泡盛曲霉β-1,3-1,4-葡聚糖酶的纯化、性质及其用于制备β-葡寡糖
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摘要
作者研究了泡盛曲霉(Aspergillus awamori)一种β-1,3-1,4-葡聚糖酶(AaBglu29)的纯化、性质及其水解燕麦麸皮制备葡寡糖。粗酶液采用硫酸铵沉淀、QSFF强阴离子柱和DEAE-52弱阴离子柱层析3步纯化,得到电泳级纯酶,经SDS-PAGE和Sephacryl-100测定的相对分子质量分别为30.7×10~3和27.6×10~3。该酶最适pH为5.0,最适温度为55℃。AaBglu29底物特异性专一,对大麦葡聚糖、燕麦葡聚糖和地衣多糖的比酶活力分别为9 500、7 950 U/mg和5 980 U/mg。该酶水解燕麦麸皮产生葡三糖和葡四糖,经优化,当底物质量分数为5%,加酶量为100 U/g时,于50℃、pH 5.0条件下水解4 h,葡寡糖转化率可达90%。AaBglu29优良的酶学特性使其在食品和饲料工业具有较大的应用潜力。
A β-1,3-1,4-glucanase(Aa Bglu29) from Aspergillus awamori was purified and its application in glucooligosaccharides production from oat bran was studied. The β-1,3-1,4-glucanase was purified to homogeneity by ammonium precipitation and anion-exchange chromatography. The molecular mass of AaBglu29 was estimated to be 30.7×10~3 and 27.6×10~3 by SDS-PAGE and gel filtration,respectively. The Aa Bglu29 was most active at pH 5.0 and 55 ℃. The enzyme displayed strict substrate specificity towards barely β-glucan(9,500 U/mg),oat β-glucan(7 950 U/mg) and lichenan(5 980 U/mg). In addition,the enzyme was suitable for production of glucooligosaccharides(GOS) from oat bran. The highest GOS yield of 90%(based on β-1,3-1,4-glucan in oat bran) was achieved from 5% oat bran after 4 h of enzymatic hydrolysis(100 U/g) at 50 ℃ and pH 5.0. The properties indicated that Aa Bglu29 is a promising candidate for application in food and feed fields.
A β-1,3-1,4-glucanase(Aa Bglu29) from Aspergillus awamori was purified and its application in glucooligosaccharides production from oat bran was studied. The β-1,3-1,4-glucanase was purified to homogeneity by ammonium precipitation and anion-exchange chromatography. The molecular mass of AaBglu29 was estimated to be 30.7×10~3 and 27.6×10~3 by SDS-PAGE and gel filtration,respectively. The Aa Bglu29 was most active at pH 5.0 and 55 ℃. The enzyme displayed strict substrate specificity towards barely β-glucan(9,500 U/mg),oat β-glucan(7 950 U/mg) and lichenan(5 980 U/mg). In addition,the enzyme was suitable for production of glucooligosaccharides(GOS) from oat bran. The highest GOS yield of 90%(based on β-1,3-1,4-glucan in oat bran) was achieved from 5% oat bran after 4 h of enzymatic hydrolysis(100 U/g) at 50 ℃ and pH 5.0. The properties indicated that Aa Bglu29 is a promising candidate for application in food and feed fields.
引文
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[2]JASKARI J,KONTULA P,SIITONEN A,et al.Oatβ-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains[J].Applied Microbiology and Biotechnology,1998,49(2):175-181.
[3]CHAARI F,BELGHITH-FENDRI L,ELLOUZ-CHAABOUNI S.Production and in vitro evaluation of oligosaccharides generated from lichenan using immobilized Penicillium occitanis lichenase[J].Journal of Molecular Catalysis B-Enzymatic,2015,116:153-158.
[4]BODE L.Human milk oligosaccharides:prebiotics and beyond[J].Nutrition Reviews,2009,67(11):S183-S191.
[5]GOLDENKOVA-PAVLOVA I V,TYURIN A D,MUSTAFAEV O N.The features that distinguish lichenases from other polysaccharide-hydrolyzing enzymes and the relevance of lichenases for biotechnological applications[J].Applied Microbiology and Biotechnology,2018,102(9):3951-3965.
[6]BI Jing.Application ofβ-glucanase in beer production[J].China Brewing,2011(9):105-106.(in Chinese)
[7]FERNANDES V O,COSTA M,RIBEIRO T,et al.1,3-1,4-beta-Glucanases and not 1,4-beta-glucanases improve the nutritive value of barley-based diets for broilers[J].Animal Feed Science and Technology,2016,211:153-163.
[8]Juanjuan,WU Bin,HE Bingfang.Enzymatic and catalytic properties ofβ-1,3-1,4-glucanase from Bacillus subtilis LC-9[J].Journal of Anhui Agricultural Sciences,2012(27):13243-13246.(in Chinese)
[9]GAO Z.Purification and characterization of a novel lichenase from Bacillus licheniformis GZ-2[J].Biotechnology and Applied Biochemistry,2016,63(2):249-256.
[10]ZHANG B,LIU Y,YANG H,et al.Biochemical properties and application of a novelβ-1,3-1,4-glucanase from Paenibacillus barengoltzii[J].Food Chemistry,2017,234:68-75.
[11]YANG S,YAN Q,JIANG Z,et al.Biochemical characterization of a novel thermostableβ-1,3-1,4-glucanase(Lichenase)from Paecilomyces thermophila[J].Journal of Agricultural and Food Chemistry,2008,56(13):5345-5351.
[12]TANG Y,YANG S,YAN Q,et al.Purification and characterization of a novelβ-1,3-1,4-glucanase(Lichenase)from thermophilic Rhizomucor miehei with high specific activity and its gene sequence[J].Journal of Agricultural and Food Chemistry,2012,60(9):2354-2361.
[13]YANG S,XIONG H,YAN Q,et al.Purification and characterization of a novel alkalineβ-1,3-1,4-glucanase(lichenase)from thermophilic fungus Malbranchea cinnamomea[J].Journal of Industrial Microbiology&Biotechnology,2014,41(10):1487-1495.
[14]HAN Bing,WANG Shilan,DE Qingmeiduo,et al.Gene cloning,expression and characterization analysis of a highly thermal stableβ-glucanase gene from Aspergillus niger[J].Food and Fermentation Industries,2018:1-11.(in Chinese)
[15]LIU Lu,CHEN Zhou,CHEN Yaoyao,et al.Optimization of fermentation conditions forβ-1,3-1,4-glucanase production by Aspergillus flavus[J].Journal of Food Science and Technology,2018:1-10.(in Chinese)
[16]DING Yemei,YUN Jianmin,WEI Long,et al.Purification and enzymatic properties ofβ-1,3-1,4-glucanase produced by Mucor petrinsularis[J].Food Science,2014,35(11):143-148.(in Chinese)
[17]ELGHARBI F,HMIDA-SAYARI A,SAHNOUN M,et al.Purification and biochemical characterization of a novel thermostable lichenase from Aspergillus niger US368[J].Carbohydrate Polymers,2013,98(1):967-975.
[18]LIU Erwei,YANG Shaoqing,YAN Qiaojuan,et al.Optimization of liquid-state fermentation conditions forβ-1,3-1,4-glucanase(lichenase)production by Aspergillus awamori[J].Food Science,2017,38(16):29-35.(in Chinese)
[19]LIU Erwei,LIU Xueqiang,YANG Shaoqing,et al.Optimization of fermentation conditions forβ-1,3-1,4-glucanase production from Aspergillus awamori CAU33 by solid state fermentation[J].Biotechnology&Business,2018(3):80-86.(in Chinese)
[20]LOWRY O H,ROSEBROUGH N J,FARR A L,et al.Protein Measurement with the folin phenol reagent[J].Journal of Biological Chemistry,1951,193(1):265-275.
[21]LAEMMLI U K.Cleavage of structural proteins during assembly of head of bacteriophage-T4[J].Nature,1970,227(5259):680.
[22]NIU Q,ZHANG G,ZHANG L,et al.Purification and characterization of a thermophilic 1,3-1,4-beta-glucanase from Bacillus methylotrophicus S2 isolated from booklice[J].Journal of Bioscience and Bioengineering,2016,121(5):503-508.
[23]YAN Q,YANG H,JIANG Z,et al.A novel thermostableβ-1,3-1,4-glucanase from Thermoascus aurantiacus and its application in oligosaccharide production from oat bran[J].Carbohydrate Research,2018,469:31-37.