摘要
β-环糊精葡萄糖基转移酶(β-Cyclodextrin Glycosyltransferase,简称β-CGTase, EC2.4.1.19)属于α-淀粉酶家族,是一种多功能型酶,能作用于淀粉后使葡萄糖基发生转移从而发生环化、偶合、歧化、水解等反应。其中环化反应生成的β-环糊精(β-Cyclodextrin,β-CD)是一种具有内疏水、外亲水的筒形结构特点,可以和许多有机物及一些无机物包埋,作为品质改良剂、稳定剂、药物的载体及吸附剂等在医药、食品、农业、化学、化妆品、环保等领域有广泛的应用,这使得β-CGTase的生产研究具有重大意义。
高温菌广泛分布于自然界之中,具有耐热嗜热的特性。所产生的酶为嗜热酶,在酶解过程中易获得高纯度的产物,无杂菌生存,提高反应速率,生产成本降低等优点,但现在对高温菌的研究还相对不足,仍然需要大量高酶活的优良高温菌株,以便能够更好地利用高温菌资源,使其在工业上具有更广阔的应用前景。
本论文以实验室保藏的一株产β-CGTase的高温芽孢杆菌(HY15)为生产菌株,以高的酶产量为目标,系统研究了发酵培养基及发酵条件对酶产量的影响,为满足工业化发展的需要,还进一步进行了5.0L发酵罐扩大培养试验研究,同时研究了酶的纯化方法和酶动力学性质。为该酶能进行工业化生产提供理论依据和技术支持。现将研究内容总结如下：
1.菌株产酶条件优化：通过单因素的摇瓶实验,确定了高温菌株HY15产β-CGTase的最佳碳源为玉米淀粉+糖蜜组成的复合碳源,最佳氮源为玉米浆+酵母浸粉组成的复合氮源。起始镁离子的浓度和磷酸盐浓度在细胞生长和促进产酶方面都有显著效果,确定起始镁离子的浓度和磷酸盐浓度分别为120mmol/L和10mmol/L。确定了高温菌株HY15产β-CGTase的最适初始pH值为7.0,最佳的温度为60℃,最适的接种量为3%,最适的摇瓶装液量为250mL三角瓶装液量为50mL；摇床转速为200r/min,发酵48h。
在单因素实验基础上,利用Plackett-Burman试验设计,考察影响高温菌株HY15发酵生产β-CGTase的10个相关因素,从中筛选出糖蜜、玉米淀粉、K2HPO4、MgSO4、玉米浆、酵母浸粉、pH值7个对菌种发酵产酶具有显著影响的因素(P<0.05)。采用Box-Behnken中心组合试验设计和响应面分析,对高温菌株HY15的发酵培养基和发酵条件进行优化和建立回归模型。得到5种成分的最佳水平组合为：糖蜜+玉米淀粉22.45g/L,K2HPO40.97g/L, MgSO4 0.18g/L,玉米浆+酵母浸粉21.82 g/L,pH值6.98。通过验证试验,测得该菌株发酵产酶的平均值为1401.81 U/mL,与理论预测值1409.70 U/mL接近,相对误差为0.56%,说明该回归模型预测准确性比较高。
2.5 L发酵罐扩大培养优化：在5 L发酵罐中从pH值、酶活力、总糖浓度、细胞生长4个方面考察了高温菌株HY15的发酵规律。研究了不同搅拌转速和不同溶氧(DO)水平对HY15菌株生长和β-CGTase的影响。当搅拌速率提高到300 r/min,相对溶氧水平控制在40%。温度控制在60℃,pH值通过流加NaOH控制在7.0,β-CGTase酶产量可以达到2411U/mL,生物量OD600达到1.88。在发酵8h后流加20g/L玉米浆和30g/L的糖蜜对菌体生物量和β-CGTase活力均有提高,β-CGTase最高活力达到2700 U/mL,生物量OD600达到1.92,是出发菌株酶活817 U/mL的3.3倍。
3.酶的提取纯化按优化条件发酵生产的β-CGTaes粗酶液,经饱和硫酸铵沉淀后,所得样品酶的比活力为566.2/mg,纯化倍数为8.1倍,回收率为58%；初步纯化的样品经SepharoseCL-6B柱层析后,纯化倍数达到16倍,比活力从粗酶液的67.4 U/mg上升至970.4U/mg,得率为16%,通过热处理的酶液纯化倍数为5.2倍,经过SDS-PAGE电泳检测只显示一条蛋白带,求得该酶的分子量为69 kDa。
4.酶学性质分别以可溶性淀粉、马铃薯淀粉、玉米淀粉为底物测得的米氏常数分别为：以可溶性淀粉为底物的米氏常数为Km=0.379mg/mL,最大反应速率Vmax=13.24mg/min;以马铃薯淀粉为底物的米氏常数为Km=0.476mg/mL,最大反应速率Vmax=6.36mg/min;以玉米淀粉为底物的米氏常数为Km=0.3125mg/mL,最大反应速率Vmax= 13.29mg/min;以玉米淀粉为底物的米氏常数最小,说明玉米淀粉与酶的亲和力最强。
β-CGTase酶反应的最适温度为60℃,在80℃以下保持30 min后的剩余酶活均在90%以上,最适pH为6.0,在pH6.0-9.0范围内剩余酶活均在90%以上。Al3+、Ag+、Fe2+、Cu2+对酶活有明显的抑制作用,Ca2+、Mn2+、Mg2+对酶活力有一定促进作用。Zn2+、K+、Cr3+对酶活力几乎没有影响。
将β-CGTase作用玉米淀粉后的水解产物采用HPLC检测方法分析可以看出,酶作用于淀粉后能够产生β-环糊精。
玉米淀粉的β-CGTase酶解过程中,还原糖含量-时间进程呈现双指数变化,温度、pH值、酶浓度、淀粉浓度对反应过程均有不同程度的影响。
综上所述,筛选得到的HY15高温细菌,可产生热稳定的、特异性较强的β-CGTase,在β-环糊精产品应用及开发方面,具有良好的应用前景。
β-Cyclodextrin glycosyltransferase(β-CGTase,EC2.4.1.19)is one unique member of a-amylase family.β-CGTase is a multifunctional enzyme. It can catalyze transglycosylation reactions:disproportionation, cyclization, coupling and hydrolysis reaction. Cyclization is (3-CGTase effect on substrate to convert starch intoβ-Cyclodextrin.β-Cyclodextrin have a hydrophilic outer surfase and nonpolar hole, it can embed many organic matter and some inorganic, as quality improver, stabilizer, drug carrier and the absorbent, etc.and leading to improvement of physical and chemical properties of the compounds. Therefore,β-Cyclodextrin products have been widely used in pharmaceutical,food, agriculture,chemical, cosmetic and environmental protection,et al.which make it very important to study the Production.
Thermophiles are abundant in the nature environment:they are thermostable and thertno-philie, and they also have excenlant degradation capability and high metabolie Effiency, and obtain easily high purity, no miscellaneous bacteria survive, which leads to the wide use of their enzymes in some industries. However, the current research of thermophiles is inadequate.Inorder to better use the thermophiles resource and facilitate broader utilization of these resources in more industries, more therinophilies with high degradation capability are needed,
thermostable strain HY15,selected and conserved in our lab,it is aim of the high yield ofβ-CGTase.In this study,β-CGTase production by this strain HY15 was systematically investigated for the purpose of high enzymatic yield.The biochemical characteristics and dynamic properties of fermentation were explored. The fermentation and fermentation conditions on the influence of enzyme production were studied systematic. In order to need the requirement of industrialization production.Still further experimental research on 5.0 L automatic fermentation tank, and study on purification methods of enzymes and enzyme kinetics properties. For this enzyme can provide theoretical basis and technical support for industrial production. The summarize research contents are as follows:
1. The optimal fermentation conditions forβ-CGTase prodtion.Through the single factor and shaking flasks experiment.Molasses and Corn starch was selected as the best carbon source for theβ-CGTase production by strain HY15. Corn steep liquor and yeast were also selected as the suitable nitrogen sources.β--CGTase yield and cell growth were significantly promoted by the addition of cations such as Mg2+,PO43-. The combined addition of 10 mmol/L Mg2+,120 mmol/L PO43-. The optimal initial pH.temperature and medium volume in 250 mL shaking flasks for enzyme fermentation were 7.0,60℃and 50 mL,The appropriate inoculum size was 3%,the rotation speed of shaker was 200r/min. Fermentation time was 48h.
On the base of single factor experiment.The Plackett-Burman design was used to evaluate the effects of ten variables which could affect theβ-CGTase production.On the basis of analyzing the steepest ascent experiment was adopted to determine the optinal region of the medium composition. Subsequently,response surface methodology was employed to determine the maximumβ-CGTase production at the optimum concentration of key factors,in which,a quadratic polynomial model for P-CGTase yield was established.The optimal fermentation conditions for P-CGTase prodction were determined.The results is theβ-CGTase production was found to correlate to the seven parameters:molasses,corn starch,K2HPO4, MgSO4, corn steep liquor and yeast extract,pH was found to be the most important factors.The optimal values of the five parameters were determined as 22.45g/L molasses and corn starch,0.97g/L K2HPO4, MgSO40.18g/L,21.82g/L corn steep liquor and yeast extract, pH 6.98. In the optimum conditions.Theβ-CGTase production was 1401.81U/mL,which shows the results are reasonable.
2. The optimal fermentation conditions of 5.0 L scale fermentor. The fermentation rule of strain HY15 were studied in a 5.0L scale fermentor for enzyme activity, total sugar concentration,cell growth,pH. The influence of various agitation speeds and dissolved oxygen (DO) levels onβ-CGTase production and cell growth. When the agitation speed was controlled at 300 r/min and DO was controlled at 40%,the temperature was controlled at 60℃, the pH was controlled at 7.0, theβ-CGTase yield reached 2411U/mL,OD600 reached 1.88. Fermentation after 8h,flow fed 20g/L corn steep liquor and molasses 30g/L,theβ-CGTase yield and OD600 were both increased, theβ-CGTase yield reached 2700U/mL,OD600 reached 1.92.which was increased 3.3 times of primitiveβ-CGTase 817 U/mL
3. Purification of P-CGTase. The specific activity of theβ-CGTase Produced by strain HY15 in liquid cultures was enhanced by16 folds after a series of purification steps including ammonium sulfate precipitation,sepharoseCL-6B chromatography.Polyacrylamide gel electro-phoresis (SDS-PAGE) analysis indicates that the purified enzyme was homogeneous with its subunit molecular mass being estimated as 69kDa in SDS-PAGE.
4. Characteristics of the purifiedβ-CGTase. The optimal conditions for reaction of the P-CGTase produced by strain HY15 was pH6 at 60℃.The enzyme was stable in a range of pH6.0-9.0 at the temperature below 80℃. The enzyme was inactivated at the presence of Al3+,Ag+, Fe2+,Cu2+ at the level of lmmol/L.Whereas it was activated at the same level of Ca2+,Mn2+, Mg2+.However,Zn2+,K+,Cr3+had insignificant effect on the activity of the P-CGTase concenred.
The km value for its reaction on soluble starch was 0.379 mg/mL,the Vmax value was 13.24mg/min. The km value for its reaction on potato starch was 0.476 mg/mL,the Vmax value was 6.36mg/min. The km value for its reaction on corn starch was 0.3125mg/mL,the Vmax value was 13.29mg/min.Its explained the affinity is strongest of the potato starch andβ-CGTase of strain HY15.
Based on HPLC analysis, hydrolysis product of the P-CGTase reacted on corn starch were researched.The starch can transformβ-CD.
In the process ofβ-CGTase reaction on the corn starch.The sugar content and the time display the relevance of double exponential. It have varying effects which temperature, pH, enzyme concentration, starch concentration reacted on the process.
In all,the thermophilic bacteria strain HY15 can produce heat-stable,great affinityβ-CGTase, which is significant potential for the application and development of P-CD
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