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.     

Xanthan Lyase of Bacillus sp. Strain GL1 Liberates Pyruvylated Mannose from Xanthan Side Chains

When the bacterium Bacillus sp. strain GL1 was grown in a medium containing xanthan as the carbon source, the viscosity of the medium decreased in association with growth, showing that the bacterium had xanthan-depolymerizing enzymes. One of the xanthan-depolymerizing enzymes (xanthan lyase) was present in the medium and was found to be induced by xanthan. The xanthan lyase purified from the culture fluid was a monomer with a molecular mass of 75 kDa, and was most active at pH 5.5 and 50°C. The enzyme was highly specific for xanthan and produced pyruvylated mannose. The result indicates that the enzyme cleaved the linkage between the terminal pyruvylated mannosyl and glucuronyl residues in the side chain of xanthan.     

Purification and Characterization of an Alginate Lyase from Marine Bacterium Vibrio sp. Mutant Strain 510-64

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.     

A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., the xalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. The xalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.     

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.     

A Pyruvated Mannose-Specific Xanthan Lyase Involved in Xanthan Degradation by Paenibacillus alginolyticus XL-1

The xanthan-degrading bacterium Paenibacillus alginolyticus XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55°C.     

Thermostable Chitosanase from Bacillus sp. Strain CK4: Cloning and Expression of the Gene and Characterization of the Enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80°C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.     

Cloning,Expression, Purification and Initial Analysis of a Novel Pectate Lyase Pcpel1 from Phytophthora capsici

Phytophthora capsici inflicts damage on numerous crop plants by secreting a series of pectinase including pectate lyase (PEL). Here, we report a pectate lyase gene (Pcpel1) from a genomic library of a highly virulent P. capsici strain SD33. Pcpel1 was identified as an open reading frame of 1233 bp encoding a protein of 410 amino acids with a predicted amino‐terminal signal sequence of 21 amino acids. The predicted protein of Pcpel1 has a calculated molecular mass of 43.8 kDa and a pI value of 6.8. Analysis of the amino acid sequence suggested that it was a member of the polysaccharide lyase family 1 that shows pectate lyase activity. Moreover, heterologous expression of Pcpel1 in Pichia pastoris produced proteins with molecular mass 66 kDa, very likely due to differential glycosylation by the yeast. By western blotting and northern blotting analysis, Pcpel1 was strongly expressed during interaction of P. capsici with the host plant, suggesting its involvement in the process of host infection. The role of Pcpel1 in cell wall disassembly and host/parasite interaction is discussed.     

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34 805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50°C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl. Received: June 10, 2000 / Accepted: October 3, 2000     

Molecular Characterization of a Fungicidal Endoglucanase from the Cyanobacterium Calothrix elenkinii

A gene responsible for fungicidal activity was identified in the cyanobacterial strain Calothrix elenkinii RPC1, which had shown promise as a biocontrol agent. Functional screening of the genomic library revealed fungicidal (against Pythium aphanidermatum) and endoglucanase activities in two clones. Sequencing revealed an open reading frame of 1,044 bp, encoding 348 amino acid residues with a predicted molecular weight of 38 kDa. Analysis of the deduced amino acid sequence of the putative gene (cael1) showed 99% similarity with the β-1,4-endoglucanase from Anabaena laxa RPAN8 and 97% with the glucanase belonging to the peptidase M20 family of Anabaena variabilis and Nostoc sp. PCC7120, respectively. The putative promoters, ribosomal binding sites and a signal peptide of 22 amino acid residues were identified, revealing the secretory nature of the protein. The phylogenetic tree indicated a close relationship of the gene with Bacillus sp. This study is the first to report on the characterization of an endoglucanase in Calothrix sp.     

Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp. strain GL1.

An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in Bacillus sp. strain GL1, which was closely related to Brevibacillus thermoruber, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765-3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular beta-D-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by beta-D-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by alpha-D-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel beta-D-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45 degrees C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.     

Purification and Characterization of Thermostable Pectate Lyase with Protopectinase Activity from Thermophilic Bacillus sp. TS 47

A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated. It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60°C on a medium containing polygalacturonate (PGA). The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies. The molecular mass of the enzyme was 50 kDa by SDS-PAGE. The isoelectric point of the enzyme was pH 5.3. The enzyme had a half-life of 13 and 1 h at 65 and 70°C, respectively, and showed optimal activity around at 70°C and pH 8.0. It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers. The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50% identity.     

Mutanase from a Paenibacillus isolate: Nucleotide sequence of the gene and properties of recombinant enzymes

A mutanase (α-1,3-glucanase)-producing microorganism was isolated from a soil sample and was identified as a relative of Paenibacillus sp. The mutanase was purified to homogeneity from culture, and its molecular mass was around 57 kDa. The gene for the mutanase was cloned by PCR using primers based on the N-terminal amino acid sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of 3651-bp open reading frame that encoded a predicted 1217-amino acid polypeptide including a 43-amino acid signal peptide. The mature enzyme showed similarity to mutanases RM1 of Bacillus sp. strain RM1 and KA-304 of Bacillus circulans with 65.6% and 62.7% identity, respectively. The predicted molecular mass of the mutanase was 123 kDa. Thus, the enzyme purified from the isolate appears to be truncated by proteolysis. The genes for the full-length and truncated mutanases were expressed in Bacillus subtilis cells, and the corresponding recombinant enzymes were purified to homogeneity. The molecular masses of the two enzymes were 116 and 57 kDa, respectively. The specific activity was 10-fold higher for the full-length enzyme than for the truncated enzyme. The optimal pH and temperature for both recombinant enzymes was pH 6.4 in citrate buffer and 45 °C to 50 °C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.     

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.     

Thermostable Flavin Reductase That Couples with Dibenzothiophene Monooxygenase,from Thermophilic Bacillus sp. DSM411: Purification,Characterization, and Gene Cloning

Flavin reductase is essential for the oxygenases involved in microbial dibenzothiophene (DBT) desulfurization. An enzyme of the thermophilic strain, Bacillus sp. DSM411, was selected to couple with DBT monooxygenase (DszC) from Rhodococcus erythropolis D-1. The flavin reductase was purified to homogeneity from Bacillus sp. DSM411, and the native enzyme was a monomer of M_r 16 kDa. Although the best substrates were flavin mononucleotide and NADH, the enzyme also used other flavin compounds and acted slightly on nitroaromatic compounds and NADPH. The purified enzyme coupled with DszC and had a ferric reductase activity. Among the flavin reductases so far characterized, the present enzyme is the most thermophilic and thermostable. The gene coded for a protein of 155 amino acids with a calculated mass of 17,325 Da. The enzyme was overproduced in Escherichia coli, and the specific activity in the crude extracts was about 440-fold higher than that of the wild-type strain, Bacillus sp. DSM411.     

Crystal structure of Bacillus sp. GL1 xanthan lyase,which acts on the side chains of xanthan

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 A resolution with final R-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal alpha-helical and C-terminal beta-sheet domains, which constitute incomplete alpha(5)/alpha(5)-barrel and anti-parallel beta-sheet structures, respectively. A deep cleft is located in the N-terminal alpha-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg(313) and Tyr(315) residues in the loop from the N-terminal domain and the Arg(612) residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.     

Characterization of a family 3 polysaccharide lyase with broad temperature adaptability, thermo-alkali stability, and ethanol tolerance

The 774-bp pectate lyase gene plyAI4 from Bacillus sp. I4 was cloned and expressed in E. coli. The gene encodes a 257-residue polypeptide (PlyAI4, 28.3 kDa) with the highest identities of 97.3% with a putative pectate lyase from Bacillus subtilis BSn5 (ADV94306) and 60.3% with an identified pectate lyase of the polysaccharide lyase family (PL) 3 from Paenibacillus amylolyticus 27C64 (ADB78774). The purified recombinant PlyAI4 (rPlyAI4) exhibited apparently optimal activity at pH 10.5 ?? 11.0 and 50°C. Compared with the majority of reported alkaline pectate lyases, rPlyAI4 exhibited more residual enzyme activity at 20°C (??45%) or at 70°C (??50%) and better thermostability at 70°C (??60 min half-life at 70°C). In the presence of 20% (v/v) ethanol, pectate lyase activity was enhanced by 0.2 fold. After incubation in 40% (v/v) ethanol at 37°C and pH 8.5 for 1 h, the purified rPelAI4 retained more than 75% of the initial activity. Sequence analysis proposed a new signature block, A-D-G-[V/I]-H, for PL 3 pectate lyases. These properties may prove to be important with regards to PlyAI4 for basic research and industrial application.     

Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1

Cells of Bacillus sp. GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa. A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies. The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies. The 260 kDa protein was purified from the cultured fluid and characterized. The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase. The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical. Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase. Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis. Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide. Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.     

Structure and Function of 2,3-Dimethylmalate Lyase, a PEP Mutase/Isocitrate Lyase Superfamily Member

The Aspergillus niger genome contains four genes that encode proteins exhibiting greater than 30% amino acid sequence identity to the confirmed oxaloacetate acetyl hydrolase (OAH), an enzyme that belongs to the phosphoenolpyruvate mutase/isocitrate lyase superfamily. Previous studies have shown that a mutant A. niger strain lacking the OAH gene does not produce oxalate. To identify the function of the protein sharing the highest amino acid sequence identity with the OAH (An07g08390, Swiss-Prot entry Q2L887, 57% identity), we produced the protein in Escherichia coli and purified it for structural and functional studies. A focused substrate screen was used to determine the catalytic function of An07g08390 as (2R,3S)-dimethylmalate lyase (DMML): k_cat = 19.2 s^− 1 and K_m = 220 μM. DMML also possesses significant OAH activity (k_cat = 0.5 s^− 1 and K_m = 220 μM). DNA array analysis showed that unlike the A. niger oah gene, the DMML encoding gene is subject to catabolite repression. DMML is a key enzyme in bacterial nicotinate catabolism, catalyzing the last of nine enzymatic steps. This pathway does not have a known fungal counterpart. BLAST analysis of the A. niger genome for the presence of a similar pathway revealed the presence of homologs to only some of the pathway enzymes. This and the finding that A. niger does not thrive on nicotinamide as a sole carbon source suggest that the fungal DMML functions in a presently unknown metabolic pathway. The crystal structure of A. niger DMML (in complex with Mg²⁺ and in complex with Mg²⁺ and a substrate analog: the gem-diol of 3,3-difluoro-oxaloacetate) was determined for the purpose of identifying structural determinants of substrate recognition and catalysis. Structure-guided site-directed mutants were prepared and evaluated to test the contributions made by key active-site residues. In this article, we report the results in the broader context of the lyase branch of the phosphoenolpyruvate mutase/isocitrate lyase superfamily to provide insight into the evolution of functional diversity.     

Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis

A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.