Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization.
Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase (CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K(M) and k(cat) were calculated 535 microM and 127 s(-1) for ABTS, 53 microM and 3 s(-1) for 2, 6-DMP and 5 microM and 20 s(-1) for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme was preincubated at 70 and 80 degrees C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation at 70 degrees C and 2.4-fold after 10 min preincubation at 80 degrees C. Preincubation of the enzyme in 70 degrees C for 30 min raised the activity four-fold with ABTS as the substrate. Also, L-dopa was used as a substrate. The enzyme was able to oxidize L-dopa with the K(M) and k(cat) of 1,493 microM and 194 s(-1), respectively.
Mohammadian
Mahdi
M
Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran.
Fathi-Roudsari
Mehrnoosh
M
Mollania
Nasrin
N
Badoei-Dalfard
Arastoo
A
Khajeh
Khosro
K
D000332:Aerobiosis; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D052160:Benzothiazoles / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015870:Gene Expression; D006835:Hydrazones / Q000378:metabolism; D007700:Kinetics; D042845:Laccase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007980:Levodopa / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011748:Pyrogallol / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013451:Sulfonic Acids / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D052160:Benzothiazoles; D006835:Hydrazones; D011994:Recombinant Proteins; D013451:Sulfonic Acids; D011748:Pyrogallol; C009370:syringaldazine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D007980:Levodopa; C010120:pyrogallol 1,3-dimethyl ether; D042845:Laccase
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