Combinatorial approach of statistical optimization and mutagenesis for improved production of acidic phytase by Aspergillus niger NCIM 563 under submerged fermentation condition.

Combination of statistical optimization and mutagenesis to isolate hypersecretory strains is studied to maximize phytase production from Aspergillus niger NCIM 563 under submerged fermentation. The overall results obtained show a remarkable 5.98-fold improvement in phytase production rates when compared to that using basal medium. Optimization of culture conditions from parent strain is studied first by the Plackett-Burman technique to evaluate the effects of 11 variables for phytase production. The results showed that glucose, MgSO(4), KCl, incubation period, and MnSO(4) are the most significant variables affecting enzyme production. Further optimization in these variables, using a central composite design technique, resulted in 3.74-fold increase in the yield of phytase production to 254,500 U/l when compared with the activity observed with basal media (68,000 U/l) in shake flask. Our experiments show that the phytase from A. niger NCIM 563 exhibits desirable activity in simulated gastric fluid conditions with low pH and also improved thermostability when compared to commercial phytase. The improved yield demonstrates the potential applicability of phytase enzyme as a source of phytase supplement for phosphorus nutrition and environmental protection in animal feed industry. Physical and chemical mutagenesis experiments were carried out in parallel to isolate hypersecretory mutants that could possibly further enhance the enzyme production. Using optimized media conditions of the parent strain, our results show that mutant strain A. niger NCIM 1359 increased the phytase activity by another 1.6-fold to 407,200 U/l.

High level phytase production by Aspergillus niger NCIM 563 in solid state culture: response surface optimization, up-scaling, and its partial characterization.

Phytase production by Aspergillus niger NCIM 563 was optimized by using wheat bran in solid state fermentation (SSF). An integrated statistical optimization approach involving the combination of Placket-Burman design (PBD) and Box-Behnken design (BBD) was employed. PBD was used to evaluate the effect of 11 variables related to phytase production, and five statistically significant variables, namely, glucose, dextrin, NaNO(3), distilled water, and MgSO(4) · 7H(2)O, were selected for further optimization studies. The levels of five variables for maximum phytase production were determined by a BBD. Phytase production improved from 50 IU/g dry moldy bran (DMB) to 154 IU/g DMB indicating 3.08-fold increase after optimization. A simultaneous reduction in fermentation time from 7 to 4 days shows a high productivity of 38,500 IU/kg/day. Scaling up the process in trays gave reproducible phytase production overcoming industrial constraints of practicability and economics. The culture extract also had 133.2, 41.58, and 310.34 IU/g DMB of xylanase, cellulase, and amylase activities, respectively. The partially purified phytase was optimally active at 55°C and pH 6.0. The enzyme retained ca. 75% activity over a wide pH range 2.0-9.5. It also released more inorganic phosphorus from soybean meal in a broad pH range from 2.5 to 6.5 under emulated gastric conditions. Molecular weight of phytase on Sephacryl S-200 was approximately 87 kDa. The K (m) and V (max) observed were 0.156 mM and 220 µm/min/mg. The SSF phytase from A. niger NCIM 563 offers an economical production capability and its wide pH stability shows its suitability for use in poultry feed.

Strain improvement and up scaling of phytase production by Aspergillus niger NCIM 563 under submerged fermentation conditions.

Combination of physical and chemical mutagenesis was used to isolate hyper secretory strains of Aspergillus niger NCIM 563 for phytase production. Phytase activity of mutant N-1 and N-79 was about 17 and 47% higher than the parent strain. In shake flask the productivity of phytase in parent, mutant N-1 and N-79 was 6,181, 7,619 and 9,523 IU/L per day, respectively. Up scaling of the fermentation from shake flask to 3 and 14 L New Brunswick fermenter was studied. After optimizing various fermentation parameters like aeration, agitation and carbon source in fermentation medium the fermentation time to achieve highest phytase activity was reduced considerably from 14 days in shake flask to 8 days in 14 L fermenter. Highest phytase activity of 80 IU/ml was obtained in 1% rice bran-3.5% glucose containing medium with aeration 0.2 vvm and agitation 550 rpm at room temperature on 8th day of fermentation. Addition of either bavistin (0.1%), penicillin (0.1%), formalin (0.2%) and sodium chloride (10%) in fermented broth were effective in retaining 100% phytase activity for 8 days at room temperature while these reagents along with methanol (50%) and ethanol (50%) confer 100% stability of phytase activity at 4 degrees C till 20 days. Among various carriers used for application of phytase in feed, wheat bran and rice bran were superior to silica and calcium carbonate. Thermo stabilization studies indicate 100% protection of phytase activity in presence of 12% skim milk at 70 degrees C, which will be useful for its spray drying.