ABSTRACT
Aims@#Bioprospecting for lipases remains limited despite its great deal of industrial application. This study reports on the purification and characterization of a novel lipase KV1 from Acinetobacter haemolyticus strain KV1. @*Methodology and results@#Strain KV1 was identified as A. haemolyticus using the 16S rDNA sequencing, phylogenetic and BIOLOG assessments. The intracellular lipase was purified to homogeneity using consecutive treatments of ammonium sulfate precipitation, dialysis and DEAE-cellulose ion exchange chromatography, affording ~3.5-fold of the purified lipase with an estimated relative molecular mass of 37 kDa. The PCR product of lipase KV1 revealed that the retrieved sequence contained the proposed complete lipase gene sequence at nucleic acid positions 1-954. The purified lipase exhibited its maximum relative activity at 40 °C and pH 8.0, respectively. Interestingly, the novel alkalophilic lipase KV1 retained its relative activities (> 50%) even up to 24 h between pH 7-11. @*Conclusion, significance and impact of study@#The findings revealed that relative activities of the intracellular lipase KV1 were the highest at 40 °C and pH 8.0, respectively. Pertinently, the remarkable stability of the lipase KV1 over a broad range of pH values (pH 7-11), as well as an optimum activity at 40 °C indicated it was an excellent enzyme for producing a wide range of industrial detergents, cleaning up enviro-agro-industrial wastes as well as catalysts in synthetic manufacturing processes. Therefore, its full characterization reported here deserves scientific and economic considerations.
ABSTRACT
Background & objectives: Available literature shows paucity of reports describing antibiotic and metal resistance profile of biofilm forming clinical isolates of Acinetobacter haemolyticus. The present study was undertaken to evaluate the antibiotic and metal resistance profile of Indian clinical isolate of A. haemolyticus MMC 8 isolated from human pus sample in planktonic and biofilm form. Methods: Antibiotic susceptibility and minimum inhibitory concentration were determined employing broth and agar dilution techniques. Biofilm formation was evaluated quantitatively by microtiter plate method and variation in complex architecture was determined by scanning electron microscopy. Minimum biofilm inhibiting concentration was checked by Calgary biofilm device. Results: Planktonic A. haemolyticus MMC 8 was sensitive to 14 antibiotics, AgNO3 and HgC12 resistant to streptomycin and intermediately resistant to netilmycin and kanamycin. MMC 8 exhibited temporal variation in amount and structure of biofilm. There was 32 – 4000 and 4 – 256 fold increase in antibiotic and metal salt concentration, respectively to inhibit biofilm over a period of 72 h as against susceptible planktonic counterparts. Total viable count in the range of 105 -106cfu / ml was observed on plating minimum biofilm inhibiting concentration on Muller-Hinton Agar plate without antimicrobial agents. Biofilm forming cells were several folds more resistant to antibiotics and metal salts in comparison to planktonic cells. Presence of unaffected residual cell population indicated presence of persister cells. Interpretation & conclusions: The results indicate that biofilm formation causes enhanced resistance against antibiotics and metal salts in otherwise susceptible planktonic A. haemolyticus MMC 8.
ABSTRACT
A lipase producing Acinetobacter haemolyticus TA106 was isolated from healthy human skin of tribal population. The maximum activity of 55 U/ml was observed after medium optimization using the “one variable at a time” and the statistical approaches. The optimal composition of the medium was determined as (% w/v or v/v): tryptone - 1, yeast extract - 0.5, sodium chloride-1, olive oil-1, Tween - 80 1, manganese sulphate - 5 mM, sucrose- 1, pH-7. It was found that maximum production occurred in late log phase i.e. after 72 h and at 200 rpm. From factorial design and statistical analysis, it was found that pH, temperature, salt, inoculum density and aeration significantly affected the lipase production. It was also noted that inoculum density of 3 % (v/v), sucrose (1% w/v) and manganese sulphate (5 mM) displayed maximum lipase activity of 55 U/ml by conventional as well as statistical method. Optimization studies also indicated the increase in specific activity from 0.2 U/mg to 6.7 U/mg.