【作者】 张小云；

【导师】 覃文庆；

【作者基本信息】 中南大学 ， 生物学， 2012， 博士

【摘要】 本研究在α-淀粉酶和葡萄糖淀粉酶(简称糖化酶)共催化的基础上,引入微波辐射,借助微波诱导催化强化淀粉水解反应。对微波辐射下整个淀粉水解反应过程进行详细研究,并与传统的加热水解方式相比较。主要研究内容和结论如下：(1)研究微波诱导双酶催化玉米淀粉水解的最佳工艺条件。首先,考察pH值、反应温度、底物浓度、酶制剂的用量、辐射时间及微波功率等因素对玉米淀粉水解率的影响。结果表明,与传统的水浴加热相比,经微波辐射的玉米淀粉的水解率均高于水浴加热20%以上；其次,运用正交试验法对微波诱导双酶催化玉米淀粉水解的工艺条件进行优化。经极差分析与方差分析后确定的微波诱导双酶催化玉米淀粉水解的最佳工艺条件为反应温度65℃,辐射时间30min,酶制剂的用量分别为α-淀粉酶的用量90U/g干淀粉,糖化酶的用量110U/g干淀粉。(2)研究微波诱导双酶催化玉米淀粉水解反应的动力学特性。考察经微波辐射和水浴加热下不同因素对玉米淀粉酶水解反应初反应速率的影响。实验结果表明该催化反应经微波辐射后的初反应速率在不同条件下均比水浴加热的要大,增大幅度20%以上；采用实验方法求取了双酶的米氏常数Km和最大初反应速度Vm,其中α-淀粉酶在微波辐射下的Km约是水浴的12倍,Vm约是水浴的14倍,糖化酶在微波辐射下的Km约是水浴的10倍,Vm约是水浴的8倍,同时指出反应产物葡萄糖对糖化酶的抑制作用经微波辐射后有所减弱。(3)SDS-聚丙烯酰胺凝胶电泳实验表明,微波辐射没有改变α-淀粉酶和葡萄糖淀粉酶的分子量大小,它们的一级结构没有发生改变,换句话说,酶蛋白的肽键没有发生断裂,微波辐射对酶蛋白的一级结构没有造成影响,这是酶蛋白维持活性的基本条件,有利于酶水解玉米淀粉反应的进行。(4)α-淀粉酶的圆二色性(CD)及二级结构研究表明,分别经微波辐射和水浴加热处理后的α-淀粉酶,其CD谱线在波长λ=193nm处的峰高均降低,微波辐射处理的峰高降低幅度在140%-220%之间,水浴加热处理的降低幅度在60%-140%之间；微波辐射处理时间为60min时,λ=193nm处谱峰消失,而水浴处理60min时在该处表现为一弱峰；在波长λ=204nm和λ=220nm处的双负峰,处理组的峰位都出现了蓝移变化,微波辐射下蓝移幅度在5-8nm之间,水浴加热下蓝移幅度在3-5nm之间；随着处理时间的延长,微波辐射和水浴加热均促使二级结构α-螺旋,p-折叠,p-转角和无规则卷曲之间的转化。糖化酶的圆二色性和二级结构的研究表明,经微波辐射和水浴加热处理后的CD谱线,在波长λ=193nm处的峰高的降幅在36.4%～68.2%之间；在λ=206nm和λ=220nm的双负峰处,处理组的峰高增幅在10.8%-31.4%之间；于λ=193nm处的峰位在微波辐射下有蓝移的趋势,幅度在0.2-3nm之间,而水浴处理下峰位变化不明显；随着处理时间的延长,微波辐射和水浴加热均促使二级结构α-螺旋,p-折叠,p-转角和无规则卷曲之间的转化,但变化趋势有所不同。结论表明,微波辐射能影响肽链中氢键的分布,导致氢键的松弛,断裂和重组,使原有维系酶蛋白二级结构的稳定性的氢键取向发生改变,使酶蛋白结构稳定性降低,柔性变大,这为微波辐射的宏观生物效应的作用机理提供了重要的理论依据。(5)α-淀粉酶和葡萄糖淀粉酶经微波辐射和水浴加热处理后的紫外吸收光谱有差异,这是微波辐射诱导酶蛋白三维结构的变化的结果,微波辐射能使酶的活性中心暴露,利于酶的活性中心和底物分子的有效结合,提高酶的催化效率。(6)分别经微波辐射和水浴加热处理后的α-淀粉酶和糖化酶,其酶活都发生了变化。在水浴加热情况下,a-淀粉酶酶活随加热温度的升高先增大后下降,60℃左右时酶有最大活性；糖化酶酶活随加热温度的升高先增大后下降,40℃左右时酶有最大活性；α-淀粉酶酶活随加热时间的延长先增大后下降,20min左右时酶有最大活性,25min之后酶的活性开始快速下降；糖化酶酶活随加热时间的延长先增大后下降,15min左右时酶有最大活性,20min之后酶的活性开始下降。经微波辐射处理的α-淀粉酶酶活随辐射温度的升高先增大后下降,60℃左右时酶有最大活性；糖化酶酶活随辐射温度的升高先增大后下降,40℃左右时酶有最大活性；α-淀粉酶酶活是随辐射时间的延长先增大后下降,20min左右时酶有最大活性；糖化酶酶活也是随辐射时间的延长先增大后下降,15min左右时酶有最大活性。a-淀粉酶微波辐射温度取55～65℃较合适,糖化酶取35-45℃为合适；α-淀粉酶微波辐射时间取15～25min较好,糖化酶微波辐射时间取15min左右较好。(7)分别在不同的水解体系(包括不加酶、加α-淀粉酶和加双酶)中,考察了辐射时间、温度、底物浓度、微波功率等因素对玉米淀粉的晶体结构,化学结构,形态结构等方面的影响。玉米淀粉溶液经过不同时间的微波辐射后,红外结晶度均有所变化。经微波辐射后的玉米淀粉经光学显微镜放大400倍后观察,可以看到,即便经过微波辐射玉米淀粉已经开始糊化,但玉米淀粉的形态结构仍为完整的颗粒,但表面变得粗糙。加酶处理后的淀粉颗粒表面甚至出现凹坑。且经双酶处理后的淀粉比单酶处理后的淀粉的效果更为明显,在红外吸收光谱中也有所显示,其结晶度更低,红外吸收峰更为宽大。研究结果表明,微波辐射能诱导酶蛋白构象改变,强化双酶之间的协同效应。与传统的淀粉酸水解法、酶水解法和酸酶水解法相比,微波诱导催化淀粉水解法具有提高反应速率和缩短反应时间等明显优点,是一种优于传统水解方法的新技术。更多还原

【Abstract】 Microwave inducement catalysis can strengthen the corn starch hydrolysis catalyzed by a-amylase and glucoamylase. Hydrolysis of corn starch by double enzyme catalyzed under microwave inducement was studied detailedly in this paper, comparing with the conventional water bath heating. The main contents and conclusions are as follows:(1) The optimum conditions of corn starch hydrolysis were studied by the microwave irradiation and double-enzyme. First, the study of the pH value, temperature, substrate concentration, enzyme’s dosage, reaction time and microwave power on the starch hydrolysis rate, compared with the conventional water bath heating, it was found that the hydrolysis rate of corn starch by microwave irradiation was more than20%higher than the water bath; Secondly, the corn starch hydrolysis process under microwave irradiation condition was optimized by orthogonal test. Range and variance analysis were used to finalize the optimal conditions of corn starch hydrolysis under microwave irradiation, the reaction temperature65℃, reaction time30min, the dosage of a-amylase90U/g dry starch, the dosage of glucoamylase110U/g dry starch.(2) The kinetics of corn starch hydrolysis by microwave irradiation and double-enzyme was studied. The influence of different factors on the initial reaction rate of corn starch hydrolysis under microwave irradiation and water bath conditions with double-enzyme, the conclusion was found that the initial reaction rate under microwave irradiation increased over20%of all cases. In addition, the two-enzyme’s Michaelis constants Km and the maximum initial reaction velocity Vm were obtained by experiments, the Km of a-amylase under microwave irradiation is12times higher than water bath, the Vm is14times higher than water bath, the Km of glucoamylase under microwave irradiation is10times higher than water bath, the Vm is about8times higher than water bath, at the same time, the influence of glucose as a inhibitor on glucoamylase decreased under microwave irradiation.(3) SDS-polyacrylamide gel electrophoresis experimental results showed that, microwave irradiation did not change the double-enzyme’s molecular size compared with water bath. That is, the peptide bonds of doubleenzymes did not break, and their primary structure did not change. That is, microwave irradiation did not have the impact on the primary structure of the double enzymes, so microwave irradiation will help the reaction of double-enzyme’s corn starch hydrolysis.(4) α-Amylase’s circular dichroism (CD) and secondary structure changes under the condition of microwave irradiation and water bath was studied by circular dichroism spectra. The results showed that, the peak height (at λ,=193nm) of the CD spectra of the samples treated by microwave irradiation and water bath both reduced, the reduced rate by microwave irradiation ranged from140%to220%, while the reduced rate by water bath ranged from60%to140%; the peak of the sample treated by microwave irradiation for60min disappeared at λ=193nm, while showed a wake peak by water bath; the peak position by microwave irradiation emerged a blue shift in the range of between5-8nm at λ=204nm and λ=220nm, while emerged in the range of between3-5nm by water bath. With time going, microwave irradiation and water bath have prompted the secondary structure of α-helix, β-sheet, β-turn and random coil’s mutual transformations, but the trends were different.Glucoamylase’s circular dichroism and secondary structure changes under the condition of microwave irradiation and water bath were studied too. Results have shown that, the peak height of the CD spectra of samples treated by microwave irradiation and water bath reduced by36.4%～68.2%at λ=193nm, and the peak height of the CD spectra of samples treated increased by10.8%～31.4%at λ=206nm and λ=220nm, and the peak position emerged a blue shift at λ=193nm in the range of between0.2-3nm nm, but the peak position chang a little by water bath. With the time going, microwave irradiation and water bath have prompted the secondary structure of a-helix, β-sheet, β-turn and random coil’s mutual transformations, but the trends were different. Conclusions indicated that microwave radiation affected the distribution of hydrogen bonds in peptide chains, resulting in relaxation, fracturing and recombinant of hydrogen bonds. The orientation of hydrogen bonds maintaining peptide secondary structure stability changed, so that the structural stability of peptide reduced, and flexibility larger, which provided an important mechanism of the theory for the macroscopic biological effects of microwave irradiation.(5) According to the difference between the UV absorption spectrums of double enzyme under microwave irradiation and water bath, it was concluded that microwave irradiation induced the changes in double enzymes’three-dimensional structure, so that microwave irradiation enables the exposure of the double enzymes’active site. This is very conducive to the combination of the active site and substrate molecules and to enhance the activity of double enzymes. It was concluded that microwave irradiation changed the micro-environment of double enzymes’active site conformation to enhance the starch hydrolysis reaction.(6) After being processed by microwave radiation and water bath respectively, the enzyme activity of both a-amylase and glucoamylase changed. While processed by water bath, the enzyme activity of a-amylase first increased and then decreased as the temperature of the water bath increased. It had the most enzyme activity around60℃. The enzyme activity of glucoamylase had the same pattern as that of the a-amylase as the temperature of the water bath kept increasing. It had the most enzyme activity around40℃. When the temperature of the water bath was kept constant, the enzyme activity of a-amylase first increased and then decreased as the time of heating increased. It had the most enzyme activity around20min and then It’s enzyme activity decreased rapidly after25min; the enzyme activity of glucoamylase had the same pattern as that of the a-amylase as the time of heating increased. It had the most enzyme activity around15min and then It’s enzyme activity decreased rapidly after20min. While processed by microwave radiation, the enzyme activity of a-amylase first increased and then decreased as the temperature of the radiation increased. It had the most enzyme activity around60℃. The enzyme activity of glucoamylase had the same pattern as that of the a-amylase as the temperature of the water bath kept increasing. It had the most enzyme activity around40℃. The enzyme activity of a-amylase first increased and then decreased as the time of radiation increased. It had the most enzyme activity around20min; the enzyme activity of glucoamylase had the same pattern as that of the a-amylase as the time of heating increased. It had the most enzyme activity around15min. The appropriate radiation temperature for a-amylase is between55～65℃and for glucoamylase is between35～45℃; the appropriate radiation time for a-amylase is between15～25min and for glucoamylase is around15min.(7)The effect of radiation time、temperature、substrate concentration and microwave power on the crystal structure、chemical structure and morphological configuration of corn starch were studied in different hydrolysis systems (with no enzyme、with a-amylase and with double enzyme) respectively. The crystallinity of corn starch had changed after its solution had been dealt with microwave radiation by different lengths of time. After the corn starch had been dealt with microwave radiation, it had been observed under400times lens of optical microscope that even it started to paste and its surface became rough, its morphology configuration was still a complete granule. Pit was also observed on the surface of the corn starch that had been dealt with enzyme. This effect was more obvious for the corn starch that had been dealt with double enzyme than that had been dealt with one, and it was also reflected in the infrared absorption spectrum:the former had low crystallinity and its infrared absorption peak was broader.Conclusions indicated that microwave irradiation can induce the structure changes in a-amylase and glucoamylase and strengthen the double-enzyme synergy effect. Compared with the traditional methods of starch hydrolysis, such as acid hydrolysis, enzymatic hydrolysis and acid-enzyme combined hydrolysis, microwave inducement catalysis can enhance the starch hydrolysis reaction and shorten the reaction time.It is a new technique superior to the tradition hydrolysis method on corn starch hydrolysis.更多还原