ABSTRACT
Stabilities of native and cross-linked crystalline forms of Streptomyces rubiginosus glucose isomerase were compared in buffer and in 45% glucose/fructose solutions. The cross-linked crystalline form of the enzyme was more stable in the presence of substrate while in a buffer solution the native enzyme was more stable. Inactivation of native enzyme in buffer did not obey first-order kinetics but proceeded with a rapid first phase followed by a stable phase. This stabilization is interpreted to be a result of a conformational change in the protein structure. Inactivation of the native enzyme in buffer was directly related to protein precipitation. In the presence of high substrate concentration, the inactivation was related to browning reactions between the enzyme and the reactive sugar, resulting in soluble sugar-protein complexes.
Subject(s)
Aldose-Ketose Isomerases/chemistry , Crystallization , Glucose/pharmacology , Streptomyces/enzymology , Temperature , Time FactorsABSTRACT
Two major endoxylanases, endo-beta-1,4-xylanase I and II (molecular mass 19 kDa and 21 kDa) from the filamentous fungus Trichoderma reesei have been crystallized by using ammonium sulphate as the precipitating agent. Both crystals were monoclinic and belonged to the space groups C2 (a = 71.9 A, b = 39.0 A, c = 59.9 A, beta = 118.0 degrees, for XYNI) and P2(1) (a = 81.6 A, b = 60.6 A, c = 38.3 A, beta = 94.4, for XYNII). The crystals diffract to at least 2.2 A and 1.5 A, respectively.
Subject(s)
Glycoside Hydrolases/chemistry , Isoenzymes/chemistry , Protein Conformation , Trichoderma/enzymology , Crystallization , Endo-1,4-beta Xylanases , Glycoside Hydrolases/isolation & purification , Isoenzymes/isolation & purification , Species Specificity , X-Ray Diffraction/methodsABSTRACT
Crystallization methods for proteins have been the subject of decades of development yet protein crystallization remains the limiting step in structural studies. We present here a new method for protein crystallization--based on the use of high pressure--that enabled us to accelerate dramatically the growth of glucose isomerase crystals. We think this method may have a more general utility.