Purification and Characterization of Agarase from &lt;em&gt;Bacillus&lt;/em&gt; sp., H12

Ghazi M Aziz,Hala M Ali

doi:10.19026/crjbs.5.5466

Abstract

The present study was conducted to study the Purification and Characterization of agarase from local isolate <em>Bacillus</em> sp., H12 to use in some industrial and pharmaceutical application. The agarase produced from local isolate <em>Bacillus</em> 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.

Highlights

Agarases are the enzymes which catalyze the hydrolysis of agar
The α-Neoagarooligosaccharide hydrolase was purified from Bacillus sp., MK03 by using ammonium sulphate fractionation with 70% saturation followed by ion exchange by using DEAEToypearl 650M gel filtration by using Superdex 200, the enzyme was purifird 49.7 fold with a yield of 3.6% and the specific activity of purified enzyme was 22.2 U/mg protein (Suzuki et al, 2002)
The fractions which had agarase activity were collected together giving specific activity of 48.5 U/mg proteins with 4.04 fold of purification and 31.25% yield (80-96) for the agarase enzyme. These result are similar to those found by Suzuki et al (2003) and Suzuki et al (2003) who purified -agarase from Bacillus sp., MKO3 by DEAE-Toyopearl 650S, and the purified agarase showed specific activity 1.57 U/mg protein and 14.3 fold of purification with a yield 21.5%

Summary

Introduction

Agarases are the enzymes which catalyze the hydrolysis of agar. They are classified into α-agarase (E.C.3.2.1.158) and ß-agarase (E.C.3.2.1.81) according to the cleavage pattern. Effect of pH on agarase stability: Equal volumes of partially purified enzyme and buffer solution with pH ranging from 4 to 10 were incubated in a water bath at 35°C for 30 min.

Results

Conclusion