RESUMEN
We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca(2+) ion. This residue lies 15-20Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate-enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca(2+) and affects the packing of the Ca(2+) binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.
Asunto(s)
Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Mutación , Serina Endopeptidasas/genética , Álcalis/química , Secuencia de Aminoácidos , Bacillus/enzimología , Bacillus/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Calcio/química , Calcio/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Estabilidad de Enzimas/genética , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteolisis , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Tensoactivos/química , Temperatura , Tirosina/genética , Tirosina/metabolismoRESUMEN
A mutanase (alpha-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 degrees C to 50 degrees C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.
Asunto(s)
Bacillus/enzimología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Microbiología del Suelo , Secuencia de Aminoácidos , Bacillus/clasificación , Secuencia de Bases , Clonación Molecular , Estabilidad de Enzimas , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Datos de Secuencia Molecular , Peso Molecular , Mutación , Polisacáridos/metabolismo , Proteínas Recombinantes/química , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
Three mutanase (alpha-1,3-glucanase)-producing microorganisms isolated from soil samples were identified as a relatives of Paenibacillus. A mutanase was purified to homogeneity from cultures of each, and the molecular masses of the purified enzymes were approximately 132, 141, and 141kDa, respectively. The corresponding three genes for mutanases were cloned by PCR using primers designed from each N-terminal amino acid sequence. Another mutanase-like gene from one strain was also cloned by PCR using primers designed from conserved amino acid sequences among known mutanases. Consequently, four mutanase-like genes were sequenced. The genes contained long open reading frames of 3411 to 3915bp encoding 1136 to 1304 amino acids. The deduced amino acid sequences of the mutanases showed relatively high similarity to those of a mutanase (E16590) from Bacillus sp. RM1 with 46.9% to 73.2% identity and an alpha-1,3-glucanase (AB248056) from Bacillus circulans KA-304 with 46.7% to 70.4% identity. Phylogenetic analysis based on the amino acid sequences of the enzymes showed bacterial mutanases form a new family between fungal mutanases (GH family 71) and Streptomycetes mycodextranases (GH family 87).
Asunto(s)
Bacterias/enzimología , Glicósido Hidrolasas/clasificación , Glicósido Hidrolasas/genética , Secuencia de Aminoácidos , Bacterias/clasificación , Glicósido Hidrolasas/química , Datos de Secuencia Molecular , Mutación , Filogenia , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Subtilisin-like serine proteases from bacilli have been used in various industrial fields worldwide, particularly in the production of laundry and automatic dishwashing detergents. They belong to family A of the subtilase superfamily, which is composed of three clans, namely, true subtilisins, high-alkaline proteases, and intracellular proteases. We succeeded in the large-scale production of a high-alkaline protease (M-protease) from alkaliphilic Bacillus clausii KSM-K16, and the enzyme has been introduced into compact heavy-duty laundry detergents. We have also succeeded in the industrial-scale production of a new alkaline protease, KP-43, which was originally resistant to chemical oxidants and to surfactants, produced by alkaliphilic Bacillus sp. strain KSM-KP43 and have incorporated it into laundry detergents. KP-43 and related proteases form a new clan, oxidatively stable proteases, in subtilase family A. In this review, we describe the enzymatic properties, gene sequences, and crystal structures of M-protease, KP-43, and related enzymes.
Asunto(s)
Detergentes/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Bacillus/enzimología , Bacillus/genética , Cristalografía por Rayos X , Detergentes/historia , Genes Bacterianos , Historia del Siglo XX , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Endopeptidasas/historiaRESUMEN
A high-molecular-mass subtilisin was found in culture broth of the alkaliphilic Bacillus sp. strain KSM-KP43. The gene encoding the enzyme (FT protease) was determined using a mixed primer designed from the N-terminal amino acid (aa) sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of a 2427-bp open reading frame (ORF) that encoded a putative prepro-peptide (152 aa) and a mature enzyme (656 aa; 68,506 Da). The deduced aa of the mature enzyme revealed a moderate homology to a subtilisin-type proteinase from Bacillus halodurans and a minor extracellular protease, Vpr, from Bacillus subtilis with 64% and 57% identity, respectively. The molecular mass of the purified recombinant FT protease was approximately 72 kDa as judged by both SDS-polyacrylamide gel electrophoresis (PAGE) and gel filtration. FT protease showed maximal activity toward glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide at pH 10.5 and at 45 degrees C. The enzyme was rapidly inactivated by incubation over 45 degrees C for 15 min at both pH 7 and 10. Calcium ions were slightly protective for thermoinactivation of the enzyme.
Asunto(s)
Bacillus/química , Subtilisina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Especificidad por Sustrato , Subtilisina/química , Subtilisina/metabolismo , TemperaturaRESUMEN
The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337-340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.
Asunto(s)
Bacillus/enzimología , Bacillus/genética , Genes Bacterianos , Serina Endopeptidasas/metabolismo , Subtilisinas/genética , Secuencia de Aminoácidos , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Serina Endopeptidasas/genética , Subtilisinas/química , Subtilisinas/metabolismoRESUMEN
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.
Asunto(s)
Glicina/análogos & derivados , Oxígeno/química , Serina Endopeptidasas/química , Animales , Bacillus/enzimología , Sitios de Unión , Catálisis , Bovinos , Cristalografía por Rayos X , Electrones , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/química , Genes Dominantes , Glicina/química , Metionina/química , Modelos Moleculares , Oxígeno/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Serina/química , Especificidad por Sustrato , Subtilisinas/química , Factores de TiempoRESUMEN
The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.
Asunto(s)
Bacillus/enzimología , Proteínas Bacterianas , Subtilisina/química , Subtilisina/genética , Ácidos Alcanesulfónicos/farmacología , Secuencia de Aminoácidos , Bacillus/genética , Bacillus/aislamiento & purificación , Bacillus/metabolismo , Dominio Catalítico/genética , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Estabilidad de Enzimas/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Subtilisina/aislamiento & purificación , Subtilisina/metabolismo , Tensoactivos/farmacología , TemperaturaRESUMEN
Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394-2,424 bp encoded prosubtilisins of 798-808 amino acids (aa) consisting of the prepropeptides of 151-158 aa and the mature enzymes of 640-656 aa. The deduced aa sequences of the mature enzymes exhibited 60-95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5-11.0, optimal temperatures of 40-45 degrees C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.
Asunto(s)
Bacillus/enzimología , Bacillus/genética , Serina Endopeptidasas/genética , Subtilisinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Calor , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Peso Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Subtilisinas/química , Subtilisinas/metabolismoRESUMEN
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.