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 thermos table chitinase (Chi40) cloned from Streptomyces thermoviolaceus in various expression vectors in Escherichia coli is described. Chi40 was overproduce d in the cytosolic and secreted forms. The cytosolic. form (Chi40c) was hig hly overproduced and purified by metal-affinity and ion-exchange chromatogr aphy in large amounts. The protein was highly active and thermostable but n ot homogeneous, since a considerable proportion of the Chi40c protein was n ot correctly folded as determined by native polyacrylamide gel electrophore sis. The Chi40 protein secreted into the culture medium (Chi40s) was purifi ed by hydrophobic interaction and ion-exchange chromatography and high amou nts of correctly folded and active Chi40 protein could be recovered in a sh ort time. The enzymatic activity of Chi40s on a synthetic and on its natura l substrate, chitin, was studied. Thermostability measurements showed that Chi40 has a T-m of 60.7 degreesC at neutral pH. C-13-N-15 double-labeled re combinant Chi40s. was also produced and purified from the pECHChi40-9 const ruct introduced into BL21trxB(DE3) cells grown in minimal medium in the pre sence of the paramagnetic elements [C-13]glucose and (NH4Cl)-N-15. The pres ented data open the possibility of an extensive structural study on Chi40s by X-ray crystallography and on enzyme-substrate interaction by NMR spectro scopy. (C) 2001 Academic Press.