Production, Purification and Optimization of cellulase by cellulolytic bacteria isolated from Hindgut of wood feeding termites Coptotermes heimi Wasmann (Blattodea: Rhinotermitidea)

Abstract

Termites are small eusocial insect that acquire the most perfect bioreactor machinery in their digestive system. Even they have small body size but they have a complete ecosystem in their gut, which help them to digest wood. This digestion is done with the help of bacteria that produces cellulase enzymes in termite gut. Due to industrial importance, the demand of cellulase enzyme is increasing day by day. The aim of current study is to extract the cellulase enzymes from the bacteria present in the gut of termite specie Coptotermes heimi. The gut of 8 termites was extracted and grinded in PBS buffer. After serial dilution these extracted bacteria was grown on the CMC supplemented agar plate. The screening of these plate was done with the help of Congo red test and only the bacteria was selected with than >1cm halo zone on agar plate. The morphological identification of this bacteria was done by the microscopy through Gram staining technique. The molecular identification of bacteria was done by 16s RNA gene by using forward 27F and reverse 1492r primers. Crude enzyme was extracted from the bacteria through broth media. Protein estimation was done by performing Bradford assay and DNSA method was performed for enzyme assay. partial purification of crude enzymes was done through the salting in and out ammonium sulphate followed by the dialysis to get the partially purified protein. Production of cellulase enzyme was also checked by growing bacteria on four types of agro-waste. Characterization of cellulase was also performed on different pH, temperature, ion concentration and solvents. Also, the kinetic parameter of the enzymes was done by the Michaelis-Menten equation. The morphological identification shows the presence of gram +ve, rod shape bacteria, indicates the presence of bacillus sp. Molecular identification confirmed the presence of 16s RNA gene when run with 1kb gene ruler. Protein content in the crude enzyme was 1.28 mg/ml and crude enzyme activity was 5.92 U/mg. Production on the sugarcane bagasse was best and produced 0.81 mg/ml of protein content. which was followed by the rice (0.67) and least production was on the rice (0.23) and corn (0.19) mg/ml, respectively. The activity was checked on different temperature range from 25-55°C and the optimum temperature for the enzyme was 40-50°C. effect of different solvents was studied, show that the relative activity of enzyme was increased by methanol (108), toluene (109) and formaldehyde (112). Acetonitrile shows a least effect on enzyme activity, but benzene and propenol decreased the activity up to 50 and 63% respectively. Also, different ions were used to study the effect the and 103%, respectively. Kinetic parameter was also studied by DRSML QAU using different substrate of CMC (0.1-1.5mg/ml) at 40°C using Michaelis-Menten equation. kinetic parameter gives the km and kmax 2.33mgml -1 and5.92µgml 1 min -1