Cloning andExpression oftheClostridium thermosulfurogenes Glucose Isomerase GeneinEscherichia coli andBacillus subtilis

was cloned by complementation ofglucose isomerase activity inaxyL4mutantofEscherichia coli. A new assaymethodfor thermostable glucose isomerase activity on agarplates, using a topagarmixture containing fructose, glucose oxidase, peroxidase, andbenzidine, was developed. Onepositive clone, carrying plasmid pCGI38, was isolated froma cosmidlibrary ofC.thermosulfurogenes DNA.Theplasmid was further subcloned into a Bacillus cloning vector, pTB523, togenerate shuttle plasmid pMLGl,whichisabletoreplicate inbothE.coli and Bacillus subtilis. Expression ofthethermostable glucose isomerase gene inbothspecies was constitutive, whereas synthesis oftheenzyme inC.thermosulfurogenes was inducible byD-xylose. B.subtilis andE.coli produced higher levels ofthermostable glucose isomerase (1.54 and0.46U/mgofprotein, respectively) thandid C.thermosulfurogenes (0.29 U/mgofprotein). Theglucose isomerases synthesized inE.coli andB.subtilis were purified tohomogeneity anddisplayed properties (subunit Mr,50,000; tetrameric molecular structure; thermostability; metalionrequirement; andapparent temperature andpHoptima) identical tothose ofthe native enzyme purified fromC.thermosulfurogenes. Simple heattreatment ofcrude extracts fromE.coli and B.subtilis cells carrying therecombinant plasmid at85°Cfor15mingenerated 80%pure glucose isomerase. Themaximumconversion yield ofglucose (35%,wt/wt) tofructose withthethermostable glucose isomerase (10.8 U/gofdrysubstrate) was 52%atpH7.0and70°C.