重组果聚糖蔗糖酶的发酵优化及应用
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摘要
为了研究不同条件下重组短小芽孢杆菌产果聚糖蔗糖酶的最适条件以及利用重组酶转化蔗糖-乳糖制备低聚乳果糖的最适转化条件。以前期构建的产果聚糖蔗糖酶重组短小芽孢杆菌Brevibacillus brevis/pNCMO2-lsc作为菌种,通过单因素试验以及正交试验确定其最适产酶的发酵培养基为:葡萄糖20 g/L、氮源(工业酵母粉∶棉籽粉=2∶1,质量比)为40 g/L、CaCl_20.5 mmol/L,最适产酶温度30℃。在最优条件下发酵培养,果聚糖蔗糖酶的酶活可达62.1 U/mL,是优化前的3.69倍。利用该重组果聚糖蔗糖酶转化蔗糖-乳糖制备低聚乳果糖,在蔗糖和乳糖质量浓度均为200 g/L情况下,确定其最适转化条件:反应温度35℃,pH 6.0,加酶量为2 U/g底物,反应8 h后低聚乳果糖转化率可达39.1%。
In order to study the optimum conditions of B.brevis producing levansucrase and the optimum transformation conditions for the conversion of sucrose-lactose to lactosucrose by recombinant enzyme.A recombinant strain containing the encoding gene of Bacillus flexus levansucrase,B.brevis/pNCMO2-Lsc had been constructed in our laboratory previously.The optimum fermentation medium was determined by single factor test and orthogonal test:20 g/L of glucose,40 g/L of nitrogen source(industrial yeast powder:cottonseed powder=2∶1),0.5 mmol/L of CaCl_2,The optimal temperature for the enzyme production was 30℃.Under these conditions,the production of levansucrases reached 62.1 U/mL,which was 3.69 times higher than the original enzyme activity.The crude enzyme was used for the preparation of lactosucrose.Under the condition of sucrose and lactose concentration of 200 g/L,the optimum conditions were as follows:reaction temperature 35℃,pH 6.0,enzyme loading 2 U/g substrate,reaction time 8 h.Reaching 39.1%.
In order to study the optimum conditions of B.brevis producing levansucrase and the optimum transformation conditions for the conversion of sucrose-lactose to lactosucrose by recombinant enzyme.A recombinant strain containing the encoding gene of Bacillus flexus levansucrase,B.brevis/pNCMO2-Lsc had been constructed in our laboratory previously.The optimum fermentation medium was determined by single factor test and orthogonal test:20 g/L of glucose,40 g/L of nitrogen source(industrial yeast powder:cottonseed powder=2∶1),0.5 mmol/L of CaCl_2,The optimal temperature for the enzyme production was 30℃.Under these conditions,the production of levansucrases reached 62.1 U/mL,which was 3.69 times higher than the original enzyme activity.The crude enzyme was used for the preparation of lactosucrose.Under the condition of sucrose and lactose concentration of 200 g/L,the optimum conditions were as follows:reaction temperature 35℃,pH 6.0,enzyme loading 2 U/g substrate,reaction time 8 h.Reaching 39.1%.
引文
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[14]曹劲松.初乳功能性食品[M].北京:中国轻工业出版社,2000.
[15]KONG Gaofei.Isolation and identification of a Bacillus pumilus and optimization of its fermentation conditions[J].Zhejiang SciTech University,2014,31(4):467-473.(in Chinese)
[16]BELGHITH K S,DAHECH I,BELGHITH H,et al.Microbal production of levansucrase for synthesis of fructooligosaccharides and levan[J].Biological Macromolecules,2012(50):451-458.
[2]TRIINU V,KARIN M,CRISTINA M,et al.Levansucrases from Pseudomonassyringaepv.tomato and P.chlororaphis subsp.aurantiaca:Substrate specificity,polymerizing properties and usage of different acceptors for Fructosylation[J].Journal of Biotechnology,2011(155):338-349.
[3]HAN Y W,WATSON M A.Production of microbial levan from sucrose,sugarcane juice and beet molasses[J].Journal of Industrial Microbiology,1992,9(3/4):257-260.
[4]ING-LUNG S,LI D C,JANE Y W.Levan production using Bacillus subtilisnatto cells immobilized on alginate[J].Carbohydrate Polymers,2010(82):111-117.
[5]BEKERS M,LAUKEVICS J,UPITE D,et al.Fructooligosaccharide and levan producing activity of Zymomonasmobilis extracellular levansucrase[J].Process Biochemistry,2002(38):701-706.
[6]CHIANG C J,WANG J Y,CHEN P T,et al.Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads[J].Applied Microbiology and Biotechnology,2009,82(3):445-451.
[7]FENG T,LOTTHIDA I,SALWA K,et al.Purification and characterization of levansucrase from Bacillus amyloliquefaciensin Intra-and extracellular forms useful for the synthesis of levan and fructooligosaccharides[J].Biosci Biotechnol Biochem,2011,75(10):1929-1938.
[8]LU Juan,XIAO Min,LU Lili.Optimization of fermentation conditions for production of levan by Bacillus licheniformis 8-37-0-1[J].Food Science,2011,32:183-187.(in Chinese)
[9]PABST M.Levan and levansucrase of Actinomycesviscosus[J].Infection&Immunity,1977,15(2):518-526.
[10]KANG H K,SEO M Y,SEO E S,et al.Cloning and expression of levansucrase from Leuconostocmesenteroides B-512 FMC in Escherichia coli[J].Biochim Biophys Acta,2005,1727(1):5-15.
[11]YOSHIKAWA J,AMACHI S,SHINOYAMA H,et al.Production of fructooligosaccharides by crude enzyme preparations ofβ-fructofuranosidase from Aureobasidiumpullulans[J].Biotechnology Letters,2007,30(3):535-539.
[12]TERADA A,HARA H,OISHI T,et al.Effect of dietary lactosucrose on faecal flora and faecal metabolites of dogs[J].Microbial Ecology in Health and Disease,1992,5(2):87-92.
[13]XIA Xiaofeng,WANG Huifei,WU Xiaoyu.Effects of six commonly used oligosaccharides on the growth of Streptococcus thermophilus in vitro[J].Journal of Food Science and Biotechnology,2016,35(3):310-317.(in Chinese)
[14]曹劲松.初乳功能性食品[M].北京:中国轻工业出版社,2000.
[15]KONG Gaofei.Isolation and identification of a Bacillus pumilus and optimization of its fermentation conditions[J].Zhejiang SciTech University,2014,31(4):467-473.(in Chinese)
[16]BELGHITH K S,DAHECH I,BELGHITH H,et al.Microbal production of levansucrase for synthesis of fructooligosaccharides and levan[J].Biological Macromolecules,2012(50):451-458.