈炼红,黄林,等.响应面法优化里氏木霉固体发酵产α-半乳糖苷酶培养条件[J].中国畜牧兽医,2016,43(8):2038-2046.
[16]NELSON M P,BOUTIN M,TSE T E,et al.The lysosomal enzyme alpha-galactosidase A is deficient in Parkinson’s disease brain in association with the pathologic accumulation of alpha-synuclein[J].Neurobiology of Disease,2018,110:68-81.
[17]EYLER N,BIKO L,HOSE D,et al.Comprehensive and differential long-term characterization of the alpha-galactosidase A deficient mouse model of Fabry disease focusing on the sensory system and pain development[J].M olecular Pain,2016,12,doi:10.1177/1744806916646379.
[18]ALCALAY R N,WOLF P,LEVY O A,et al.Alpha galactosidase A activity in Parkinson’s disease[J].Neurobiology of Disease,2018,112:85-90.
[19]张波,王继强,张宝彤.α-半乳糖苷酶水解底物的特异性及其在保育猪日粮中的应用[J].中国饲料,2013(1):18-20.
[20]许尧兴,许少春,李艳丽,等.在玉米-豆粕型日粮中添加α-半乳糖苷酶对生长猪生产性能的影响[J].饲料工业,2008,29(4):9-11.
[21]WANG C L,LU W Q,LI D F,et al.Effects of alphagalactosidase supplementation to corn-soybean meal diets on nutrient utilization,performance,serum indices and organ weight in broilers[J].Asian-Australasian Journal of Animal Science,2005,18(12):1761-1768.
[22]缪志军,王向荣,蒋桂韬,等.复合非淀粉多糖酶制剂中补充α-半乳糖苷酶对番鸭生产性能的影响[J].中国饲料,2010(12):17-19.
[23]沈仁权,陈中孚,顾其敏,等.嗜热脂肪芽孢杆菌中一株对分解代谢物不敏感突变型的α-半乳糖苷酶的纯化及其性质[J].复旦学报(自然科学版),1981,20(2):177-185.
[24]SHANKAR S K,MULIMANI V H.α-galactosidase production by Aspergillus oryzae in solid-state fermentation[J].Bioresource Technology,2007,98(4):958-961.
[25]GAO X L,YIN Y Y,ZHOU C S.Purification,characterisation and salt-tolerance molecular mechanisms of aspartyl aminopeptidase from Aspergillus oryzae 3.042[J].Food Chemistry,2018,240:377-385.
[26]KE Y,HUANG W Q,LI J Z,et al.Enzymatic characteristics of a recombinant neutral proteaseⅠ(r NpⅠ)from Aspergillus oryzae expressed in Pichia pastoris[J].Journal of Agricultural and Food Chemistry,2012,60(49):12164-12169.
[27]LEE S K,HWANG J Y,CHOI S H,et al.Purification and characterization of Aspergillus oryzae LK-101salt-tolerant acid protease isolated from soybean paste[J].Food Science and Biotechnology,2010,19(2):327-334.
[28]ZAAK H,SASSI M,FERNANDEZ-LAFUENTE R.A newheterofunctional amino-vinyl sulfone support to immobilize enzymes:application to the stabilization ofβ-galactosidase from Aspergillus oryzae[J].Process Biochemistry,2018,64:200-205.
[29]REZESSY-SZABJ M,NGUYEN Q D,HOSCHKE A,et al.A novel thermostableα-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS395.62/b:purification and characterization[J].Biochimica et Biophysica Acta:General Subjects,2007,1770(1):55-62.
[30]CHANG S W,LEE G C,SHAW J F.Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase[J].Journal of Agricultural and Food Chemistry,2006,54(3):815-822.
[31]KATROLIA P,JIA H Y,YAN Q J,et al.Characterization of a protease-resistantα-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides[J].Bioresource Technology,2012,110:578-586.
[32]HUANG Y,ZHANG H,BEN P P,et al.Characterization of a novel GH36α-galactosidase from Bacillus megaterium and its application in degradation of raffinose family oligosaccharides[J].International Journal of Biological M acromolecules,2018,108:98-104.
[33]KAPNOOR S,MULIMANI V H.Production ofα-galactosidase by Aspergillus oryzae through solid-state fermentation and its application in soymilk galactooligosaccharide hydrolysis[J].Brazilian Archives of Biology and Technology,2010,53(1):211-218.
[34]VIANA S D F,GUIMARES V M,JOSI C,et al.Hydrolysis of oligosaccharides in soybean flour by soybeanα-galactosidase[J].Food Chemistry,2005,93(4):665-670.