摘要
单宁酶是一种酰基水解酶,是在单宁酸等诱导剂存在下合成的诱导酶,已广泛应用于食品饮料、化妆品和饲料工业等方面。但由于目前酶法的生产水平较低,限制了其在工业生产中的应用。同时,没食子酸及其没食子酸酯类化合物是重要的医药和化工原料,市场上的这类产品基本是由五倍子单宁及塔拉单宁酸水解生产而来的,因为酸碱法水解会造成的环境污染、设备腐蚀等问题。多年来,人们一直在寻求用单宁酶法来生产没食子酸及其没食子酸酯类化合物方法来替代原有工艺。因此,开展相关研究以提高单宁酶生产水平及酶法转化没食子酸及其没食子酸酯类化合物具有积极的现实意义。
本文采用实验室保藏的黑曲霉菌株为产单宁酶的出发菌株,采用紫外和氮离子注入方式对其进行诱变选育,并对黑曲霉发酵产单宁酶条件、黑曲霉发酵动力学、单宁酶的固定化及酶学性质进行研究,对固定化单宁酶转化没食子酸和没食子酸丙酯条件进行优化。主要研究结果如下：
产单宁酶菌株选育以黑曲霉产单宁酶菌株为试验菌株,经筛选培养得到酶活为86U/mL的菌株N5。以N5为出发菌株经过紫外诱变,得到酶活为158U/mL的菌株N5-U5-3。用N+离子对N5-U5-3进行诱变选育,经过筛选后得到编号为03号的菌株,酶活力为198U/mL。该菌株具有较好的遗传稳定性,是一株比较理想的单宁酶高产菌株,命名为黑曲霉111(Aspergillus niger111)。
产单宁酶条件优化通过单因素试验、中心组合实验设计及响应面法对黑曲霉的产单宁酶条件进行优化。确定对发酵产酶影响最大的因素依次为单宁浓度、培养温度、培养时间,以及其最适产酶条件为：30℃,转速180r/min,单宁浓度1.76%,初始pH6.0,接种量10%.,装液量20%,培养时间82.2h。在此条件下,发酵单宁酶活力可达380.34U/mL。
黑曲霉发酵动力学研究以Aspergillus niger111为实验菌种,对其进行分批发酵动力学研究。以该菌株在5L发酵罐中分批发酵的数据为依据,建立分批发酵生产单宁酶的菌体生长、单宁酶合成以及基质消耗动力学模型。
单宁酶固定化筛选LX-1000HA树脂作为载体,对游离单宁酶进行固定化,通过中心组合实验设计和RSM,对LX-1000HA树脂固定化单宁酶的条件进行优化,得出最佳工艺条件为酶液/载体比17.6(v/m)、pH6.9、时间24h、温度36.5℃,该条件下固定化酶活力达到(18495±200)U/g,对温度和pH的适应性更广,酶活回收率为45.25%。
固定化酶制备没食子酸及其丙酯固定化单宁酶作为水解五倍子单宁转化制备没食子酸的催化剂,优化工艺条件,在pH6.5、温度50℃、固定化酶酶量4.5g、时间30h的条件下,没食子酸浓度达到27.93mg/mL,单宁酸转化率为93.1%。筛选环己烷为有机介质,在非水相中用固定化酶催化合成没食子酸丙酯,优化工艺条件,在温度52℃、环己烷和正丙醇比例4：1、没食子酸浓度1.8%、时间40h条件下,没食子酸丙酯得率90.64%。
Tannase as an acyl hydrolase, which is produced by certain microorganisms with inducers, for example tannic acid, has been widely applied in such areas as food and beverages, cosmetics and feed industries. Gallic acid and its ester derivatives are important pharmaceutical and chemical raw materials, which are manufactured by the Tala acid and tannic acid hydrolysis. For acid hydrolysis causes environmental pollution, corrosion problems, the replacement of acid method by tannase method to produce gallic acid and its esters has been explored for many years. But the current low yield of tannase method limited its application in industrial production, therefore to undertake research to raise the acticity of tannase then enzymatic conversion yield of gallic acid and gallic acid esters have positive practical significance.
This thesis uses Aspergillus strains preserved in laboratory as original strains to screen by UV and nitrogen ion injection. And the conditions of fermentation of tannase from Aspergillus niger, Aspergillus niger fermentation kinetics, tannase immobilization and enzyme properties were studied. Tannase enzymatic conversion conditions for gallic acid and its ester were also optimized. Main findings are as follows:
Ten tannase-producing strains were selected as test strains from Aspergillus strains preserved in laboratory. Strain N5with86U/ml of enzymic activity was screened out. Through3min UV mutation, a mutant strain named N5-U5-3was obtained. The tannase activity of which reached158U/ml,83.7%higher than that of original strain. Though the genetic stability verification, its enzyme performaned relatively stable.
N5-U5-3was treated with10KeV and3×1015N+/cm2. After prescreening and rescreening experiments in rotation-flask, a high-yield strain Aspergillus nigerlll was obtained. The tannase activity of which reached198U/mL, almost25.3%higher than that of N5-U5-3. The genetic stability of Aspergillus niger111was comparative stable.
Single factor experiments, central composite design and response surface method(RSD) were used for optimization on Aspergillus niger tannase producing conditions. The most three important factors of determining the tannase production were tannic acid concentration, temperature and incubation time. The optimum conditions for enzyme production are as follows:30℃; speed,180r/min; tannic acid concentration,1.76%; initial pH,6.0; inoculum,10%; bottling volume,20%and culture time,82.2h. In these conditions, the fermentation tannase activity reached380.34U/ml.
Based on the data gotten from Aspergillus niger111batch fermentation in the5L fermenter, kinetic models of cell growth, synthesis of tannase and substrate consumption were established.
Compared with various carriers, LX-1000HA were chosen to immobilize tannase. Then Box-Behnken experimental design and RSD were used to optimize the conditions for immobilizing tannase by LX-1000HA resin. The results showed that the best conditions were17.6(v/m) of the enzyme solution/carrier ratio, pH6.9,24h and36.5℃. Under these conditions, the activity of the immobilized enzyme was (18495±200) U/g with activity recovery of45.25%. The optimal reaction temperature, pH for immobilized enzyme were50℃,6.5, respectively.
Immobilized tannase by LX-1000HA was applied to hydrolyse tannic acid to produce gallic acid. The best conditions were got by the monofactorial experiment which were pH,6.2-6.8; temperature range,48℃-52℃; immobilized enzyme,3.5-4.5g; time range,25-35h. Conditions were optimized by orthogonal experimental design and the best converting conditions were:pH6.5,50℃,4.5g of immobilized tannse and30h. Under these conditions, the concentration of gallic acid could reach27.93mg/ml and the conversion of tannin could reached93.1%.
Immobilized tannase by LX-1000HA was applied to synthetize propyl gallate in the system of organic reaction. Firstly, cyclohexane was chosen as the best organic reaction media, then the best synthesize conditions were obtained through monofactorial experiment and orthogonal experimental design as follow:52℃,4:1of cyclohexane to propanol,1.8%of gallic acid and40h. Under these conditions, the production of propyl gallate could reach90.64%.
引文
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