The xylose isomerase gene from Thermoanaerobacterium saccharolyticum strain B6A-RI was cloned by complementation using Escherichia coli xyl-5 mutant strain HB101. One positive clone was detected and the recombinant plasmid, pZXI6, was isolated. The clone contained the vector pUC18 and an insert fragment of 4.5 kb. The cloned xylose isomerase gene (xylA) was expressed constitutively in E. coli. The gene contained one open reading frame (ORF) of 1317 bp, which corresponds to 439 amino acid residues. The molecular mass of the gene product was calculated to be 50474Da from the deduced amino acid sequence. A putative promoter region (Pribnow box), TATAATATATAAT, which repeated twice at the -10 region in E. coli, was found 25 bp upstream of the ribosomal binding site. The deduced amino acid sequence of T. saccharolyticum strain B6A-RI xylose isomerase exhibited very high homology to those from Thermoanaerobacterium thermosulfrcrigenes 4B (formerly Clostridium thermosulfuvogenes 4B) and Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E). Codon usage in xynA, xynB and xylA showed a clear propensity for AT-containing isocodons. The native molecular mass of the purified recombinant thermostable xylose isomerase was 200 kDa, and the enzyme was a tetramer comprised of identical subunits. The apparent temperature and pH optima for activity of the cloned xylose isomerase were 80 "C and 7.0 to 7.5, respectively.

An elastinolytic serine proteinase produced byAspergiUusflavus 28 that was isolated from a patient who died of aspergillosis has been purified and characterized. The enzyme was inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. The metal-chelating agents EDTA and EGTA [ethylene glycol-bis(1-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not severely inhibit the enzyme. A cDNA and a 2.95-kb segment of genomic DNA containing the proteinase gene were sequenced. The open reading frame that would code for a protein containing 403 amino acids was interrupted by three introns. The mature protein lacks 121 N-terminal amino acids including a putative 21-amino-acid signal peptide. The purified mature protein showed a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas that calculated from the deduced protein sequence was 30 kDa. This elastinolytic serine proteinase ofA. flavus has 83 and 82% sequence homology to the similar proteinases from A. fumigatus and A. oryzae. The catalytic properties and the sequence homology around the putative catalytic amino acids suggest that this enzyme belongs to the serine proteinases of the subtilisin family.

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The serine proteinase gene (sep) in Aspergillus flavus was disrupted by homologous recombination with a hygromycin resistance gene as the marker. The gene-disrupted mutant GR-2 contained a single-copy insertion of the marker gene and did not express the sep gene. Serine proteinase activity, 36-kDa protein labeled by 3 H-diisopropylfluorophosphate, and immunologically detectable proteinase were not detected in the culture fluid of GR-2. Despite the absence of the serine proteinase, the total elastinolytic activity levels in the mutant and the wild-type A. flavus were comparable. Immunoblots revealed that the mutant secreted greater amounts of an elastinolytic metalloproteinase gene (mep20) product than did the wild type. Furthermore, mep20 mRNA levels, measured by RNase protection assay, in the mutant were higher than those in the wild type. Inhibition of the serine proteinase by Streptomyces subtilisin inhibitor (SSI) in the culture medium of wild-type A. flavus also resulted in an elevation of mep20 gene products. Although no serine proteinase activity could be detected, the level of elastinolytic activity of the SSI-treated culture was comparable to that of the control. Immunoblots revealed that the addition of SSI caused an elevation in the levels of metalloproteinase and its mRNA. These results suggest that the expression of the genes encoding serine and metalloproteinases are controlled by a common regulatory system and the fungus has a mechanism to sense the status of extracellular proteolytic activities.Extracellular hydrolases are produced by many microbes to degrade and obtain nutrients from polymers found in their environment. In addition, microbial proteinases have been implicated in the colonization and virulence of a number of bacteria and fungi (9,12,21,30,32). In the invasive lung infections of immunocompromised hosts, aspergilli must penetrate proteinaceous barriers such as elastin, collagen, and laminin (45), and thus proteinases are thought to be virulence factors (5,20,27,35,37,44). Aspergillus fumigatus and A. flavus produce serine proteinase and metalloproteinases that are capable of hydrolyzing such polymers (19,25,28,(33)(34)(35)(36)(37). A serine proteinase is produced as a major extracellular protein when these fungi are grown in a medium containing either elastin or collagen as the sole nitrogen source (19,20,35,36). The serine proteinase genes (sep) in both fungi show about 80% amino acid sequence identity (15, 33). In addition, A. fumigatus produces a 42-kDa metalloproteinase and A. flavus produces a 23-kDa metalloproteinase (25,28,34,36,42). In A. flavus, the levels of the 23-kDa metalloproteinase and serine proteinase are almost comparable (33,34,36), while in A. fumigatus, the 42-kDa metalloproteinase is produced in amounts less than those of the serine proteinase (25, 28). The gene for the 23-kDa metalloproteinase in A. flavus would encode a protein with a theoretical molecular mass of 20 kDa, and, therefore, this gene was designated mep20 and the protein was designated MEP20 (34)...

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an Mr of 142,000 + 2,000 and had the same catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.