Title of Thesis

Production and characterization of alkaline phosphatase from psychrphilic bacteria

Author(s)

Bashir Ahmad

Abstract
The normal flora entombed in ice of glaciers and freezers may have adapted the severe physiological conditions and scarce source of macronutrients for their survival.Aim of this study was to isolate, identify and characterize psychrophilic bacteria from glacial and non glacial samples, and to purify and characterize alkaline phosphatase from a selected strain.Three cold active bacteria, morphophysiologically, identified as Bacillus subtilis MRLBA7, Bacillus licheniformis MRLBA8 and Bacillus megaterium MRLBA9 were isolated from -20°C freezer of Microbiology Research Laboratory (MRL), Quaid-i-Azam University, Islamabad, Pakistan.These strains were able to grow aerobically at 6°C but not at 40°C except MRLBA8 that could grow at 48°C.None of the isolates showed inhibition of growth in presence of glycerol. Isolate MRLBA7, bearing central spore, grew in the presence of 30% glycerol at 0°C after 48 hrs of incubation and showed maximum growth without glycerol at 25°C after 24 hrs. Isolate MRLBA8 showed growth in the presence of 50% glycerol at 4°C after 72 hrs of incubation and maximum growth was observed at 20°C in the absence of glycerol. Isolate MRLBA9 showed growth at 6°C in the presence of 40% glycerol after 48 hrs of incubation and maximum growth was observed at 25°C in the absence of glycerol. Isolates were susceptible to antibiotics except Bacillus subtilis MRLBA7 that exhibited antibiotic resistance against penicillin and fosphomycin, Bacillus licheniformis MRLBA8 against aztreonam and fosphomycin, and Bacillus megaterium MRLBA9 against vancomycin and penicillin.The growth profile and biochemical characteristics of all three isolates were rather similar to that of mesophilic counterparts except adaptation to low temperature.
On the basis of morphology, biochemical tests and 16S rRNA analyses, six cold active bacteria identified as Pseudomonas sp. MRLBA1, Pseudomonas sp.MRLBA2, Pseudomonas sp.MRLBA3 Pseudomonas sp.MRLBA4, Arthrobacter sp.MRLBA5 and Stenotrophomonas sp. MRLBA6 were isolated from ice, water and soil samples obtained from Batura, Hopper and Passu glacier, Northern Areas of Pakistan.All of the glacial isolates were aerobic, asporous, non-motile and Gram-negative rods except MRLBA5 that was Gram variable, motile and exhibited rod-coccus growth cycle.Pseudomonas sp. MRLBA1 was capable of growing at 2-30°C, and pH 4-11; Pseudomonas sp. MRLBA2 at 4-30°C, and pH 4-10; Pseudomonas sp.MRLBA3 at 4-35°C, and pH 5-10; Pseudomonas sp. MRLBA4 at 4-37°C, and pH 5-10; Arthrobacter sp. MRLBA5 at 4-37°C, and pH 4- 9; and Stenotrophomonas sp. MRLBA6 at 4-30°C, and pH 4-11.The glacial Isolates were susceptible to antibiotics except Pseudomonas sp. MRLBA1 that exhibited antibiotic resistance against vancomycin and penicillin, Pseudomonas sp. MRLBA2 against aztreonam and fosphomycin, Pseudomonas sp. MRLBA3 against vancomycin and penicillin, Pseudomonas sp. MRLBA4 against fosphomycin to vancomycin and penicillin, Arthrobacter sp. MRLBA5 against aztreonam and fosphomycin, and Stenotrophomonas sp. MRLBA6 against aztreonam and vancomycin.
Pseudomonas sp. MRLBA1 selected for the production of alkaline phosphatase showed highest extracellular alkaline phosphatase activity at pH 8.0 and 18°C when inoculated with 24 hrs old inoculum (5%), after 48hrs of incubation in shake flask experiments. After precipitation with 60% ammonium sulfate, the enzyme was purified with gel permeation (134.81 U/mg) and ion exchange chromatography (225 U/mg) with 9.75 and 16.27 fold of purification, respectively.A single active peak of 54-58 KDa was estimated by gel permeation column and a single band of ~54-56 KDa was determined from SDS-polyacrylamide gel electrophoresis.The purified alkaline phosphatase was stable between pH 4-13 and 0-55°C but maximally active at pH 9.0 and 37°C. The enzyme was concluded as the thermo-labile in nature. The chloride salts of calcium zinc, magnesium, mercury and copper increased the specific activity of alkaline phosphatase but iron and potassium decreased it to some extent.The enzyme was stable when assayed along with 45% of glycerol but showed decrease in activity from 50-90% glycerol, sharply.The kinetic constants, Km and Vmax, were calculated as 122 μmol and 28 μmol.min-1 from Eadie-Hofstee plot. The potassium ferricyanide did not have any inhibitory or stimulatory effect on alkaline phosphatase whereas potassium ferrocyanide showed uncompetitive inhibition.

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