β-甘露聚糖酶产生菌的筛选及魔芋低聚糖制备工艺的研究
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摘要
以魔芋粉为唯一碳源,从种植魔芋土壤中定向筛选一株高产胞外β-甘露聚糖酶的菌株,进行形态观察、生理生化试验和16S r DNA序列分析鉴定,并研究了该β-甘露聚糖酶水解魔芋胶制备魔芋低聚糖的工艺。结果表明,筛选出一株高产胞外β-甘露聚糖酶的菌株,编号为G1,被鉴定为枯草芽孢杆菌(Bacillus subtilis)。确定魔芋低聚糖制备的酶解条件为酶添加量50 U/g魔芋葡甘聚糖(KGM),酶解p H值6.5,酶解温度55℃;当KGM质量浓度为10 g/L,酶解时间2 h时,还原糖转化率为51.6%;当KGM质量浓度为30 g/L,酶解时间4 h时,还原糖转化率仍可达到46.9%,表明该酶具有较高的催化效率。利用薄层层析(TLC)定性分析酶解产物主要为三糖及三糖以上的低聚糖。该研究为实现酶法制备魔芋低聚糖的工业化生产奠定了基础。
Using konjac powder as single carbon source, a strain with high-yield extracellular β-mannanase was screened from soil planted konjac and identified by morphological observation, physiological and biochemical tests and 16 S r DNA sequence analysis. At the same time, preparation technology of konjac oligosaccharides was studied by using β-mannanase enzymatic hydrolysis amorophophallus konjac. Results showed that strain G1 with high-yield extracellular β-mannanase was screened and identified as Bacillus subtilis. The enzymatic hydrolysis conditions of preparation of konjac oligosaccharides were determined as follows: β-mannanase addition 50 U/g konjac glucomannan(KGM), enzymatic hydrolysis p H value 6.5,temperature 55 ℃. When the concentration of KGM was 10 g/L and the hydrolysis time was 2 h, the conversion rate of reducing sugar was 51.6%.When the concentration of KGM was 30 g/L and the hydrolysis time was 4 h, the conversion rate of reducing sugar could still reach 46.9%, indicating that the β-mannanase has higher catalytic efficiency. Enzymolysis hydrolysis products were qualitatively analyzed by thin layer chromatography(TLC) and revealed that enzymolysis hydrolysis products were mainly composed of oligosaccharide with trisaccharide and more than trisaccharide.This study laid the foundation for industrial production of konjac oligosaccharides which were prepared by enzymatic hydrolysis.
Using konjac powder as single carbon source, a strain with high-yield extracellular β-mannanase was screened from soil planted konjac and identified by morphological observation, physiological and biochemical tests and 16 S r DNA sequence analysis. At the same time, preparation technology of konjac oligosaccharides was studied by using β-mannanase enzymatic hydrolysis amorophophallus konjac. Results showed that strain G1 with high-yield extracellular β-mannanase was screened and identified as Bacillus subtilis. The enzymatic hydrolysis conditions of preparation of konjac oligosaccharides were determined as follows: β-mannanase addition 50 U/g konjac glucomannan(KGM), enzymatic hydrolysis p H value 6.5,temperature 55 ℃. When the concentration of KGM was 10 g/L and the hydrolysis time was 2 h, the conversion rate of reducing sugar was 51.6%.When the concentration of KGM was 30 g/L and the hydrolysis time was 4 h, the conversion rate of reducing sugar could still reach 46.9%, indicating that the β-mannanase has higher catalytic efficiency. Enzymolysis hydrolysis products were qualitatively analyzed by thin layer chromatography(TLC) and revealed that enzymolysis hydrolysis products were mainly composed of oligosaccharide with trisaccharide and more than trisaccharide.This study laid the foundation for industrial production of konjac oligosaccharides which were prepared by enzymatic hydrolysis.
引文
[1]KATSURAYA K,OKUYAMA K,HATANAKA K,et al.Constitution of konjac glucomannan:chemical analysis and C NMR spectroscopy[J].Carbohyd Polym,2003,53(2):183-189.
[2]钟燕,索化夷.魔芋葡甘聚糖的功能及在食品领域的应用[J].中国酿造,2014,33(8):6-9.
[3]BIN L,BIJUN X.Study on gel formation mechanism of konjac glucomannan[J].Agr Sci China,2003,2(4):424-428.
[4]MANOM,NERINUMA I A,DA J S,et al.Oligosaccharide biotechnology:an approach of prebiotic revolution on the industry[J].Appl Microbiol Biot,2017,102(1):1-21.
[5]SRIVASTAVA P K,KAPOOR M.Production,properties,and applications of endo-beta-mannanases[J].Biotechnol Adv,2017,35(1):1-19.
[6]张蕊,朱虹,周峻沛,等.β-甘露聚糖酶分子生物学研究进展[J].中国农业科技导报,2018,20(5):34-46.
[7]JIANG B,SUNZ,HOU Y,et al.Isolation and properties of an endo-mannanase-producing Bacillus sp LX114 capable of degrading guar gum[J].Prep Biochem Biotech,2016,46(5):495-500.
[8]李云程,林娟,梁燕辉,等.产甘露聚糖酶海洋微生物的筛选及酶学性质研究[J].中国食品学报,2015,15(12):66-73.
[9]HUANG J L,BAO L X,ZOU H Y,et al.High-level production of a coldactiveβ-mannanase from Bacillus subtilis Bs5 and its molecular cloning and expression[J].Mol Genet Microbiol Virol,2012,27(4):147-153.
[10]LU H,LUO H,SHI P,et al.A novel thermophilic endo-beta-1,4-mannanase from Aspergillus nidulans XZ3:functional roles of carbohydrate-binding module and Thr/Ser-rich linker region[J].Appl Microbiol Biot,2014,98(5):2155-2163.
[11]SHIMIZU M,KANEKO Y,ISHIHARA S,et al.Novel beta-1,4-mannanase belonging to a new glycoside hydrolase family in Aspergillus nidulans[J].J Biol Chem,2015,290(46):27914-27927.
[12]李剑芳,邬敏辰,程科,等.β-甘露聚糖酶制备魔芋葡甘露低聚糖的研究[J].食品与发酵工业,2007,33(1):21-24.
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:43-65.
[14]布坎南,吉本斯.伯杰细菌鉴定手册(第8版)[M].北京:北京科学出版社,1984:729-758.
[15]赵梅,王春娟,董运海,等.重组β-甘露聚糖酶制备低聚葡甘露糖工艺条件的优化[J].食品与生物技术学报,2016,35(7):704-708.
[16]成莉凤,冯湘沅,段盛文,等.新型β-甘露聚糖酶制备葡甘寡糖工艺优化[J].食品科学,2016,37(6):34-38.
[17]吴长菲,董岩岩,李俊俊,等.魔芋葡甘露低聚糖的酶法制备工艺的初步研究[J].生物技术通报,2010(1):118-122.
[18]何丹,郭熊,杨双莲,等.酶法制备魔芋甘露寡糖和产物分析[J].中国酿造,2013,32(5):85-88.
[2]钟燕,索化夷.魔芋葡甘聚糖的功能及在食品领域的应用[J].中国酿造,2014,33(8):6-9.
[3]BIN L,BIJUN X.Study on gel formation mechanism of konjac glucomannan[J].Agr Sci China,2003,2(4):424-428.
[4]MANOM,NERINUMA I A,DA J S,et al.Oligosaccharide biotechnology:an approach of prebiotic revolution on the industry[J].Appl Microbiol Biot,2017,102(1):1-21.
[5]SRIVASTAVA P K,KAPOOR M.Production,properties,and applications of endo-beta-mannanases[J].Biotechnol Adv,2017,35(1):1-19.
[6]张蕊,朱虹,周峻沛,等.β-甘露聚糖酶分子生物学研究进展[J].中国农业科技导报,2018,20(5):34-46.
[7]JIANG B,SUNZ,HOU Y,et al.Isolation and properties of an endo-mannanase-producing Bacillus sp LX114 capable of degrading guar gum[J].Prep Biochem Biotech,2016,46(5):495-500.
[8]李云程,林娟,梁燕辉,等.产甘露聚糖酶海洋微生物的筛选及酶学性质研究[J].中国食品学报,2015,15(12):66-73.
[9]HUANG J L,BAO L X,ZOU H Y,et al.High-level production of a coldactiveβ-mannanase from Bacillus subtilis Bs5 and its molecular cloning and expression[J].Mol Genet Microbiol Virol,2012,27(4):147-153.
[10]LU H,LUO H,SHI P,et al.A novel thermophilic endo-beta-1,4-mannanase from Aspergillus nidulans XZ3:functional roles of carbohydrate-binding module and Thr/Ser-rich linker region[J].Appl Microbiol Biot,2014,98(5):2155-2163.
[11]SHIMIZU M,KANEKO Y,ISHIHARA S,et al.Novel beta-1,4-mannanase belonging to a new glycoside hydrolase family in Aspergillus nidulans[J].J Biol Chem,2015,290(46):27914-27927.
[12]李剑芳,邬敏辰,程科,等.β-甘露聚糖酶制备魔芋葡甘露低聚糖的研究[J].食品与发酵工业,2007,33(1):21-24.
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:43-65.
[14]布坎南,吉本斯.伯杰细菌鉴定手册(第8版)[M].北京:北京科学出版社,1984:729-758.
[15]赵梅,王春娟,董运海,等.重组β-甘露聚糖酶制备低聚葡甘露糖工艺条件的优化[J].食品与生物技术学报,2016,35(7):704-708.
[16]成莉凤,冯湘沅,段盛文,等.新型β-甘露聚糖酶制备葡甘寡糖工艺优化[J].食品科学,2016,37(6):34-38.
[17]吴长菲,董岩岩,李俊俊,等.魔芋葡甘露低聚糖的酶法制备工艺的初步研究[J].生物技术通报,2010(1):118-122.
[18]何丹,郭熊,杨双莲,等.酶法制备魔芋甘露寡糖和产物分析[J].中国酿造,2013,32(5):85-88.