Pectinase production by bacteria from the caeco-colic region of the rabbit hind gut
thesisposted on 03.03.2017, 01:26 by Awe, Francis Adeyinka
Several bacterial species in the caeco-colic region of rabbit gut have been identified as pectinases producers that degrade pectin substances. Pectinases are used in food, pulp/paper and textile industries. This study aimed to isolate, identify pectinolytic bacteria in rabbit gut, select and optimize best producers followed by partial purification and characterization of extracellular crude pectinases produced. Initially, pectinolytic bacterial species from caecum and colon of high fiber fed rabbits were isolated and characterized. Bacillus subtilis, B. pumilus, B. ginsengihumi, B. megaterium, B. licheniformis and Paenibacillus spp. were identified. Profile of extracellular pectinases showed that pectin-methyl esterase (PME) was rarely produced, followed by polygalacturonase (PG) while pectin lyase (PL) was most commonly and substantially produced by strains isolated. B. subtilis 1E7 was the best producer of PG and PL. Pectinase production by B. subtilis 1E7 was optimized using response surface methodology for influence of cultural temperature and agitation as independent variables in a central composite design for allocation of treatment combinations which generated 13 experimental runs. The PG and PL responses were analysed using Design Expert 8.0 and Stat-Ease statistical software to validate the responses by graphical and numerical approaches. Second order polynomial was fitted and adequate for PG (p<0.01) and PL (p<0.01) productions while R2 values were 0.9215 and 0.9746 respectively. Predicted PG and PL productions were 0.56U/ml and 111.49U/ml respectively under cultural conditions of 38°C; 190rpm which agreed with practical experiment. Pectinase produced by B. subtilis 1E7 in yeast pectin broth at predicted conditions was purified using ammonium sulphate (40 – 90% saturation) with subsequent dialysis, gel filtration and anion exchange chromatography, desalting was by gel filtration on sephadex C-25. The partially purified pectinase was characterised for pectinolytic performance and stability under certain physico-chemical conditions. Catalytic performance of the pectinase was influenced by temperature, pH and substrate concentrations of the reaction mixtures with maximum PG activity at 60°C, pH 9 and 10mg/ml respectively while PL activity optimised at 60°C, pH 10 and 16mg/ml respectively. The pectinase showed stability at broad range of temperature (40°C-70°C) and pH (7-12). Vmax and Km values were 0.53U/ml, 1.29mg/ml respectively for PG and 64.52U/ml, 4.13mg/ml respectively for PL. PG and PL activities were significantly enhanced in the presence of Ca2+. However, while PG showed no effect in the presence of Mg2+, Co2+, Glycine and Mercapto-ethanol, slight inhibition was observed in Hg2+, Fe3+ and EDTA. Similarly, PL showed no effect in Mg2+, Co2+, glycine and Mercapto-ethanol but the inhibitory effects of Hg2+ and Fe3+ were significant while the extreme inhibitory effect of the chelating EDTA on the lyase suggested that pectate lyase was present. Liquid chromatography-Mass spectroscopy analysis confirmed presence of polygalacturonase and pectate lyases produced by a B. subtilis in our partially purified pectinase. In summary, a novel B. subtilis 1E7 strain that produced alkaline and thermo-stable pectinase with alkaliphilic and thermophilic activity showing resistance towards some known potent inhibitors such as Hg2+, glycine and mercapto-ethanol were identified from rabbit gut. These characteristics indicated structural strength of the pectinase and its suitability for industrial use.