Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase fromAspergillus nigerATCC 20611

Abstract

The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO3, Zn+2, and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R 2) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO3, 1.5 mM (v/v) Zn+2, and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM)

“…The correlation between the two compounds shows that inulinase is produced during invertase biosynthesis phase. Inulinase serves as a precursor of invertase, hence, increasing inulinase content leads to an elevation of invertase production 12 . On the one side, there was a significant positive correlation ( r ) coefficient at p  ≤ 0.01 between the production of inulinase ( r  = 0.98) and invertase ( r  = 0.99) with fungi growth (Fig.…”

“…The spores were harvested and suspended in sterile distilled water containing 0.01% (v/v) Tween 80 to obtain approximately 2.0 × 10 6  spores/mL. Preliminary experiments were performed by using a production medium consisting of: sucrose 10% (w/v), yeast extract 2.5% (w/v), NaNO 3 2% (w/v), Zn +2 1.5 mM (v/v), Triton X-100 1% (v/v) 12 . The initial pH of the basal medium was adjusted to 6.0 with HCl (5 M) and NaOH (5 M), prior to sterilization at 121 °C for 15 min.…”

“…For the extraction of intracellular enzymes, the pellet was resuspended in 50 mL of sodium acetate buffer (200 mM, pH 5.0) with vigorous vortexing followed by incubation at 30 °C and 150 rpm for 30 min. Then, it was sonicated on ice in glass tubes using a Branson Sonic Power Sonicator (48 Bransonic Power, 40 W, 30 s with 30 s cooling periods) for 5 min followed by centrifugation at 4 °C (20 min, 10,000 ×g ) 12 . Cell debris was removed by centrifugation at 4 °C (20 min, 10,000  ×   g ).…”

“…The optimum conditions can be used for future upscale productions of the intra-extracellular inulinase, invertase and biomass. The maximum enzymes production and growth rate were obtained in a short time (96 h) 12 . From an economic stand-point, it would be favorable to use the minimum time and maximum enzyme production.…”

“…It is a collection of statistical techniques for design of experiments, building models, measured responses, evaluating the effects and relationship between clusters of controlled experimental factors and searching for the optimum conditions, has successfully been used in the optimization of bioprocesses 11 . The benefit of employing RSM is to decrease the number of experimental trials that are needed to assess numerous parameters and their interactions 12 . Also, the method of RSM makes it possible to study several factors simultaneously, to quantify the individual effect of each factor and to investigate their possible interactions 13, 14.…”