COMPARATIVE ASSESSMENT OF PULLULOLYTIC ACTIVITY OF BACILLUS PARAMYCOIDES RESIDENTS IN RUMINANTS’ GUT AND ITS CHEMICAL MUTANTS

Abstract

This study aimed at screening bacterial isolates associated with the rumen of cow and goat for pullulanase production, purifying and characterizing pullulanase from the best pullulanaseproducing bacterium and its most improved mutant. The identities of previously isolated bacteria from the rumen of cow and goat were authenticated by standard microbiological technique. The isolate were qualitatively screened on pullulan agar medium using plate assay technique. Furthermore, the quantitative screenings of the isolates were performed in pullulanase production medium and the pullulanase activities were determined by standard assay procedure. The isolates were cultivated on pullulan agar medium at elevated temperatures to determine their thermotolerance. The isolate with the highest activity was identified by 16S rRNA Gene Sequence analysis. The best pullulanase producing bacterium, Bacillus paramycoides was subjected to chemical mutagenesis using Ethyl Methyl Sulfonate (EMS) for possible mutant generation. Afterward, the crude enzyme from the parent strain and the best pullulanase producing mutants were purified by ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sephadex A- 50 and gel-filtration on Sephacryl S-200, and characterized. The partially purified pullulanase from the parent type and its mutant were optimally active at pH 7.0 and pH 8.0 and at temperature 50˚C and 40˚C respectively. The purified pullulanase from the parent type was relatively stable at pH 7.0 for 30 minutes and retained approximately 80% of its residual activity after 120 minutes of incubation, while it was 90% relatively thermostable at 40°C for 30 minutes and approximately 40 to 80% residual activities were retained at 40 to 60°C after 1hour of incubation. However, the purified pullulanase from the mutant was relatively stable at pH 8.0 for 30 minutes and retaining approximately 80% of its residual activity after 120 minutes of incubation, while it was above 90% relatively thermostable at 40°C for 30min and approximately 50 to 80% residual activities at 40 to 50°C after 1hour of incubation. The activity of purified pullulanase from the parent type was stimulated by Ca2+, Na+, Co2+ , K3 Fe and Mn2+ but inhibited by Mg2+, Hg2+ and Urea while the mutant’s pullulanase activity was also stimulated by Al3+and Ca2+ but inhibited by Cu2+, Hg2+, EDTA and Urea. Apparent michaelis-menten constant (Km) and maximum velocity (Vmax) for the purified pullulanase from the parent type and the mutant were 1.6 mM and 0.121 μmol/min/mg and 6.12 mg/mL and 59.64 μmol/min/mg respectively. Single changes were observed in the neucleotides sequences of each mutant which gives rise to changes in the codon thereby changing the amino acids they code for. The remarkable biochemical properties such as thermal stability, chemical and metal tolerant, ability to tolerate neutral and alkaline conditions including high affinity to substrates makes it an employable candidate for biotechnological applications in pullulan-utilizing industries.