OPTIMIZATION AND CHARACTERIZATION OF FREE AND CROSS-LINKED ENZYMES AGGREGATE FROM SOLID-STATE FERMENTED MATERIALS

The fermentation process is well known which can lead to a selective strain that produces a large-scale optimum enzyme. In this study, the enzymes used were thermophilic resistant and obtained from solid-state fermented material. However, another technique was approached in this research to obtain higher stability of enzymes. Cross-linked Enzyme Aggregate (CLEA) technology was applied to provide higher tolerance of enzymes under harsh industrial environments. The enzymes assayed were protease, cellulase, xylanase, and pectinase. Maximizing the extraction of enzymes and optimizing the overall process before the immobilization was significant. Moreover, free, and immobilized enzymes were characterized to evaluate their stability and reusability. While the face-centered central composite design (FCCCD) under the response surface methodology (RSM) was applied to optimize the immobilized enzymes. The influence of independent parameters i.e., the concentration of (precipitant) acetone, (cross-linker) glutaraldehyde, and (additive) bovine serum albumin, (BSA) were studied. This was followed by the further characterization of free and CLEA enzymes based on the optimum pH and temperature rooted in the stability and reusability of CLEA enzymes. Overall, the result obtained from optimization showed that CLEA-enzymes have a high recovery at 60% glutaraldehyde, 60% acetone, and 2.25mg/ml BSA for all the selected enzymes. In conclusion, the results gained from the characterization of free and immobilized enzymes showed higher resistance at a temperature of 60°C for free enzymes and 65°C for CLEA-enzymes. On the other side, the pH value was found at pH 8 for protease, xylanase, and pectinase, and pH 7 for cellulase. The application of CLEAs influenced the pH value, where pH 10 for xylanase and protease, pH9 for pectinase, and cellulase had the same pH of 7 as the free and immobilized cellulase.  Overall, the activity was found to be 28.24% after six cycles.