ASSESSMENT OF SELECTED HEMICELLULOLYTIC ENZYME ACTIVITIES OF BACTERIA ASSOCIATED WITH THE RUMEN OF COW AND GOAT

Abstract

This research aimed at screening bacterial isolates associated with the rumen of cow and goat for cellulase, xylanase and mannanase production, partially purifying and characterizing them. Ruminal samples from cows and goats were collected from the abattoirs in Akure, Nigeria. Bacteria were isolated and identified by standard microbiological techniques. The isolates were screened for cellulase, mannanase and xylanase production in enzyme production media and assayed for by standard assay procedure. The incubation period was optimized and crude enzymes were partially purified by ammonium sulphate and biochemical properties were determined. The isolates were presumptively identified as Bacillus cereus, Micrococcus sp., Streptococcus sp., Staphylococcus aureus, Enterobacter sp., Psychrobacter sp. and Pseudomonas aeruginosa. Among the bacterial isolates initially screened, isolates represented by G15, C12 and C14 identified by16S rRNA Gene Sequence as Providencia helmbachae, P. vermicola and Psychrobacter pulmonis had highest specific cellulase, xylanase and mannanase activities, respectively. The optimal cellulase, xylanase and mannanase activities were achieved at 30, 6 and 12 hours of incubation, respectively. The partially purified cellulase, xylanase and mannanase were optimal at pH 7.0, 9.0 and 4.0, and at temperature 40, 40 and 60 oC, respectively. The partially purified cellulase was relatively stable at pH 6.0 for 30 minutes and retained 50% of its residual activity after 150 minutes of incubation, while it was 100% relatively thermostable at 40 oC for 30 minutes and approximately 53 to 86% residual activities were retained at 40 and 60 oC after 1 hour of incubation. The maximum pH stability of xylanase and mannanase were achieved at pH 6.0 and 5.0 respectively, while both enzymes were thermally stable with residual activity of 86% for xylanase at 60 oC after 30 minutes and 67 to 83% for mannanase at 40 to 60 oC after 1 hour. Apparent Km and Vmax for cellulase, mannanase and xylanase were 12.28 mg/ml and 56.8 μmol/min/ml, 4.60 mg/ml and 214 μmol/min/ml and 0.76 mg/ml and 3.08 mmol/min/ml respectively. The activities of the enzymes were stimulated by metal ions, while xylanase was exceptionally stimulated by enzyme inhibitors such as Pb2+, SDS and EDTA. Novel biochemical properties such as thermal stability, surfactant and metal tolerant, ability to tolerate alkali and acidic conditions including high affinity for substrates of cellulase, xylanase and mannanase from bacteria in the rumen of ruminants have made them candidates for many industrial processes especially pulp-bleaching