Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520.
A new procedure for the large-scale purification of the recombinant thermostable chitinase (Chi40) cloned from Streptomyces thermoviolaceus in various expression vectors in Escherichia coli is described. Chi40 was overproduced in the cytosolic and secreted forms. The cytosolic form (Chi40c) was highly overproduced and purified by metal-affinity and ion-exchange chromatography in large amounts. The protein was highly active and thermostable but not homogeneous, since a considerable proportion of the Chi40c protein was not correctly folded as determined by native polyacrylamide gel electrophoresis. The Chi40 protein secreted into the culture medium (Chi40s) was purified by hydrophobic interaction and ion-exchange chromatography and high amounts of correctly folded and active Chi40 protein could be recovered in a short time. The enzymatic activity of Chi40s on a synthetic and on its natural substrate, chitin, was studied. Thermostability measurements showed that Chi40 has a T(m) of 60.7 degrees C at neutral pH. (13)C-(15)N double-labeled recombinant Chi40s was also produced and purified from the pECHChi40-9 construct introduced into BL21trxB(DE3) cells grown in minimal medium in the presence of the paramagnetic elements [(13)C]glucose and (15)NH(4)Cl. The presented data open the possibility of an extensive structural study on Chi40s by X-ray crystallography and on enzyme-substrate interaction by NMR spectroscopy.
Christodoulou
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Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, 157 84, Greece.
Duffner
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F
Vorgias
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CE
D002247:Carbon Isotopes; D002686:Chitin / Q000302:isolation & purification / Q000378:metabolism; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D004355:Drug Stability; D004926:Escherichia coli; D009587:Nitrogen Isotopes; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature; D014170:Transformation, Genetic
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases