Abstract

The present study was conducted to study the Purification and Characterization of agarase from local isolate Bacillus sp., H12 to use in some industrial and pharmaceutical application. The agarase produced from local isolate Bacillus sp., H12 was purified by precipitation with 70% saturation ammonium sulphate, followed by ion-exchange chromotography and Gel filtration. Results showed appearance of two protein peaks, once in the wash step without enzyme activity and the other in the elution step have enzyme activity at this step by using Ion-exchanger DEAE-cellulose and separate one protein peak contain enzyme activity at gel filtration step by using the gel Sephadex G-100, the enzyme was purified to 5.67 times with an enzymes yields of 21.45%. Enzyme characterization of the enzyme indicated that the optimum pH for the enzyme activity and stability was 7. The maximum activity for enzyme appeared at 45°C and lost approximately 60% of its activity at rang above 65°C. Enzyme characterization results showed that the chlorides of silver and mercury had inhibitory effect on enzyme activity, the remaining enzyme activity for the enzyme was 25%, for silver ions and 12.5% for mercury ions at 5 mM and 13.75% for silver ions and 7.5% for mercury ions at 10 mM. The enzyme was affected by chelating agent Ethylene Diamine Tetra Acetic Acid (EDTA) at concentration 2, 5 mM the remaining activity 43.75 and 25%, respectively and the enzyme referred to metalloenzyme the enzyme was kept their activity when treated. by reducing agent (2-mercaptoethanol) at 2 mM while the enzyme kept 83.75% of its activity at 5 mM of (2-mercaptoethanol). The enzyme was kept their activity when treated by Phenyl Methyl Sulphonyl Fluoride (PMSF) at concentration 1, 5 mM, the remaining activity was 97.5 and 91.25%, respectively and this indicated that this enzyme did not refer to serine enzyme group.

Keywords:

Agarase, Bacillus sp, characterization, gel filtration, ion-exchange, purification,

References

Competing interests

The authors have no competing interests.

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