Immobilization of microbial multienzyme preparation on calcium alginate beads as well as lyophilization with mosambi peel matrix improved its shelf-life and stability.

The purpose of the current study was to evaluate the functional activity and storage viability (at 4 °C and 35 °C) of an immobilized as well as lyophilized multienzyme, viz., pectinase, cellulase, and amylase (PCA) that was produced by Bacillus subtilis NG105 under solid state fermentation (SSF) at 35 ℃ for 10 days using mosambi peel as a substrate. After SSF, the culture media was divided into two aliquots. From the first aliquot, the produced ME was extracted, precipitated, and further immobilized on calcium alginate beads (MEICA). In order to immobilize on mosambi peel matrix, the second aliquot was mixed with acetone and subsequently lyophilized (MELMP). Thus, ready MEICA and MELMP extracted 87.5 and 91.5% juice from mango pulp, respectively. In the reusability study, after 5 cycles, MEICA exhibited 23.8%, 24.4%, and 36.5% PCA activity, respectively. The PCA activity of MEICA and MELMP was examined after 60 days of storage at 4 ℃. The result revealed that the PCA for MEICA declined from 100 to 66%, 58.2%, and 64.5%, respectively, while for MELMP, it dropped from 100 to 84.2%, 82.1%, and 69.7%, respectively. Further, after 60 days of storage, the reduction of total protein content (TPC) in free multienzyme (FME), MEICA, and MELMP was 92.2%, 91.5%, and 36.3% observed, respectively. In the localization study, the maximum levels of multienzyme activity were found in cell exudates. This study demonstrated that immobilizing of multienzyme through lyophilization on waste substrates like mosambi peel boosted its stability and shelf-life along with greatly reducing the cost of products.

Microbial production of multienzyme preparation from mosambi peel using Trichoderma asperellum.

Fruit and vegetable wastes create unhygienic conditions and pose a environmental pollution. The utilization of such wastes as carbon sources for production of enzyme with microbial intervention could be an ecofriendly and profitable approach, apart from diminishing the waste load. The present investigation focused on the feasibility of using mosambi (Citrus limetta) peel as substrate for multienzyme production (pectinase, cellulase and amylase) through microbial intervention. Fifteen fungi were isolated from organic waste and screened in vitro their potential of biodegradation of mosambi peel through enzymes production. The best performing isolate was selected and identified as Trichoderma asperellum NG-125 (accession number-MW287256). Conditions viz. temperature, pH, incubation time and nutrient addition were optimized for efficient enzymes production. The maximum enzyme activity (U ml-1 min-1) of pectinase (595.7 ± 2.47), cellulase (497.3 ± 2.06) and amylase (440.9 ± 1.44) were observed at pH 5.5, incubation temperature of 30 °C after 10 days of fermentation. Moreover, macro-nutrients such as ammonium sulfate (0.1%) and potassium-di-hydrogen-ortho-phosphate (0.01%) further also enhanced the production of enzymes. The SDS-PAGE analysis of purified pectinase, cellulase and amylase using showed molecular mass of 43, 66 and 33 kDa, respectively. The enzyme retention activity (ERA) of aforesaid enzymes was also tested with four different natural fiber matrices viz., bagasse, rice husk, paddy straw and wheat straw. Among these, the maximum ERA was observed on bagasse matrix (pectinase-56.35%, cellulose-77.68% and amylase 59.54%). Enzymatic juice clarification yield obtained with test enzyme was 75.8%, as compared to 80.5% of commercial enzyme. The result indicates that T. asperellum may be exploited as multifaceted biocatalysis.