RESUMO
We have previously indicated that three single mutations (Leu144-->Ser, Asp150-->Glu, and Ile168-->Ala) in the site-directed mutagenesis of thermolysin increase the activity and two single (Ser53-->Asp and Leu155-->Ala) and one triple (Gly8-->Cys/Asn60-->Cys/Ser65-->Pro) mutations increase the stability. In the present study, aiming to generate highly active and stable thermolysin variants, we combined these mutations and analyzed the effect of combinations on the activity and stability of thermolysin. The combination of the mutations of Leu144-->Ser and Asp150-->Glu yielded the most significant increase in the hydrolytic activities for N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide (FAGLA) and N-carbobenzoxy-L-Asp-L-Phe methyl ester (ZDFM), while that of Leu144-->Ser and Ile168-->Ala abolished the activity. The combination of Ser53-->Asp and Leu155-->Ala yielded the greatest increase in the thermal stability, while that of Ser53-->Asp and Gly8-->Cys/Asn60-->Cys/Ser65-->Pro increased the stability as high as the individual mutations do. The combination of three mutations of Leu144-->Ser, Asp150-->Glu, and Ser53-->Asp yielded a variant L144S/D150E/S53D with improved activity and stability. Its k(cat)/K(m) values in the hydrolysis of FAGLA and ZDFM were 8.6 and 10.2 times higher than those of wild-type thermolysin (WT), respectively, and its rate constant for thermal inactivation at 80 degrees C was 60% of that of WT.
Assuntos
Mutagênese/genética , Mutação/genética , Termolisina/genética , Termolisina/metabolismo , Acrilatos/metabolismo , Caseínas/metabolismo , Dipeptídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Escherichia coli , Hidrólise/efeitos dos fármacos , Cinética , Mutagênese/efeitos dos fármacos , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Cloreto de Sódio/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Temperatura , Termolisina/química , Termolisina/isolamento & purificação , Transformação Genética/efeitos dos fármacosRESUMO
The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.
Assuntos
Peptídeos/genética , Termolisina/química , Catálise , Domínio Catalítico , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Termolisina/genética , Termolisina/metabolismoRESUMO
Thermolysin [EC 3.4.24.27] is a thermostable neutral zinc metalloproteinase originally identified in the culture broth of Bacillus thermoproteolyticus Rokko. Since the discovery in 1962, the enzyme has been extensively studied regarding its structure and catalytic mechanism. Today, thermolysin is a representative of zinc metalloproteinase and an attractive target in protein engineering to understand the catalytic mechanism, thermostability, and halophilicity. Thermolysin is used in industry, especially for the enzymatic synthesis of N-carbobenzoxy L-Asp-L-Phe methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame. Generation of genetically engineered thermolysin with higher activity in the synthesis of ZDFM has been highly desired. In accordance with the expansion of studies on thermolysin, various strategies for its expression and purification have been devised and successfully used. In this review, we aim to outline recombinant thermolysins associated with their engineering, expression, purification, and production.
Assuntos
Bacillus/fisiologia , Escherichia coli/fisiologia , Biossíntese de Proteínas/genética , Engenharia de Proteínas/métodos , Termolisina/biossíntese , Termolisina/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Termolisina/genética , Termolisina/isolamento & purificaçãoRESUMO
The aim of this study was to improve the performance of affinity gels containing glycyl-D-phenylalanine (Gly-D-Phe) as a ligand to thermolysin. Gly-D-Phe was immobilized to the resin through spacers of varying chain lengths. The resulting affinity gels had spacer chain lengths of 2 carbon atoms and 11 and 13 carbon-and-oxygen atoms (designated T2, T11, and T13), and were characterized for their binding abilities to thermolysin. Measurement of adsorption isotherms showed that the association constants to thermolysin were in the order T13 > T11 > T2. In affinity column chromatography, in which 5 mg thermolysin was applied onto 1-ml volumes of the gels, the adsorption ratios of thermolysin were also in the order T13 > T11 > T2. These results indicate that the performance of affinity gels is improved by increasing the spacer chain length to 13 carbon-and-oxygen atoms.
Assuntos
Cromatografia de Afinidade/métodos , Géis/química , Termolisina/isolamento & purificação , Cromatografia de Afinidade/normas , Reagentes de Ligações Cruzadas/química , Dipeptídeos , Géis/normas , Ligantes , Ligação Proteica , Resinas Sintéticas , TemperaturaRESUMO
Thermolysin, a representative zinc metalloproteinase from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of thermolysin containing an NH(2)-terminal pelB leader sequence and the pre-prosequence of thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes thermolysin. Unlike the conventional expression method, not only the wild-type thermolysin but also mutant thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in thermolysin.
Assuntos
Escherichia coli/genética , Termolisina/biossíntese , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Bacillus/enzimologia , Bacillus/genética , Caseínas/metabolismo , Fermentação , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Termolisina/química , Termolisina/genética , Termolisina/isolamento & purificaçãoRESUMO
Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.
Assuntos
Asparagina/genética , Mutagênese Sítio-Dirigida , Engenharia de Proteínas/métodos , Termolisina/genética , Substituição de Aminoácidos/genética , Asparagina/metabolismo , Sítios de Ligação/genética , Caseínas/metabolismo , Catálise , Eletroforese em Gel de Poliacrilamida , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Histamina/genética , Histamina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Termodinâmica , Termolisina/química , Termolisina/metabolismo , Zinco/metabolismoRESUMO
Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.
Assuntos
Bacillus/genética , Proteínas de Bactérias/biossíntese , Escherichia coli , Expressão Gênica , Proteínas Recombinantes/biossíntese , Termolisina/biossíntese , Bacillus/enzimologia , Bacillus/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Catálise , Cromatografia Líquida/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Termolisina/genética , Termolisina/isolamento & purificaçãoRESUMO
In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.