Optimization And Scaling Up Of Fermentation Conditions On A-L-Rhamnosidase Production By Aspergillus Niger

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.