Production, Characterization and Immobilization of Laccase Enzyme from Bacillus cereus Local Isolate

In this study, fifty five soil samples were collected from different locations in Baghdad city. Many Gram positive and negative bacterial isolates were obtained;of which 39 were identified as Bacillus spp. upon subjecting to morphological and microscopic tests. Laccase enzyme activity was determined by quantitative methods using a syringaldazine as substrate for growth of these isolates. Results indicated that 17 of these isolates were laccase producer with different specific activity ranged between 98-600U/mg  protein. Results of the biochemical test and Vitek 2 system showed that isolate B5 of  Bacillus cereus was the most efficient in production of laccase when its specific activity reached 600 U/mg protein. Therefore, Bacillus cereus B5 was chosen to determine the optimum conditions for laccase production. Maximum laccase production was achieved after supplementation of the minimal salt medium(pH7) with 0.5% dextrose, 0.5% yeast extract with on inoculums size 10 5  after incubation at 35 o C in a shaker incubator (200 rpm) for 24h. Under these conditions, the specific activity of laccase produced in culture supernatant was increased to 7000 U/mg protein.Laccase  produced under the optimum conditions was purified by precipitation with 70% saturation of ammonium sulfate, ion exchange chromatography by DEAE-Cellulose and gel filtration chromatography throughout Sephacryl S-200 column. After the last purification step, specific activity of the purified enzyme was jumped to 230.000 U/mg with 32.8 purification fold and 49.2% overall yield.when biochemical characteristics of the purified enzyme were studied, results showed that the molecular weight of laccase  produced by Bacillus cereus B5 was about 66000 Dalton, and an optimum pH of 7 for enzyme activity with a wide range of pH stability (7-9). The optimum temperature for enzyme activity was 35 o C, while the enzyme was stable with its full activity at a range of between 3040 o C.The effect of chelating, reducing agents and heavy-metal ions on the purified laccase  activity was investigated. Results indicated that the enzyme activity was inhibited by Zn +2  and Cu +2 , EDTA and Sodium azide. Two immobilization supports (polyacrylamide and agarose) gel were used for entrapment of purified laccase enzyme. Result showed there were two fold increase in the laccase activity with agarose gel as compared to the original enzyme activity.When  storage stability of the free and immobilized laccase was studied, the immobilized enzyme showed good stability by  retaining  66% of activity compared to only 5% for the  free laccase after 61 days storage.