Isolasi, Produksi dan Karakterisasi Protease Kapang Halofilik Asal Tanah Ladang Garam Nusa Tenggara Barat.

Abstract

Protease is an enzyme commonly used to hydrolyze protein peptide chains. Its various applications in the textile, biopharmaceutical, and cosmetic sectors make protease one of the most popular enzymes. To date, Indonesia still imports 5,464 tons of enzymes worth US$45,380 from China, Finland, and Denmark in 2019. Therefore, Indonesia needs to meet its own industrial needs by producing local enzymes. In doing so, Indonesia can also support the global demand for unique enzymes. Unique protease enzymes can be obtained from microorganisms that have the ability to adapt to extreme environments. These extreme environments include salt fields, which have been reported as habitats for halophilic microorganisms such as archaea, algae, bacteria, and fungi with unique physicochemical properties. The halophilic fungus Aspergillus reticulatus SK1-1, capable of producing protease, has been isolated from seawater in the Sekwang salt pond, Republic of Korea. Based on several studies, halophilic and halotolerant fungi can be new sources of unique proteases, which form the scientific basis for isolating fungi from the soil of salt fields in NTB, Indonesia. Protease enzyme production using halotolerant fungi has been carried out, namely Penicillium goetzii from mangrove sediments in Kerala, India, which produced protease activity of 81 U/mL for 6 days in a medium containing 3% NaCl. However, the production results were relatively low compared to the latest study, which found protease activity of 174 U/mL for 5 days with 4% NaCl by the halophilic mold Penicillium chrysogenum from the sediments of the Marakkanam salt fields in Tamil Nadu, India. Currently, there are no studies related to the production of protease enzymes from fungi from Indonesian salt fields. Therefore, this study aims to isolate halophilic fungi from the NTB salt fields that are capable of producing extracellular protease and to carry out its production and characterization. This study consisted of four stages, namely: 1) isolation and physiological growth testing; 2) screening of extracellular protease and molecular identification; 3) optimization and production of protease enzymes; and 4) purification and characterization of protease enzymes. Salt pond soil was collected from the center and edges of the ponds. Isolation was then carried out using a physiological solution in PDA with 3% NaCl. The isolated fungi were tested for salt tolerance based on the diameter of growth during 7 days of incubation to obtain fungi that were halotolerant and/or halophilic by varying 0, 3, and 10% NaCl. The isolates were tested for their ability to produce protease using the enzymatic index (EI) parameter and were molecularly identified using ITSrDNA. Optimization of incubation time and protease production will be performed using one potential mold based on the highest EI results. Protease enzyme activity will be tested quantitatively using the colorimetric method with folin dye at 660 nm. Protease enzymes will be produced and concentrated to 70% with ammonium sulfate, followed by purification using dialysis membranes. The protein yield was purified by SDS-PAGE. The protease enzyme was characterized based on the effects of pH 5-10, temperature 30-70°C, and the effects of PMSF and EDTA inhibitors to determine its type. Based on this study, 17 isolated fungi indicate that salt pond have diverse fungal communities capable of producing protease. Physiological testing revealed that 9 isolates were halophilic and the rest were halotolerant. Testing of the 17 isolates resulted in 7 isolates capable of producing extracellular protease in 2% SMPDA, and 4 of them were identified molecularly. Among the four isolates, LT0214 was the superior isolate with an enzymatic index of 1,82 and was identified as Beauveria sp. which is capable of surviving in 10% NaCl. This enriches the information that enzyme stability in high salinity conditions makes it very valuable for seafood processing and other industrial biotechnology applications. Optimization and production of protease enzymes in 3% PDB resulted in a best time of 9 days and the highest specific enzyme activity from 70% ammonium sulfate concentration, namely 186.2 U/mg with a purity value of 2,3x and a protein molecular weight of ~35 kDa. Characterization revealed that isolate LT0214 is optimal at pH 8 and 40°C, and strongly inhibits PMSF by 92% and EDTA by 100%. This indicates that the extracellular protease from the halophilic fungus Beauveria sp, isolate LT0214 consists of alkaline serine which requires metal ions as cofactors. Collectively, these findings support the existence of protease information produced by halophilic fungi from salt pond soil Beauveria sp., which previous reports had only limited to testing its adaptability in 10% NaCl and the possibility that most serine metalloprotease proteases have other industrial applications.