Production Of Cellulases By Rhizopus Stolonifer Using Starch Subjected To Enzymolysis As The Carbon Source

Although cellulase has been widely used in many industrial fields, including food, medicine, feed, papermaking and bioenergy, its application is still partly limited to the high production cost. This phenomenon mainly due to the following two reasons:Firstly, the low level of enzyme activity produced by lignocellulosic strains is a urgent problem for mass production, which is the major factor that restrict the enlarged production and application of cellulase; secondly, filamentous fungi currently used in cellulase production is hardly to be fast and efficient because the characteristics of strains and the factors of culture conditions that results a long fermentation time and the high production costs.For the first time reported, here we studied and characterized the media and culture conditions for Rhizopus stolonifer to produce cellulase utilizing starch as the carbon source, including starch percentage, nitrogen sources, inorganic salts, shake bottled liquid volume, temperature, initial pH and other factors effected on the production of enzyme. The filter paper activity was selected as the main examine item. By means of adding yeast to medium for reduce the large number of organic acids generated in the cellulase production of Rhizopus stolonifer, thus avoiding the impact of organic acids. Optimization of carbon processing method, the results showed that the optimum condition is starch 10%, α-amylase 0.3%, hydrolysis 60℃ for 30 min. By screening single inorganic nitrogen and composite organic nitrogen source, it was discoveried that NH4Cl and yeast extract added in a certain percentage could result a higher filter paper activity (6.81 IU/mL). Response surface design was adopted to optimize the media, which finally confirmed as follows:starch hydrolyzate 10%, wheat bran extract juice 5%, NH4Cl 0.5%, KH2PO4 0.5%, MgSO4·7H20 0.4%, CaCl2 0.4%, yeast extract 0.6%, PEG4000 0.02%, trace elements was 0.0002%. Under this condition, the filter paper activity peaked 7.46 IU/mL after fermentation 54h. Optimization of culture conditions, the results showed that the optimum condition is as follows:culture temperature 30℃,250 mL flask installed with a liquid volume of 80 mL, inoculation amount 8%, initial pH 5.0. Under this condition, the filter paper activity peaked 9.66 IU/mL that increased nearly 2 fold than before.Based on the optimum conditions previously optimized in shake flask, amplification experiments for Rhizopus stolonifer to produce cellulase were studied in 5 L fermenter, including the temperature control segment, segment control pH, and dissolved oxygen control on fermentor experiments. By controlling the dissolved oxygen content in about 35%, FPA peaked at 15.02 IU/mL, BG peaked at 5.13 IU/mL, CBH peaked at 13.45 IU/mL, and CMC peaked at 10.86 IU/mL, respectively. Compared with the start condition, the cellulase activity of those after optimized was obviously improved. By segment controlling temperature to produce cellulose, the best fermentor temperqture was confirmed to be 30℃ in the first 36 h and 29℃ for the rest of time. Under this condition, FPA peaked at 16.51 IU/mL, BG peaked at 6.17 IU/mL, CBH peaked at 15.39 IU/mL, and CMC peaked at 11.47 IU/mL, respectively. By segment controlling pH to produce cellulose, the best fermentor pH was confirmed to be 4.8 in the first 36 h and 4.6 for the rest of time, while the dissolved oxygen content was maintained about 35%. Under this condition, FPA peaked at 16.51 IU/mL, BG peaked at 6.17 IU/mL, CBH peaked at 15.39 IU/mL, and CMC peaked at 11.47 IU/mL, respectively. Compared with fermentation in shake flask, the filter paper activity of that in 5L fermentor were increased nearly 1.95 fold with and obviously optimization. By comparing pretreatment method of starch, it was confirmed that utilization of starch that hydrolyzed by a-amylase could greatly improved the pruduction cellulase. Compared with conventional production that directly induces cellulase by cellulose, it was observed that utilization of starch could greatly shorten the fermentation time and improved total cellulase activity.In this paper, we for the first time utilized starch to produce cellulase from Rhizopus stolonifer, which different from conventional production that directly induce cellulase by cellulose. Currently, researchs on induction of cellulase by cellulose have been widely reported. Cellobiose was generally considered to induce the production of cellulase. However, fast and efficient synthetic of cellulase by using starch hydrolysis has yet to be revealed, of which the induction mechanism should to be further studied. Compared with the induction of straw, the component of cellulase that induced by starch was observably different. In details, the filter paper activity increased of 25.5%, endoglucanase activity was slightly decreased, the exoglucanase activity was increased nearly 28-fold, β-glucosidase activity changed little, and thus the enzyme composition becomes more reasonable. Moreover, the maximum activity of cellulase peaked at 84 h when straw was used as substrate for Rhizopus stolonifer to produce cellulase, while the fermentation time of that induced by starch only required 60 h. It indicated that utilization of starch could greatly shorten the fermentation time and improved total cellulase activity and digestion efficiency, which providing a significant opportunity for the future industrial applications.