2. State Key Laboratory of Rice Biology and Institute of Insect Science, College of Agriculture and Biotechnology, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China.
Background: Polygalacturonase is an important pectin degrading enzyme. The western honey bee (Apis mellifera L.) collects pollens from different flowers which are rich source of pectin. The microbiota in the gut of honey bee, release polygalacturonase enzyme and help in pectin digestion. This study aims to isolate and characterize novel polygalacturonase producing bacterial strain from honey bee’s gut.
Methods: The bacterial strain was isolated by using pectin agar plate assay. The bacterial strain was identified on the basis of morphology and 16S rDNA sequence analysis. The enzyme assay was performed by using pectin as a substrate. Biomass of different fruits/vegetables was also used as a source of carbon during fermentation. The protein gel was run using SDS-PAGE for molecular weight determination of the polygalacturonase.
Results: The bacterial strain showed the maximum growth, protein and polygalacturonase production at 72 hours of incubation. This bacterial strain was identified as new Bacillus sp. HD2. The sequence of this strain was successfully uploaded in NCBI Genbank database (Accession no. KP676929). The exo polygalacturonase produced by this strain of Bacillus was optimal at 40°C and exhibited enzyme activity in a wide range of pH from pH 5-12. The polygalacturonase production was enhanced by using yeast extract (3%) in the production medium and the enzyme activity was stimulated by Ca2+ (2 mM) and SDS (200 mM). Biomass of apple’s peel (1%) was found as an excellent source of carbon for the polygalacturonase production in fermentation medium (17.11±0.46 μmol ml-1min-1). In SDS-PAGE gel, the two clear bands of polygalacturonase were found at ~36 kDa and ~72 kDa.
Conclusions: A new bacterial strain Bacillus sp. HD2 was isolated from the gut of honey bee. This strain produced the exo polygalacturonase enzyme. This enzyme was characterized under different pH and temperature and found to have maximum activity in pH 11 at 40°C. Apple peel’s biomass was found as a good source of carbon during fermentation for polygalacturonase production. The SDS-PAGE analysis confirmed two bands of protein with polygalacturonase activity at ~36 kDa and ~72 kDa.
Keywords: Pectin, polygalacturonase, isolation, Bacillus, biomass