Engineering, expression, purification, and production of recombinant thermolysin.

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.

A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli.

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.

Cloning, sequencing, expression, and characterization of protealysin, a novel neutral proteinase from Serratia proteamaculans representing a new group of thermolysin-like proteases with short N-terminal region of precursor.

The gene of Serratia proteamaculans proteinase, protealysin, was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a precursor of 341 amino acids (AAs) with a significant homology to thermolysin-like proteinases (TLPs). The molecular weight of the purified mature active recombinant protein was 32 kDa, the N-terminal amino acid sequence was AKTSTGGEVI. The optimum pH for azocasein hydrolysis by protealysin was seven and it was completely inhibited by o-phenanthroline. The enzyme hydrolyzed 3-(2-furyl)acryloyl-glycyl-L-leucine amide, the standard substrate for TLPs, with k(cat)/K(m) ratio of (2.52 +/- 0.02) x 10(2) M(-1) s(-1). Protealysin maturation removes 50 AA from the N-terminus of the precursor. The removed region had no similarity with the preprosequence of thermolysin (232 AA) but was homologous to some other TLPs. These proteins shared a conserved 7-AA motif near the initial methionine. Such motif was also found in some nonproteolytic putative proteins; two of them were eukaryotic.

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization.

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.

Engineering an enzyme to resist boiling.

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

Intramolecular processing of prothermolysin.

Thermolysin, an extracellular zinc endopeptidase from Bacillus thermoproteolyticus, is synthesized as a pre-proenzyme and the prosequence has been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (O'Donohue, M. J., and Beaumont, A. (1996) J. Biol. Chem. 271, 26477-26481). To determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether it is an intermolecular or intramolecular process, N-terminal histidine-tagged prothermolysin was expressed in Escherichia coli. Although partial processing to mature enzyme occurred, most of the proenzyme was recovered intact from inclusion bodies. This was then solubilized in guanidinium hydrochloride, immobilized on a cobalt-containing resin, and after dialysis against renaturation buffer, was quantitatively transformed to mature enzyme. However, when a mutation was introduced into the mature sequence to inactivate thermolysin, the proenzyme was not processed either in vivo or in vitro. In addition, mutated prothermolysin was not processed by exogenous thermolysin under a variety of experimental conditions. The results demonstrate that thermolysin maturation can proceed via an autocatalytic intramolecular pathway.

Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding.

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.