The peptide sequences near the bound pyridoxal phosphate are conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli

The blocked amino-terminal residue of rat liver serine dehydratase was shown to be acetylalanine by analysis of an isolated amino-terminal peptide after digestion with acylamino acid-releasing enzyme. Digestion of the borohydride-reduced, carboxymethylated enzyme with lysyl endopeptidase yielded a single epsilon-N-pyridoxyllysine-containing peptide, whose sequence is Met-Asp-Ser-Ser-Gln-Pro-Ser-Gly-Ser-Phe-Lys(Pxy)-Ile-Arg-Gly- His-Leu-Cys(Cm)-Lys. This peptide comprises residues 30-49 of the cDNA-deduced amino acid sequence. The sequence of seven amino acids around the bound pyridoxal phosphate is highly conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli.     

Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.     

Thermostable aspartate aminotransferase from a thermophilic Bacillus species. Gene cloning, sequence determination, and preliminary x-ray characterization

The gene encoding aspartate aminotransferase of a thermophilic Bacillus species, YM-2, has been cloned and expressed efficiently in Escherichia coli. The primary structure of the enzyme was deduced from nucleotide sequences of the gene and confirmed mostly by amino acid sequences of tryptic peptides. The gene consists of 1,176 base pairs encoding a protein of 392 amino acid residues; the molecular mass of the enzyme subunit is estimated to be 42,661 daltons. The active site lysyl residue that binds the coenzyme, pyridoxal phosphate, was identified as Lys-239. Comparison of the amino acid sequence with those of aspartate aminotransferases from other organisms revealed very low overall similarities (13-14%) except for the sequence of the extremely thermostable enzyme from Sulfolobus solfataricus (34%). Several amino acid residues conserved in all the compared sequences include those that have been reported to participate in binding of the coenzyme in three-dimensional structures of the vertebrate and E. coli enzymes. However, the strictly conserved arginyl residue that is essential for binding of the distal carboxyl group of substrates is not found in the corresponding region of the sequences of the thermostable enzymes from the Bacillus species and S. solfataricus. The Bacillus aspartate aminotransferase has been purified from the E. coli clone cell extracts on a large scale and crystallized in the buffered ammonium sulfate solution by the hanging drop method. The crystals are monoclinic with unit cell dimensions a = 121.2 A, b = 110.5 A, c = 81.8 A, and beta = 97.6 degrees, belonging to space group C2, and contain two molecules in the asymmetric unit. The crystals of the enzyme-alpha-methylaspartate complex are isomorphous with those without the substrate analog.     

The primary structure of thermostable D-amino acid aminotransferase from a thermophilic Bacillus species and its correlation with L-amino acid aminotransferases

The gene for thermostable D-amino acid aminotransferase from a thermophile, Bacillus species YM-1 was cloned and expressed efficiently in Escherichia coli. The entire covalent structure of the enzyme was determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product. The polypeptide is composed of 282 amino acid residues with a calculated molecular weight of 32,226. Comparison of the primary structure with those of various proteins registered in a protein data bank revealed a significant sequence homology between D-amino acid aminotransferase and the L-branched chain amino acid aminotransferase of E. coli (Kuramitsu, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 993-999); the active site lysyl residue is located in an equivalent position in both enzyme sequences of similar size. Despite the difference in subunit composition and no immunochemical cross-reactivity, the sequences of the two enzymes show similar hydropathy profiles, and spectrophotometric properties of the enzyme-bound cofactor are also similar. The sequence homology suggests that the structural genes for D-amino acid and L-branched chain amino acid aminotransferases evolved from a common ancestral gene.     

Biosynthetic alanine racemase of Salmonella typhimurium: purification and characterization of the enzyme encoded by the alr gene

An alanine racemase, encoded by the alr (dal) gene and believed to be the biosynthetic source of D-alanine for cell wall formation, was purified to homogeneity from an overproducing strain of Salmonella typhimurium (dadB), and the enzymological properties of this enzyme were compared with those of the dadB alanine racemase that functions in the catabolism of L-alanine [Wasserman, S. A., Daub, E., Grisafi, P., Botstein, D., & Walsh, C. T. (1984) Biochemistry 23, 5182]. The alr-encoded enzyme has a monomeric structure with a molecular weight of about 40 000. One mole of pyridoxal 5'-phosphate is bound per mole of enzyme, which is essential for catalytic activity of the enzyme. After the internal Schiff base with pyridoxal 5'-phosphate was reduced with NaB3H4, followed by carboxamidomethylation and tryptic digestion of the enzyme, the amino acid sequence of the pyridoxal 5'-phosphate binding peptide was determined. The sequence of 10 amino acid residues around the lysine residue, to which pyridoxal 5'-phosphate is bound, was identical with that of the dadB racemase. No homology was found in the amino-terminal amino acid sequence between the two enzymes. The enzyme was inactivated with D- and L-beta-fluoroalanine, D- and L-beta-chloroalanine, and D-O-acetylserine in a mechanism-based fashion with a common partition ratio of about 150. The enzyme was labeled with an equimolar amount of [14C]-D-beta-chloroalanine. The inactivator-pyridoxal 5'-phosphate adduct was isolated and shown to be the same structure formed in the dadB racemase inactivation [Roise, D., Soda, K., Yagi, T., & Walsh, C. (1984) Biochemistry 23, 5195].     

Deduced amino-acid sequence of a calcium-free alpha-amylase from a strain of Bacillus: implications from molecular modeling of high oxidation stability and chelator resistance of the enzyme.

Alkaline alpha-amylase (AmyK38) from the alkaliphilic Bacillus sp. strain KSM-K38 is a unique enzyme in that it is highly chelator-resistant and oxidatively stable [Hagihara, H., Igarashi, K., Hayashi, Y., Endo, K., Ikawa-Kitayama, K., Ozaki, K., Kawai, S. & Ito, S. (2001) Appl. Environ. Microbiol. 67, 1744-1750]. This enzyme was found to contain no Ca and require Na (or monovalent cations) for manifestation of activity. The nucleotide sequence of the gene for the novel enzyme was determined, and it harbored an ORF of 1503 bp encoding the enzyme of 501 amino acids, including a 21-amino-acid signal peptide. The deduced amino-acid sequence of the mature enzyme (55 097 Da) showed moderate homology to those of alpha-amylases from Bacillus licheniformis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, with approximately 63% identity. A methionine residue, which is conserved and susceptible to chemical oxidation, was replaced with leucine in AmyK38. Moreover, many conserved residues that are crucial ligands for Ca were replaced with other amino acids, thereby leading to loss of the Ca coordination geometries. By building a molecular model, we showed the calcium-independent, oxidatively stable active-site topology and structural integrity of AmyK38.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH2-terminal amino acid sequence analysis of the G6-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102,598. Identity of the NH2-terminal amino acid sequences among each component of the multiform G6-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G6-amylase gene showed no homology with those of other bacterial alpha-amylases although the consensus amino acid sequences of the active center were well conserved.     

Purification and properties of the aromatic amino acid aminotransferase from gramicidin S-producing Bacillus brevis

The aromatic amino acid aminotransferase was purified to a homogenous state from a gramicidin S-producing strain of Bacillus brevis. The enzyme shows a molecular weight of about 71,000 on gel-filtration. The subunit molecular weight is about 35,000 as determined by sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is a dimer. The enzyme exhibits absorption maxima near 425 and 330 nm at neutral pH. One mole of pyridoxal phosphate is bound per subunit. The enzyme has amino donor specificity for aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophan, and utilizes 2-oxoglutarate as the amino acceptor. This enzyme activity was separated from both the aspartate aminotransferase activity and the branched chain amino acid aminotransferase activity by chromatography on DEAE-Sephadex.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G₆-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH₂-terminal amino acid sequence analysis of the G₆-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102598. Identity of the NH₂-terminal amino acid sequences among each component of the multiform G₆-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G₆-amylase gene showed no homology with those of other bacterial α-amylases although the consensus amino acid sequences of the active center were well conserved.     

Molecular identification of family 38 alpha-mannosidase of Bacillus sp. strain GL1, responsible for complete depolymerization of xanthan

When cells of Bacillus sp. strain GL1 were grown in a medium containing xanthan as a carbon source, alpha-mannosidase exhibiting activity toward p-nitrophenyl-alpha-D-mannopyranoside (pNP-alpha-D-Man) was produced intracellularly. The 350-kDa alpha-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzed pNP-alpha-D-Man (Km = 0.49 mM) and D-mannosyl-(alpha-1,3)-D-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway of Bacillus sp. strain GL1. The gene for alpha-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of alpha-mannosidases belonging to glycoside hydrolase family 38.     

Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene

A chitosanase produced constitutively by Bacillus sp. MET 1299 was purified by SP-Sephadex column chromatography. The molecular weight was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzyme activity was observed at a pH of 5.5 and temperature of 60 degrees C. The purified chitosanase showed high activity on 90% deacetylated colloidal chitosan and beta-glucan, but not on hydrolyzed colloidal chitin, CMC, or their derivatives. The N-terminal amino acid sequence of the enzyme was determined. The cloned full length gene, 1362 bp in size, encoded a single peptide of 453 amino acids and had a conserved amino acid sequence of glycosyl hydrolase family 8. A search of the cDNA sequence with NCBI BLAST showed homology with chitosanase of Bacillus sp. KTCC 0377BP and Bacillus sp. No. 7-M. The recombinant protein was expressed in Escherichia coli, purified using affinity chromatography and characterized.     

Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca²⁺. However, Hg²⁺ and Ni⁺ ions strongly inhibited the enzyme. The enzyme produced, GlcN_2–4, were the major products from a soluble chitosan.     

Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus

A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.     

Monoacylglycerol lipase from moderately thermophilic Bacillus sp. strain H-257: molecular cloning, sequencing, and expression in Escherichia coli of the gene

Monoacylglycerol lipase [MGLP, EC 3.1.1.23] is produced intracellularly by the moderately thermophilic Bacillus sp. strain H-257. The gene encoding MGLP was cloned, sequenced, and expressed in Escherichia coli. A genomic library of Bacillus sp. strain H-257, prepared in the plasmid vector pACYC184, was screened with a 0.2-kbp DNA fragment amplified by the polymerase chain reaction (PCR) with oligonucleotide primers designed based on the amino acid sequence of a purified MGLP. The plasmid pMGLP31, identified by hybridization with the amplified DNA fragment, contained a 5.3-kbp insert from Bacillus sp. strain H-257 DNA. Sequence analysis of the MGLP gene revealed an open reading frame encoding MGLP consisting of 250 amino acids, with a calculated molecular mass of 27.4 kDa. The deduced amino acid sequence of MGLP contained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conserved among lipases, esterases, and serine proteases. The MGLP is homologous to a putative esterase/lipase from Streptomyces coelicolor (41.8% homology). When pMGLP31 was introduced into E. coli DH1, the transformants produced MGLP intracellularly as an active form to an approximately 13.8-fold greater extent than Bacillus sp. strain H-257. The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic behavior, isoelectric point, and physicochemical and catalytic properties.     

Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2

A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C.     

A new high-alkaline and high-molecular-weight pectate lyase from a Bacillus isolate: enzymatic properties and cloning of the gene for the enzyme

A pectate lyase (Pel; pectate transeliminase: EC4.2.2.2.), designated Pel-15H, was found in an alkaline culture of Bacillus sp. strain KSM-P15 and purified to homogeneity by sequential column chromatographies. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 70,000 and the pI was around pH 4.6. Pel-15H randomly trans-eliminated polygalacturonate in the presence of Ca2+ ions, and the maximum activity was observed at pH 11.5 and at 55 degrees C in glycine-NaOH buffer. The gene for Pel-15H was cloned and sequenced, and the structural gene contained a 2,031-bp open reading frame that encoded 677 amino acids including a possible 28-amino-acid signal sequence. The mature enzyme (649 amino acids, molecular weight 69,550) showed very low similarity to Pels from Bacillus with 12.7-18.2% identity. Interestingly, part of the amino acid sequence of Pel-15H had fairly high similarity only to an N-terminal half of PelL and a C-terminal half of PeIX from Erwinia chrysanthemi 3937, and a C-terminal half of PeIX from E. chrysanthemi EC16 (approximately 35% identity for all).     

Primary structure and chemical modification of some amino acid residues of bifunctional alginate lyase from a marine bacterium Pseudoalteromonas sp. strain no. 272

The entire amino acid sequence of bifunctional alginate lyase from Pseudoalteromonas sp. strain No. 272 were determined by two approaches, Edman degradation of the peptides obtained from protease digestion of the enzyme protein and analysis of PCR products of the structural gene. The former resulted in incomplete amino acid sequence in the entire sequence, due to lacking of the proper peptides from the protease digestion. To compensate for this lack of sequences we applied the method of PCR of the structural gene that was initially elucidated from the primers designed from N- and C-terminal amino acid sequences of the enzyme. The results of the amino acid sequences from these two approaches showed good agreement. The enzyme consisted of 233 amino acid residues with a molecular mass of 25,549.5, including the sole W and cystine residue. The sequence homology search among the other alginate lyases from different origins indicated that they were very weakly homologous, with the exception of the sequence homology (80.3%) of Pseudoalteromonas elyakovii alginate lyase. The consensus sequence, YFKhG + Y-Q (Wong, T. Y., Preston, L. A., and Schiller, N. L. 2000. Annu. Rev. Microbiol. 54: 289–340) in the C-terminal regions was conserved. The kinetic analyses of chemical modification of some amino acid residues of the enzyme showed that W, K, and Y appeared to be important in the enzyme function.     

Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4-beta-glucanase from Bacillus sp. KSM-330.

The gene encoding an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The recombinant plasmid contained a 3.1 kb HindIII insert, 1.8 kb of which was sufficient for the expression of endoglucanase activity in E. coli HB101. Nucleotide sequencing of this region (1816 bp) revealed an open reading frame of 1389 bp. The protein deduced from this sequence was composed of 463 amino acids with an Mr of 51882. The deduced amino acid sequence from amino acids 56 through 75 coincided with the amino-terminal sequence of the endoglucanase, Endo-K, purified from culture of Bacillus sp. KSM-330. The deduced amino acid sequence of Endo-K had 30% homology with that of the celA enzyme from Clostridium thermocellum NCIB 10682 and 25% homology with that of the enzyme from Cellulomonas uda CB4. However, the Endo-K protein exhibited no homology with respect to either the nucleotide or the amino acid sequences of other endoglucanases from Bacillus that had been previously characterized. These results indicate that the gene for Endo-K in Bacillus sp. KSM-330 has evolved from an ancestral gene distinct from that of other Bacillus endoglucanases.