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A thermostable xylanase gene ( stx I) obtained from Streptomyces thermonitrificans NTU-88 on domain analysis revealed an N-terminal catalytic domain featuring homology to a known xylanase within the glycoside hydrolase family 11. Recombinant STXI retained more than 60% of its activity following its incubation for at 60 °C for 24 h. These characteristics were close to thermophile and mesophile Streptomyces strains. The main hydrolysis products of xylan degraded by STXI included large xylooligosaccharide fragments. These results indicated that STXI was a typical endoxylanase. As regards the phylogenetic relationships of GH11, STXI and the other xylanase deriving from Streptomyces were included in a subgroup of the aerobic bacterial group. This result implied that the evolutionary relationships between the various xylanases deriving from Streptomyces strains were convergent.

A thermostable D-xylose–glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by γ-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE–Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12™ columns. The N-terminal amino acid sequence and amino acid analysis shows 73–92% homology with xylose–glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12™ column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60–85°C at pH 7.0, using D-glucose, and up to 50–60°C at pH 7.6, using D-xylose as substrates. The activation energy (Ea= 46 kJ·mol–1) and the critical temperature (Tc= 60°C) were determined by fluorescence spectroscopy. Tcis in close coincidence with the melting temperature of denaturation (Tm= 59°C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 kJ·mol–1. The specific activity (kmvalues) for D-xylose-glucose isomerase at 70°C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, recpectively.Key words: D-xylose-glucose isomerase, protein sequencing, protein stability, protein denaturation.

Industrially, the use of high temperatures (40-60 degrees C) in the L: -malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50 degrees C and highly stable even after 24 h of incubation at 40 degrees C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30 degrees C and was rapidly denatured at 50 degrees C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10 degrees C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C.