β-葡萄糖苷酶转化葡萄糖制备低聚龙胆糖的研究
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摘要
低聚龙胆糖是葡萄糖通过β-1,6-糖苷键组成的功能性低聚糖,是高效的双歧杆菌增殖因子,有众多优良的生理功能,市场前景广阔。低聚龙胆糖的研究和应用起步晚,但发展很快。工业化生产低聚龙胆糖主要是通过酶法生产,但目前国内仍未能大规模工业化生产。
本论文系统地研究了低聚龙胆糖的酶法生产工艺,以高浓度的葡萄糖为原料,利用高活性重组β-葡萄糖苷酶转糖苷和缩合得到低聚龙胆糖浆,再进行分离纯化获得高纯度的低聚龙胆糖成品。主要研究内容如下:
1.将黑曲霉(Aspergillus niger CMI CC324262)β-葡萄糖苷酶的基因bgl连接到载体pPIC9K上,在毕赤酵母(Pichia pastoris) KM71中表达,成功构建了基因工程菌KM 71/bgl/pPIC 9K,表达产物经HPLC、MS鉴定具有转苷活性,可作为低聚龙胆糖的生产用酶。
2.考察基因工程菌KM 71/bgl/pPIC 9K的摇瓶发酵情况,确定适宜的发酵条件:生长阶段接种量10%,甘油初始浓度30 g/L,生长pH6.0,温度30℃。诱导阶段甲醇初始浓度4%,每24 h添加0.5%甲醇,诱导表达周期120 h,诱导pH 7.5,温度30℃,250 mL三角瓶诱导培养基装液量50 mL,酶活可达到245 U/mL。本研究为低聚龙胆糖大规模生产奠定了坚实的基础。
3.基因工程菌KM 71/bgl/pPIC 9K发酵获得β-葡萄糖苷酶粗酶液后,用于转化葡萄糖制备低聚龙胆糖,优化其反应条件。确定最佳条件:底物浓度为80%,反应pH4.5,温度60℃,每克葡萄糖加酶60 U,添加1 mmol/L的K+,转化周期为48 h,低聚龙胆糖浓度为50 g/L。
4.初步研究酶反应产物中低聚龙胆糖的分离纯化。比较结晶、色谱柱两种分离方法,结果表明色谱柱分离法效果较好,最佳分离条件是利用阳离子树脂DTF-02,控制分离柱柱高500 mm,内径16 mm,分离温度50℃,进料量0.25 g低聚龙胆糖,以水为洗脱剂,2 mL/min的洗脱流速,可使低聚龙胆糖含量达到73%,回收率为43%,有望进一步分离得到更为理想的结果,使生产高纯度低聚龙胆糖成为可能。
Gentiooligosaccharides are one of functional oligosaccharides, composed of glucose units linked byβ-1,6-xylosidete bonds. They are highly-effective Bifid bacteria proliferation factor and have many excellent physiological activities and broad markert prospect.The research and application of Gentiooligosaccharides started late, but developing rapidly. The industrial production of Gentiooligosaccharides is mainly through the enzymatic production, but currently not been able to product in large scale in china.
In this paper, the enzymatic production process of Gentiooligosaccharides has been systemically studied. The Gentiooligosaccharide syrup, producted from high concentrations of glucose by using highly active recombinantβ-glucosidase, was then purified to obtain high-purity products. The main research contents are as follows:
1. Aspergillus niger (CMI CC 324626)β-glucosidase encoding gene, bgl, was cloned into the expression vector pPIC9K to construct the recombinant plasmid pPIC9K- bgl.The plasmid was then transformed into Pichia pastoris KM71 to achieve the secretive extracellular overproduction ofβ-glucosidase.The activity of the expressed protein was performed by the assay of transglucosidation reaction and the transglucosidation product was identified by HPLC and MS.The results showed that this recombinant enzyme can be used in the production of Gentiooligosaccharides.
2. The required nutrients forβ-glucosidase production by recombinant P. pastoris in shaking flasks were investigated.The optimized ferment condition of the recombinant P. pastoris has been determined: initial concentration of glycerol 30g/L, inoculating amount 10%, initial pH 6.0, growth temperature 30℃, initial methanol concentration 4%, methanol supplementation quantity 0.5%(in every 24h), duration of induction 120h initial pH 7.5, inducing temperature 30℃,liquid volume 50mL in 250mL flask, ,and the maximum yield of BGL was achieved to 245U/L. This study laid a solid foundation for large-scale production of Gentiooligosaccharides. 3. In addition, the main operation parameters of this enzymatic conversion were optimized.At 80% glucose,60℃,pH 4.5,1 mmol/L K+,60 Uβ-glucosidase per gram substrate, and 48 h reaction time, the gentiooligosaccharide produced reached its maximum of 50 g/L.
4. Compared with crystallization and and cation exchange chromtography ,we found the second way was better.The purification of gentiooligosaccharide were studied with the resin DTF-01.and correponding operat techniques were researched too.The experiments date showed that the optimal purifiration result could be acieved on the resin column ofф15mm×600mm with eluting rate 2mL/min at 50℃,when 5mL syrup used.The content of active component would be increased from 9.3% to 73% and recovery were 43%. It would be possible to produce high-purity Gentiooligosaccharides for further separation.
本论文系统地研究了低聚龙胆糖的酶法生产工艺,以高浓度的葡萄糖为原料,利用高活性重组β-葡萄糖苷酶转糖苷和缩合得到低聚龙胆糖浆,再进行分离纯化获得高纯度的低聚龙胆糖成品。主要研究内容如下:
1.将黑曲霉(Aspergillus niger CMI CC324262)β-葡萄糖苷酶的基因bgl连接到载体pPIC9K上,在毕赤酵母(Pichia pastoris) KM71中表达,成功构建了基因工程菌KM 71/bgl/pPIC 9K,表达产物经HPLC、MS鉴定具有转苷活性,可作为低聚龙胆糖的生产用酶。
2.考察基因工程菌KM 71/bgl/pPIC 9K的摇瓶发酵情况,确定适宜的发酵条件:生长阶段接种量10%,甘油初始浓度30 g/L,生长pH6.0,温度30℃。诱导阶段甲醇初始浓度4%,每24 h添加0.5%甲醇,诱导表达周期120 h,诱导pH 7.5,温度30℃,250 mL三角瓶诱导培养基装液量50 mL,酶活可达到245 U/mL。本研究为低聚龙胆糖大规模生产奠定了坚实的基础。
3.基因工程菌KM 71/bgl/pPIC 9K发酵获得β-葡萄糖苷酶粗酶液后,用于转化葡萄糖制备低聚龙胆糖,优化其反应条件。确定最佳条件:底物浓度为80%,反应pH4.5,温度60℃,每克葡萄糖加酶60 U,添加1 mmol/L的K+,转化周期为48 h,低聚龙胆糖浓度为50 g/L。
4.初步研究酶反应产物中低聚龙胆糖的分离纯化。比较结晶、色谱柱两种分离方法,结果表明色谱柱分离法效果较好,最佳分离条件是利用阳离子树脂DTF-02,控制分离柱柱高500 mm,内径16 mm,分离温度50℃,进料量0.25 g低聚龙胆糖,以水为洗脱剂,2 mL/min的洗脱流速,可使低聚龙胆糖含量达到73%,回收率为43%,有望进一步分离得到更为理想的结果,使生产高纯度低聚龙胆糖成为可能。
Gentiooligosaccharides are one of functional oligosaccharides, composed of glucose units linked byβ-1,6-xylosidete bonds. They are highly-effective Bifid bacteria proliferation factor and have many excellent physiological activities and broad markert prospect.The research and application of Gentiooligosaccharides started late, but developing rapidly. The industrial production of Gentiooligosaccharides is mainly through the enzymatic production, but currently not been able to product in large scale in china.
In this paper, the enzymatic production process of Gentiooligosaccharides has been systemically studied. The Gentiooligosaccharide syrup, producted from high concentrations of glucose by using highly active recombinantβ-glucosidase, was then purified to obtain high-purity products. The main research contents are as follows:
1. Aspergillus niger (CMI CC 324626)β-glucosidase encoding gene, bgl, was cloned into the expression vector pPIC9K to construct the recombinant plasmid pPIC9K- bgl.The plasmid was then transformed into Pichia pastoris KM71 to achieve the secretive extracellular overproduction ofβ-glucosidase.The activity of the expressed protein was performed by the assay of transglucosidation reaction and the transglucosidation product was identified by HPLC and MS.The results showed that this recombinant enzyme can be used in the production of Gentiooligosaccharides.
2. The required nutrients forβ-glucosidase production by recombinant P. pastoris in shaking flasks were investigated.The optimized ferment condition of the recombinant P. pastoris has been determined: initial concentration of glycerol 30g/L, inoculating amount 10%, initial pH 6.0, growth temperature 30℃, initial methanol concentration 4%, methanol supplementation quantity 0.5%(in every 24h), duration of induction 120h initial pH 7.5, inducing temperature 30℃,liquid volume 50mL in 250mL flask, ,and the maximum yield of BGL was achieved to 245U/L. This study laid a solid foundation for large-scale production of Gentiooligosaccharides. 3. In addition, the main operation parameters of this enzymatic conversion were optimized.At 80% glucose,60℃,pH 4.5,1 mmol/L K+,60 Uβ-glucosidase per gram substrate, and 48 h reaction time, the gentiooligosaccharide produced reached its maximum of 50 g/L.
4. Compared with crystallization and and cation exchange chromtography ,we found the second way was better.The purification of gentiooligosaccharide were studied with the resin DTF-01.and correponding operat techniques were researched too.The experiments date showed that the optimal purifiration result could be acieved on the resin column ofф15mm×600mm with eluting rate 2mL/min at 50℃,when 5mL syrup used.The content of active component would be increased from 9.3% to 73% and recovery were 43%. It would be possible to produce high-purity Gentiooligosaccharides for further separation.
引文
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[2] Marcel Roberfroid,Nondigestible oligosaccharides[J]. Critical reviews in food science and nutrition,2000,40(6):461-480
[3]袁涛.低聚糖在动物营养学上的研究进展[J].粮食与饲料工业,2003,5:26-27
[4]魏新林.甲壳低聚糖的生理活性研究进展[J].中国药理学通报,2003,19(6):614-617
[5]刘冬梅.一种新型的功能性低聚糖一蔗果低聚糖及其生产方法[J].食品工业,1998,3:14-16
[6]侯东军.菊粉低聚糖生产的研究进展[J].中国食品添加剂,2003,5:88-91
[7]尤新.功能性发酵制品[M].北京:中国轻工业出版社,2001
[8]包怡红,生庆海.低聚糖的种类及其应用[J].粮油食品科技,2002,10(6):14-17
[9]郑建仙.功能性低聚糖[M].北京:化学工业出版社,2004,280-290
[10]尤新.功能食品配料—新型低聚糖[J].食品科学,1995,16(11):41-44
[11]唐传核,朱高翔.功能性食品添加剂-龙胆低聚糖[J].江苏食品与发酵,2003,(1):29-31
[12]黄琼华.带苦味的低聚龙胆糖玉米糖浆[J].中国食品添加剂,2002,(1):52-54
[13]谷利伟,赵金兰.日本低聚糖开发新动态[J].功能食品基料,1999,1:27-28
[14]邹辉,黎锡流,罗明姬.低聚龙胆糖的酶法生产技术[J].中国食品添加剂,2004,13:97-101
[15]顾卫民.β-葡萄糖苷酶的特性及其在食品工业中的应用[J].江苏食品与发酵,2003,(1): 5-7
[16] Heather F.Seidle,Ira Marton, Oded Shoseyov, et al.Physical and Kinetic Properties of the Family 3β-glucosidase from Aspergillus niger Which is Important for Cellulose Breakdown[J].The Protein Journal, 2004, 23(1):11-23
[17] Sinnott M L.Catalytic mechanisms of enzymic glycosyl transfer[J].Chemical Reviews,1990,90: 1171-1202
[18] Vulfson, E. N., Patel, R., Law, B. A.Alkyl.glucoside synthesis in a water-organic two-phase system[J]. Biotechnol.Lett.,1990,12:397-402
[19] Heather F.Seidle,Reuben E.Huber.Transglusidic reactions of the Aspergillus niger Family 3β-glucosidase Qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH[J]. Archives of Biochemistry and Biophysics,2005,436:254-264
[20] Saxena,I.M., Brown, R. M., Jr., Fevre, M., Geremia, R. A., et al.Multidomain architecture of I-glycosyl transferases: implications for mechanism of action[J]. Bacterial, 1995,177:1419-1424
[21]闫会平,陈士华,吴兴泉.黑曲霉β-葡萄糖苷酶的进展[J].纤维素科学与技术,2007, 15(1):59-63
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