海藻糖合酶高产菌株的选育及酶学特性研究
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摘要
海藻糖作为一种非特异性的保护剂,它具有对生物大分子和生物体组织非特异性的保护作用,从而在分子生物学、医学、食品工业、化妆品工业、农业等领域有着广阔的应用前景,但由于其制取上的困难限制了它的广泛应用。目前海藻糖有多种制取方法,而酶转化法被认为是一种最有前途的海藻糖生产方法。本研究立足于利用海藻糖合酶酶法合成海藻糖,研究内容包括产海藻糖合酶菌株的选育及其培养特性的研究,海藻糖合酶的提取和纯化、酶学特性的研究,海藻糖的提取等。
通过高温、高渗等条件从自然界筛选到一株产海藻糖合酶活性较高的菌株No.62,并以其为出发菌株,通过物理和化学诱变方法,得到一株优良的诱变株P-23-37作为本实验的生产菌株。并对其形态特征及生理、生化特性的研究。通过正交实验及响应面法优化确定了其产酶最佳诱导培养基的组成及培养条件,在此优化条件下对菌株进行培养,所得酶活力为48.82U/g湿菌体,比原始条件下的酶活力(36.75U/g湿菌体)提高了32.93%。
海藻糖合酶属于胞内酶,必须将细胞破碎使其释放到发酵液中。本实验确定了脂溶法(甲苯破碎法)为破碎方法,得到最优的破碎处理条件:甲苯浓度2%、破壁温度35℃、料液比1:20和150r/min搅拌条件下反应2.5h。粗酶液经过Sephadex- G75和Sephadex-G200凝胶过滤柱,通过SDS-PAGE电泳验证其纯度,得到两条亚基带,并测定它们的分子量分别为55kDa和50kDa,并对其酶学特性进行了研究。以米氏方程为基础,求得海藻糖合酶的固有参数米氏常数Km为8×10~(-4)mol/L,最大反应速度Vm为3.24×10~(-5) mol/(L﹒S)。
本文还对实验所得的海藻糖粗液进行了分离纯化。将海藻糖粗液进行酸解,使麦芽糖分解成单糖,海藻糖保留率达91.9%;而后采用活性炭柱分离,用去离子水和5%乙醇分级洗脱,可将单糖、麦芽糖和海藻糖分开,然后通过离子交换等步骤提取出海藻糖,其结晶纯度为97.5%。另外,通过旋光仪、傅立叶红外光谱仪等测试手段,最终可以确认海藻糖合成酶将麦芽糖转化α,α-型海藻糖。
Trehalose(α-D-glucopyranosyl-α-D-glucopyranoside) exists in nature widely, especially lower plants, alga, bacteria, fungi, yeasts, insects, and invertebrates. Because trehalose exhibit non specialized protective action against damage of biologic molecule, it could be widely used in molecular biology, pharmaceuticals, foodstuff, cosmetic and agriculture etc.. Trehalose’s uses are restricted due to the difficulty in production. At present, there are many ways to produce trehalose, but the enzyme method is regarded as the most promising one. This study mainly discussed the synthesis of trehalose by trehalose synthase. The main research contents include the screen of a strain producing trehalose synthase, the investigation of culture condition, the separation and purification of trehalose synthase enzyme and separation and purification of trehalose.
A strain, No.62, producing trehalose synthase highly was screened using improved method according to the interaction of trehalose with high temperature and infiltration. Based on No.62, we screened and got the P-23-37 strain that was treated with ultraviolet inducement. The strain′s modality and biochemical character are researched. Using orthogonal experiment and Respond Surface Analysis(RSA)to optimize the constitute and culture condition of inducement culture medium which is most adapt to produce enzyme, then we culture Bacillus under the optimal condition .We can find that the harvest of the enzyme come up to 48.82U/g wet thalli. The activity of the enzyme is 32.93% more than that under originality condition (36.75U/g wet thalli).
Trehalose synthase is a kind of intercellular enzyme, so cell disruption is important for releasing it. Toluene disruption was chosen as our experiment method. The optimal disruption conditions were as follows: toluene dosage 2%, thalli?toluene2% 1:20, 35℃and 150r/min for 2.5h. The crude enzyme was purified by using gel filtration chromatography of Sephadex-G75 and Sephadex-G200. The enzyme appeared to be a dimmer when determined by SDS Polyacrylamide gel electrophoresis (SDS-PAGE).Their molecular weight were 55kDa and 50kDa respectively. And then work over the characteristic of the enzyme.
Following Michaelis-Menten kinetics we get the inherent parameter of trehalose synthase enzyme, Km is 8×10~(-4)mol/L,Vm is 3.24×10~(-5) mol/(L.S).
The separation and purification of trehalose was also researched. The maltose that existed in enzymatic reaction liquid was hydrolyzed into glucose by maltase, with the trehalose remained 91.9%. And then glucose and trehalose can be separated by successive displacement with H2O and 5% CH3CH2OH through a charcoal column. Trehalose was purified by ion-exchange etc and purity is 97.5%. At last we canaffirmed that trehalose synthase enzyme cantransform maltose to a,a-trehalose by infrared spectrometry etc determined means.
通过高温、高渗等条件从自然界筛选到一株产海藻糖合酶活性较高的菌株No.62,并以其为出发菌株,通过物理和化学诱变方法,得到一株优良的诱变株P-23-37作为本实验的生产菌株。并对其形态特征及生理、生化特性的研究。通过正交实验及响应面法优化确定了其产酶最佳诱导培养基的组成及培养条件,在此优化条件下对菌株进行培养,所得酶活力为48.82U/g湿菌体,比原始条件下的酶活力(36.75U/g湿菌体)提高了32.93%。
海藻糖合酶属于胞内酶,必须将细胞破碎使其释放到发酵液中。本实验确定了脂溶法(甲苯破碎法)为破碎方法,得到最优的破碎处理条件:甲苯浓度2%、破壁温度35℃、料液比1:20和150r/min搅拌条件下反应2.5h。粗酶液经过Sephadex- G75和Sephadex-G200凝胶过滤柱,通过SDS-PAGE电泳验证其纯度,得到两条亚基带,并测定它们的分子量分别为55kDa和50kDa,并对其酶学特性进行了研究。以米氏方程为基础,求得海藻糖合酶的固有参数米氏常数Km为8×10~(-4)mol/L,最大反应速度Vm为3.24×10~(-5) mol/(L﹒S)。
本文还对实验所得的海藻糖粗液进行了分离纯化。将海藻糖粗液进行酸解,使麦芽糖分解成单糖,海藻糖保留率达91.9%;而后采用活性炭柱分离,用去离子水和5%乙醇分级洗脱,可将单糖、麦芽糖和海藻糖分开,然后通过离子交换等步骤提取出海藻糖,其结晶纯度为97.5%。另外,通过旋光仪、傅立叶红外光谱仪等测试手段,最终可以确认海藻糖合成酶将麦芽糖转化α,α-型海藻糖。
Trehalose(α-D-glucopyranosyl-α-D-glucopyranoside) exists in nature widely, especially lower plants, alga, bacteria, fungi, yeasts, insects, and invertebrates. Because trehalose exhibit non specialized protective action against damage of biologic molecule, it could be widely used in molecular biology, pharmaceuticals, foodstuff, cosmetic and agriculture etc.. Trehalose’s uses are restricted due to the difficulty in production. At present, there are many ways to produce trehalose, but the enzyme method is regarded as the most promising one. This study mainly discussed the synthesis of trehalose by trehalose synthase. The main research contents include the screen of a strain producing trehalose synthase, the investigation of culture condition, the separation and purification of trehalose synthase enzyme and separation and purification of trehalose.
A strain, No.62, producing trehalose synthase highly was screened using improved method according to the interaction of trehalose with high temperature and infiltration. Based on No.62, we screened and got the P-23-37 strain that was treated with ultraviolet inducement. The strain′s modality and biochemical character are researched. Using orthogonal experiment and Respond Surface Analysis(RSA)to optimize the constitute and culture condition of inducement culture medium which is most adapt to produce enzyme, then we culture Bacillus under the optimal condition .We can find that the harvest of the enzyme come up to 48.82U/g wet thalli. The activity of the enzyme is 32.93% more than that under originality condition (36.75U/g wet thalli).
Trehalose synthase is a kind of intercellular enzyme, so cell disruption is important for releasing it. Toluene disruption was chosen as our experiment method. The optimal disruption conditions were as follows: toluene dosage 2%, thalli?toluene2% 1:20, 35℃and 150r/min for 2.5h. The crude enzyme was purified by using gel filtration chromatography of Sephadex-G75 and Sephadex-G200. The enzyme appeared to be a dimmer when determined by SDS Polyacrylamide gel electrophoresis (SDS-PAGE).Their molecular weight were 55kDa and 50kDa respectively. And then work over the characteristic of the enzyme.
Following Michaelis-Menten kinetics we get the inherent parameter of trehalose synthase enzyme, Km is 8×10~(-4)mol/L,Vm is 3.24×10~(-5) mol/(L.S).
The separation and purification of trehalose was also researched. The maltose that existed in enzymatic reaction liquid was hydrolyzed into glucose by maltase, with the trehalose remained 91.9%. And then glucose and trehalose can be separated by successive displacement with H2O and 5% CH3CH2OH through a charcoal column. Trehalose was purified by ion-exchange etc and purity is 97.5%. At last we canaffirmed that trehalose synthase enzyme cantransform maltose to a,a-trehalose by infrared spectrometry etc determined means.
引文
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