Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties

Two extracellular alkaline proteases produced by an alkalophilic Bacillus isolate were purified and characterized using acetone precipitation, DEAE- and CM-Sepharose CL-6B ion exchange and Sephacryl S-200 gel filtration chromatographic techniques. Analysis of the purified proteases by SDS–PAGE revealed that both proteases, AP-1 and AP-2 were homogenous with molecular weight estimates of 28 and 29 kDa, respectively. The optimum activity of AP-1 and AP-2 were at temperatures of 50 and 55°C and pHs of 11 and 12, respectively. The enzymes were also stable in the pH range of 6.0–12.0 for a period of 4 h with and without Ca²⁺ (5 mM) and temperatures of up to 50°C. The half-lives of the enzymes recorded at 50°C were 50 and 40 min for proteases AP-1 and AP-2, respectively. The inhibition profile of the enzymes by phenylmethanesulphonyl fluoride, confirmed these enzymes to be alkaline serine proteases. The purified proteases hydrolysed native protein substrates such as casein, elastin, keratin, albumin and the synthetic chromogenic peptide substrates Glu-Gly-Ala-Phe-pNA and Glu-Ala-Ala-Ala-pNA. The K_m values for the purified proteases were calculated as 1.05 mM and 1.29 mM, respectively, for Glu-Gly-Ala-Phe-pNA, and 3.81 mM and 4.79 mM, respectively, for Glu-Ala-Ala-Ala-pNA as substrates. The kinetic data also indicated that small aliphatic and aromatic amino acids were the preferred residues at the P₁ position.     

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.     

Subtilases: the superfamily of subtilisin-like serine proteases.

Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719-731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca(2+)-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling.     

A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences

A highly alkaline endo-1,4-beta-glucanase (Egl) was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-N252. The optimal pH for activity was as high as 10, and the optimal temperature was 55 degrees C. The molecular mass and isoelectric point were around 50 kDa and pH 4.2, respectively. The enzyme hydrolyzed carboxymethyl cellulose in a random fashion. Unlike previously reported Egls, the enzyme was highly active on p-nitrophenyl cello-oligosaccharides and acid-swollen cellulose, and its activity was stimulated by cellobiose at high concentrations. The entire gene for the enzyme contained a 1,476-bp single open reading frame encoding 492 amino acids, including a 29-amino-acid signal peptide. The mature enzyme (463 amino acids: 51,174 Da) exhibited moderate homology to other family 5 alkaline Egls. In the C-terminal region, a carbohydrate-binding module that belongs to family XII was repeated. Furthermore, four and six repeats of Pro-Pro-Ser/Thr-Glu/Asp-Pro-(Glu) were found immediately before the first and second carbohydrate-binding modules, respectively.     

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.     

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal beta-barrel domain

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering. The native form was refined to a crystallographic R factor of 0.134 (Rfree of 0.169) at 1.30-A resolution. KP-43 consists of two domains, a subtilisin-like alpha/beta domain and a C-terminal jelly roll beta-barrel domain. The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal beta-barrel domain both differ in each case. Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role. KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases. To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (Rfree of 0.212) at 1.73-A resolution. The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN'. Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70-80% activity against proteinaceous substrates. These results, as well as the beta-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.     

Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis

A serpin homologue (Tk-serpin) from the hyperthermophilic archaeon Thermococcus kodakaraensis was overproduced in E. coli, purified, and characterized. Tk-serpin irreversibly inhibits Tk-subtilisin (TKS) from the same organism with the second-order association rate constants (k(ass)) of 5.2×103 M?1 s?1 at 40°C and 3.1×10? M?1 s?1 at 80°C, indicating that Tk-serpin inhibits TKS more strongly at 80°C than at 40°C. It also irreversibly inhibits chymotrypsin, subtilisin Carlsberg, and proteinase K at 40°C with the k(ass) values comparable to that for TKS at 80°C. Casein zymography showed that Tk-serpin inhibits these proteases by forming a SDS-resistant complex, which is typical to inhibitory serpins. The ratio of moles of Tk-serpin needed to inhibit 1 mol of protease (stoichiometry of inhibition, SI) varies from 40 to 80 at 20°C, but decreases to the minimum values of 3-7 as the temperature increases. The inhibitory activities of Tk-serpin for these proteases increase as the stabilities of these proteases decrease, suggesting that a flexibility of the active-site of protease is one of the determinants for susceptibility of protease to inhibition by Tk-serpin. This report showed for the first time that Tk-serpin inhibits both chymotrypsin- and subtilisin-like serine proteases and its inhibitory activity increases as the temperature increases up to 100°C.     

Nucleotide sequences of serine tRNAs from Bacillus subtilis.

Three B. subitilis serine tRNAs were sequenced including modified nucleosides. All the serine tRNAs contained 1-methyl-adenosine in the D-loop. As other characteristic modified nucleosides, 5-methoxyuridine was found in the first letter of the anticodon in the tRNA(UGA).     

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.     

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.     

Alkaline cellulases from alkaliphilic Bacillus: Enzymatic properties, genetics, and application to detergents

We have isolated a number of alkaliphilic Bacillus that produce alkaline exoenzymes and found a possible use for alkaline cellulase (carboxymethylcellulase) as an additive for improving the cleaning effect of detergents. Enzymatic properties of some candidate cellulases fulfilled the essential requirements for enzymes to be used practically in laundry detergents. Here I describe the properties and possible catalytic mechanism of the hydrolytic reaction and the gene for the industrial alkaline cellulase produced by one of the isolates, Bacillus sp. KSM-635. Received: October 4, 1996 / Accepted: December 2, 1996     

Multiple proteases from Streptomyces moderatus. II. Physicochemical and enzymatic properties of the extracellular proteases

The physicochemical and enzymatic properties of five different extracellular proteases of Streptomyces moderatus were studied. The first protease was found to be a metal chelator sensitive protease with a Mr of 21,000 +/- 1000 a and a pI of 4.6. The second enzyme was an anionic trypsin-like protease (Mr 19,000 +/- 1000; pI 3.8) with a Km value of 4.76 X 10(-4) M on N-benzoyl-L-arginine-p-nitroanilide. A Km value of 1.52 X 10(-4) M was obtained when N-benzoyl-L-arginine ethyl ester was used as the substrate. The other three enzymes were found to be serine alkaline proteases with Mr's of 22,000, 29,000, and 23,000 +/- 1000 and with respective pI's of 7.8, 8.4, and 9.2. All the proteases showed optimum activity in the alkaline pH range. One of the three proteases was found to possess chymotrypsin and elastase-like properties. All five proteases were found to be unstable at temperatures above 60 degrees C. Except the trypsin-like protease, which was stable only in acidic pH, all other enzymes were found to be stable over a wide range of pH.     

The phylogeny of trypsin-related serine proteases and their zymogens. New methods for the investigation of distant evolutionary relationships.

The sequence of all presently known trypsin-related serine proteases and their zymogens of animal and bacterial origin were optimally aligned on the basis of three different scoring schemes for amino acid comparisons. Sequence homology was found to extend into the activation peptides. The gaps resulting from the alignment of the sequences of the active enzymes formed the basis for a new procedure based on position and number of gaps, which allowed the correct topology of the evolutionary relationship of thrombin and the pancreatic enzymes trypsin, chymotrypsin and elastase to be determined. The procedure was applied in an analogous manner to changes in disulfide bridges as well as to a selected set of amino acid positions.Evolutionary distances between proteins were estimated by minimum, base differences as well as according to the stochastic model of evolution. These distances were used successfully to find the best topology of evolutionary relationships. The fact that the branch lengths in evolutionary trees were less affected by the number of sequences considered when evolutionary distances between contemporary sequences were measured in minimum base differences than when measured according to the stochastic model of evolution, suggested in our specific case, that minimum base differences yielded estimates of evolutionary distance closer to reality than the stochastic model of evolution.All these techniques combined yielded the following picture for the evolution of the four protease families. Prothrombin and the zymogens of the pancreatic serine proteases had a common ancestor with tryptic specificity. After the initial divergence, the gene for trypsinogen duplicated. Evidence was found that the duplicated gene underwent drastic changes for a short period of time to become eventually the common ancestor of chymotrypsin and elastase. The phylogenetic tree elaborated for these enzyme families and the methods introduced to determine its topology, should readily allow determination of the attachment site of branches leading to newly sequenced serine proteases, provided their amino acid sequence can be aligned fairly unambiguously. In addition, the consequences of the alignment of the different serine proteases for the relationship of zymogen to enzyme are discussed.     

Structure determination of an exopolysaccharide from an alkaliphilic bacterium closely related to Bacillus spp.

An exopolysaccharide obtained from an alkaliphilic bacterium closely related to Bacillus spp. was found to contain D-galactopyranuronic acid (GalpA), 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose (QuipNAc4NAc), 2-acetamido-2-deoxy D-mannopyranuronic acid (ManpNAcA) and one uncommon unit of D-galactopyranuronic acid with the carboxyl group amide-linked to glycine [GalpA(Gly)]. The polysaccharide was studied by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy both on native polysaccharide and on monosaccharides and oligosaccharides obtained from methanolysis and from anhydrous HF solvolysis. The following linear structure of the repeating unit was established: -->3)-alpha-D-GalpA(Gly)-(1-->4)-beta-D-ManpNAcA-(1-->4)-alp ha-D-Galp A-(1-->3)-alpha-D-QuipNAc4NAc-(1-->. A preliminary phylogenetic assignment for the bacterium is also reported.     

Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp. 221.

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus subtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6-61.7%). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Nucleotide and amino-acid sequences of a new-type pectate lyase from an alkaliphilic strain of Bacillus.

A pectate lyase (pectate transeliminase; EC 4.2.2.2), designated Pel-15E, was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-P15. The purified enzyme had a molecular mass of approximately 33 kDa, as determined by SDS/PAGE, and a pI of approximately pH 9.2. Pel-15E exhibited optimum activity at pH 10.5 and 50-55 degrees C in glycine/NaOH buffer. Pel-15E had an absolute requirement for Ca2+ ions for manifestation of the enzymatic activity and trans-eliminated poly(galacturonic) acid, most likely by endo-type cleavage. A gene for the enzyme, which was cloned using the shotgun method and sequenced, contained a 960-bp ORF encoding 320 amino acids. The mature enzyme (286 amino acids, 32 085 Da) from the deduced amino-acid sequence showed quite low homology to known Pels from various microorganisms with 16.1-20.4% identity. Furthermore, we were not able to find any conserved regions in the sequence of Pel-15E when aligned with the sequences of other enzymes from the established Pel superfamily. However, Pel-15E had some regions that were homologous to PelA from Azospirillum irakense with 39.8% identity. Based on their amino-acid sequence homology, Pel-15E and PelA appear to belong to a new class of Pel family, although the enzymatic properties of both enzymes were quite different.     

zmsbt1 and zmsbt2, two new subtilisin-like serine proteases genes expressed in early maize kernel development

Planta - Two subtilisin-like proteases show highly specific and complementary expression patterns in developing grains. These genes label the complete surface of the filial–maternal...