Purification and Characterization of Cellulase Enzyme from Bacteria Isolated from Seaweed Waste

Abstract

Seaweedwasteis a sourceof bacteria thatcanproduce cellulaseenzyme. PMP0126yisolateis one collection isolateof the Research and Development Center for Marine and Fisheries Product Proecessing and Biotechnology Agency for Marine and Fisheries Research and Development Ministry of Marine Affairs and Fisheries obtainedfromseaweed wastefrom Pameungpeukarea, Garut, West Java. The aims this research were to purify, characterize the cellulase enzyme, and identify the bacteria producing the enzyme using 16S-rRNA. PMP 0126yisolatewasa Gram-negativeshortrodshape bacteria. Based on thesequencingof the 16S-rRNA genfrom 1282base pairPMP0126y isolate had96%similaritywith Chryseobacteriumindologenesstrain McR-1. Theisolate had 1.9 cellulolytic index onCarboxymethyl Cellulose(CMC) agar medium. The highestcellulaseactivityobtained onthe thirdday offermentation timewith acellulaseactivityof0.108U/mLandspecificactivityof0.120U/mg. Initialpurificationof cellulasebyultrafiltrationproducedactivityof0.112U/mL. Purificationthe enzyme byanionexchange chromatographyproducedthe highestpeak of proteininthefraction no. 48withcellulaseactivityof0.154U/mLat 37.3mMNaCl.Optimumactivity ofthe cellulaseenzymeafterultrafiltrationwaspH 5and300C,while optimumactivity ofthe cellulaseenzymebyanionexchange chromatographywaspH 5and400C. At 300C, the enzymeremainedstableup to4hourincubation.Theactivity ofthe cellulasewasincreasedbyaddition ofCaCl2ions by 53%anddecreased bythe additionof ZnCl2 ions by 78%. Thecellulase showed the highest activity i.e. 0.149U/mL using treated seaweed wasteGlacilariasp. as substrate. Using SDS-PAGE and zimogram analysis, the molecular weight of the cellulase was estimated to be 39 kDa, 30 kDa, and 14 kDa.