【作者】 马骏；

【导师】 汪钊；

【作者基本信息】 浙江工业大学 ， 发酵工程， 2013， 硕士

【摘要】 a-L-鼠李糖苷酶(E.C.3.2.1.40)是一种可作用于末端含有a-L-鼠李糖残基化合物的水解酶,其在柑橘汁脱苦、酒类饮料香味改善以及天然化合物结构改性方面具有重要的应用价值。黑曲霉WZ001能同时高产a-L-1,2-鼠李糖苷酶和a-L-1,6-鼠李糖苷酶。为了考察菌体生理状态对黑曲霉WZ001发酵产酶的影响,首先优化了菌丝体活力测定方法——TTC-脱氢酶法。在此基础上,在5 L发酵罐水平研究了不同控制参数对a-L-鼠李糖苷酶产量及菌丝体活力的影响。单因素实验结果表明,种龄、pH和搅拌转速是影响反应器水平上a-L-鼠李糖苷酶产量的三个关键因素。建立了最佳的发酵生产工艺：摇瓶种龄42 h；发酵罐通气强度0.5 VVM；培养温度30℃；0～36 h,搅拌转速控制于600 r/min, pH控制于5.5；36-120 h,搅拌转速控制于400 r/min, pH控制于4.5。在此条件下,a-L-1,2-鼠李糖苷酶和a-L-1,6-鼠李糖苷酶最大产量分别为2438 U/mL和3594 U/mL,比优化前分别提高了112%和297%。菌丝体活力研究结果表明,摇瓶种子的菌丝体活力高时,a-L-鼠李糖苷酶产量也高；当搅拌转速或通气量过低时,发酵24～36 h的菌丝体活力明显低于正常水平,a-L-鼠李糖苷酶产量也较低。因此,菌丝体活力可以作为a-L-鼠李糖苷酶发酵优化和过程控制的一个重要监测指标。进一步研究了5L发酵罐至30 L发酵罐的工艺放大。结果表明,以搅拌转速按搅拌桨叶尖线速度相等,通气量按5L发酵罐中空气表观线速度的1.5倍的放大原则,放大后的α-L-1,2-鼠李糖苷酶和α-L-1,6-鼠李糖苷酶产量分别为2515 U/mL和3612 U/mL,达到5 L罐的发酵产酶水平。发酵液经过高速离心、膜分离和喷雾干燥工艺,制得固体酶制剂,α-L-1,2-鼠李糖苷酶和α-L-1,6-鼠李糖苷酶的总收率分别为71.2%和73.6%。更多还原

【Abstract】 a-L-Rhamnosidase (E.C.3.2.1.40) is a kind of hydrolase that can act on a family of nature compounds containing terminal a-L-rhamnose residues. It has turned out to be a biotechnologically important enzyme due to its applications in a variety of processes like debittering of citrus fruit juices, enhancement of wine aromas and modification structure of nature compounds.This research had focused on Aspergillus niger WZ001 which could produce both a-L-1,2-rhamnosidase and a-L-1,6-rhamnosidase. To investigate the influences of physiological status of thallus to Aspergillus niger WZ001 producing enzyme, the first step was to optimize the method for measuring the viability of mycelium——TTC-dehydrogenase method.Based on this method, the influences of different conditions on the yield of a-L-rhamnosidase and the viability of Aspergillus niger mycelium in 5 L fermentation process were researched. Through the single factor experiment, we found that the age of cell, pH and agitation speed were the three key factors in the reactor level to effect the yield of a-L-rhamnosidase. The optimal fermentation process was obtained:the age of cell and aeration intensity were 42 h and 0.5 VVM respectively. The cultivate temperature was stayed at 30℃. pH and agitation speed were controlled in 5.5 and 600 r/min before 36 h, respectively, and then controlled in 4.5 and 400 r/min during 36～120h, respectively. Under the condition, the maximum enzyme production of α-L-1,2-rhamnosidase and α-L-1,6-rhamnosidase were 2438 U/mL and 3594 U/mL, respectively. They were improved by 112% and 297% than that before, respectively.The results showed that the yield of a-L-rhamnosidase was high while the viability of mycelium in shake flask was high. When it fermented for 24～36 h, the viability of mycelium was remarkable lower than normal and the yield of a-L-rhamnosidase was significantly drop than before with the agitation speed or the throughput was much too low. Therefore, the viability of mycelium could be used as an important index in fermentation optimization and process control of a-L-rhamnosidase.The next step was to research the technique of magnify the fermentation tank from 5 L to 30 L. The results showed that when the agitation speed was equal and the throughput was 1.5 times of the apparent air velocity in 5 L fermentation tank, the magnified enzyme yield of a-L-1,2-rhamnosidase and a-L-1,6-rhamnosidase were 2515 U/mL and 3612 U/mL, respectively. After high speed centrifugation, membrane separation and spray drying process, the fermentation broth would produced to solid enzyme preparation. And the total recovery of α-L-1,2-rhamnosidase and α-L-1,6-rhamnosidase were 71.2% and 73.6%, respectively.更多还原