Crystallization and preliminary investigation of xylose isomerase from Bacillus coagulans

Abstract

SHORT COMMUNICATIONS 231 effect on the starting point of crystallization, which occurred 40 times faster. In addition, the mutation may have increased the crystal growth rate. This point was difficult to establish, however, because the growing surfaces of the numerous smaller mutant crystals were larger than those of the single wild-type crystals, which may well explain the observed rate difference of a factor of about three in this series of experiments (Figs. 3a,b).The gas-diffusion progress in the crystallization drops (Fig. 3c) indicated that the mutant enzyme succeeds in nuclei formation shortly after the PEG-10000 concentration crosses the solubility and crystal stability limits of about 5.3%, requiring almost no supersaturation. In contrast, the wild-type enzyme requires a high and enduring supersaturation for nucleation. Wild-type nuclei form 60 h after the drop crosses the solubility and crystal stability limit, which is 25 h after the drop has equilibrated at a high level of supersaturation (Fig. 3c). Most surprising is the uniform timing in all eight successful crystallization trials of the series (Figs. 3a,b).In conclusion, we find that the addition of contact C' accelerated the crystal nucleation process, shortening the crystallization time by a factor of 40. The engineered contact may have increased the crystal growth rate. It did not affect protein solubility, crystal stability and crystalline order. The experiments demonstrate that contact engineering can have a strong influence on crystallization, nurturing the hope that it can be used for improving the crystalline order in the future. References AbstractXylose isomerase from Bacillus coagulans has been crystallized in two different crystal forms. One crystal form is in space group P2~2~2, cell dimensions a = 462, b = 165, c = 82 A. The other is in space group I422, cell dimensions a b= 113, c= 153~,.

Molecular and industrial aspects of glucose isomerase.

“…The anomerism and stereospecificity of the enzyme are shown to be fully consistent with the proposed hydride shift mechanism (46). Crystallization and characterization of GI from Bacillus coagulans (132) and Actinoplanes missouriensis (92) is also reported.…”

Restoration of a Defective Lactococcus lactis Xylose Isomerase

“…Although they share only 20 to 30% amino acid sequence identity with group I XIs, the active-site residues in group I and group II enzymes are highly conserved ( Table 1). The structures of monomeric, homodimeric, and homotetrameric XIs from a number of microbial sources including Streptomyces (5-7), Actinoplanes (1,21,29,36), Arthrobacter (37), Bacillus (28), and Thermus (9) were solved by X-ray crystallography. A structural feature of monomeric XIs is that they consist of an (␣/␤) 8 barrel having an active site and a C-terminal loop region.…”

“…The average length of their polypeptide chains is 380 to 390 amino acids, and they have about 60% amino acid sequence identity, with the active-site residues being highly conserved. The enzymes from Klebsiella (14), Escherichia (22,34), Lactobacillus (3,4,24,40), Lactococcus (13), Clostridium (25), Bacillus (28), Staphylococcus (33), and Thermoanaerobacter (23) are classified in group II. They are 440 to 460 amino acids long and show more than 50% amino acid sequence identity among the group members.…”