Optimization of cultural conditions for biosynthesis, partial purification and characterization of Invertase by Aspergillus species

Enzyme production is a growing field of biotechnology. Invertase (β-D (fructofuranosidase, EC 3.2.1.26) is one of the important commercial enzymes used in food industry and belongs to first proteins that were identified as biocatalysts. It catalyses the reaction of detachment of the terminal non-reducing β-D-fructofuranoside residue in β-D-fructofuranosides. A wide range of microorganisms produce Invertase and can, thus, utilize sucrose as the only carbon source and as inducer of such enzyme. Food processing industry produces an enormous amount of carbohydrate wastes, which pose increasing disposal costs and environmental challenges, approximately 45% of total organic industrial pollution generates from this sector. Beside their pollution and hazardous aspects, these organic wastes are rich in biodegradable materials, and have potential to be used as suitable substrates for biotechnological productions.

Extensive studies have been done by using synthetic medium for preparation of Invertase while a little attention has been paid on its production from less expensive sources. In Pakistan food industry is spreading rapidly and the demand of processed enzymes is very high. A lot of foreign exchange is spent for the import of these enzymes. Need of the time is to improve the processes for the better production of these enzymes at domestic level by using waste products. The present study was planned to produce Invertase from indigenous wastes/byproducts including carrot and potato peels, as substrates for Aspergillus species through solid state fermentation (SSF). For this purpose, five different species were screened. Higher activities of Invertase were produced by Aspergillus niger on carrot peels and A.terreus on potato peels at 30°C over 72h of incubation.

The process parameters influencing the production of Invertase by A.niger and A.terreus were optimized in SSF. 90% of moisture content was found to be best for maximum production of Invertase (7.95±0.13 and 6.69±0.04 U/ml) on carrot and potato peels. The results explicated that 72 and 60 h of incubation period for A.niger and A .terreus respectively exhibited more potential for enzyme synthesis. However, 2.5% of Inoculum's size gave highest units of Invertase (10.24±0.06 U/ml) from A.niger while in case of A.terreus maximum activity (7.09±0.04 U/ml) was noted at 2%.

Enzyme purification was carried out, and 512 IU of crude Invertase from A.niger was partially purified to maximum specific activity (11.5 IU/mg protein) after 40-80% ammonium sulphate precipitation. There was a decrease in recovery to 65.6% after precipitation with 1.42 purification fold. Similarly, in A.terreus enzyme purification was carried out, and 354.8 IU of crude Invertase was partially purified to maximum specific activity (9.46 IU/mg protein) after 40-80% ammonium sulphate precipitation. There was a decrease in recovery to 55.9% after precipitation with 1.37 purification fold.

During characterization, it was observed that Invertase from A.niger was stable from pH 5.5 to 6.5 while it exhibited maximum activity at pH 5.5. However, enzyme show highest activity at 50°C whereas, it show stability from 20 to 40°C. When the effect of different substrates on Invertase activity was assessed, it was found that sucrose showed highest activity both at higher and lower concentrations. Enzyme was treated with various metal ions, Cobalt and sodium showed highest activity while mercury significantly inhibited Invertase activity. While Invertase from A.terreus was stable from pH 4.0 to 6.0 while it exhibited maximum activity at pH 6.0. Whereas, enzyme shows highest activity at 60°C whereas, it shows stability from 30 to 60°C. Similar results were observed in case of substrate specificity. However, when the effect of various metals ions on Invertase activity was evaluated, it was found that Cobalt and calcium showed highest activity while mercury and nickel considerably inhibited Invertase activity.