M.L. Shinohara; M.Ihara; M.Abo; M.Hashida; S.Takagi; T.C.Beck.
"A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis".
Appl Microbiol Biotechnology, 57, 653-659 (2001)
Abstract
A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular weight of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the E. coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E.coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0 - 6.5 and 65 °C. The enzyme was stable after 30 min at 80 °C, and retained 50 % of activity at 80 °C after 16 hours. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.
F. Xu; E.J. Golightly; C.C. Fuglsang; P. Schneider P, K.R. Duke. L. Lam; S. Christensen; K.M. Brown; C.T. Jorgensen; S.H. Bro wn.
"A novel carbohydrate : acceptor oxidoreductase from Microdochium nivale".
Eur. J. Biochem., 268, 1136-1142 (2001)
Abstract
A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (less than or equal to 25% identity or 65% similarity) to other known flavin-containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino-acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono-, oligo-, or polymeric saccharides, and transfer their electrons to O-2 or other accepters. When D-glucose served as electron-donating substrate, an activity of 2 s(-1) was observed at pH 5.5 and 23 degreesC. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications.
F. Xu; E.J. Golightly; P. Schneider; R.M. Berka; K.M. Brown; J.A. Johnstone; D.H. Baker; C.C. Fuglsang; S.H. Brown; A.V. Klotz.
"Expression and characterization of a recombinant Fusarium spp. galactose oxidase."
Appl. Biochem. Biotechnol. 88, 23-32 (2000 )
Abstract
The Fusarium spp. (Dactylium dendroides) galactose oxidase was expressed in Aspergillus oryzae and Fusarium venenatum hosts. Under the control of an A. niger á-amylase or a Fusarium trypsin promoter, high level galactose oxidase expression was achieved. The recombinant oxidase expressed in the A. oryzae host was purified and characterized. The purified enzyme had a molecular weight of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 0.4 mol copper atom per mole protein. The stoichiometry increased to 1.2 after Cu saturation. Based on peroxidase-coupled assay, the enzyme preparation showed an activity of 440 turnovers per second toward D-galactose (0.1 M) at pH7 and 20°C. The enzyme had an optimal temperature of 60°C at pH6.0 and an activation free Gibbs energy of 33 kJ/mol. A series of D-galactose derivatives was tested as the reducing substrate for the oxidase. The difference in activity was interpreted by the stereospecificity of the oxidase toward the substituents in the pyranose substrate, particularly on the C5 and cyclic hemiacetal O sites. The recombinant oxidase could act on some galactose-containing polysaccharides, such as guar gum, but was not able to oxidize several common redox compounds that lacked a primary alcohol functional group.
R.M. Berka; P. Schneider; E.J. Golightly; S.H. Brown; M. Madden, K.M. Brown, T. Halkier, K. Mondorf; F. Xu.
"Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme produced in Aspergillus oryzae."
Appl. Environ. Microbiol. 63, 3151-3157 (1997)
Abstract
A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion- exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase.