Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-12-07
2003-02-11
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S183000, C435S212000, C435S252300, C435S320100, C435S221000, C536S023200
Reexamination Certificate
active
06518054
ABSTRACT:
The present invention relates to metallo-endopeptidases, sometimes also called neutral proteases, that are produced, processed and secreted by e.g. members of the bacterial genus Bacillus. The present invention provides genes encoding variants of metallo-enopeptidases that have been engineered to be resistant to prolonged boiling while maintaining their enzymatic performance at much lower temperatures. In addition, thermal stability of the metallo-endopeptidases is highly dependent on calcium at concentrations in the mM range. The invention thus further provides active metallo-endopeptidase variants whose stability depending on calcium concentration can be changed so as to provide metallo-endopeptidases that are calcium dependent or independent. The invention also provides genes that encode boiling-resistant metallo-endopeptidases whose stability depending on calcium concentration can be changed. The invention also provides vectors and cells comprising these genes and proteases produced through these genes, vectors and/or cells.
Denaturation of proteins at elevated temperatures is usually the result of unfolding which is followed by an irreversible process, most often aggregation. The notion that the unfolding processes involved in irreversible denaturation often have a partial (as opposed to global) character has been confirmed experimentally in several cases. We have studied the thermal stability and denaturation of a broad-specificity metalloprotease produced by
Bacillus stearothermophilus
CU21 (called TLP-ste) which shares 85% sequence identity with its more stable and better known counterpart
Bacillus thermoproteolyticus
(hereinafter also referred to as “thermolysin”). Thermolysin-like proteases (TLPs) are a family of homologous metalloproteases or neutral proteases that contain a catalytically important zinc ion in their active site. Thermal denaturation of thermolysin-like proteases (TLPs) depends on partial unfolding processes as well, which, however, are not followed by aggregation but by autolytic degradation starting at unknown sites in the partially unfolded molecule (Vriend, G. & Eijsink, V. G. H. J.
Comput
.-
Aided Mol. Des.
7, 367-396, 1993). An extensive mutation study in which residues in TLP-ste were replaced via site-directed mutagenesis by the corresponding amino acid in thermolysin showed that only a few of the 43 substitutions between the two enzymes are important for stability (Veltman, O. R., Vriend, G., Middelhoven, H., Van den Burg, B., Venema, G. & Eijsink, V. G. H.
Protein Engn.
9, 1181-1189, 1996). All important substitutions are clustered in the N-terminal domain of the protein, in particular in a weak region comprising the 55-69 surface loop (European patent application 94200182.7). Remarkably, stabilizing substitutions or combinations of several substitutions either (i) at the positions of SEQUENCE ID No. 2: 4, 56, 58, 63, 65 or 69 (for example A4T, T56A, G58A, T63F, S65P, A69P) or (ii) the positions of SEQUENCE ID No. 1: 4, 59, 61, 66, 68 or 72 (for example A4T, T59A, G61A, T66F, S68P, A72P) result in enzyme variants that are more stable than thermolysin. The three-dimensional structure of thermolysin is known (Holmes, M. A. and Matthews, B. W. (1982) J. Mol. Biol, 160, 623-639) and this enzyme was shown to bind four calcium atoms which contribute to thermal stability. Two calcium ions are bound in the so-called double-calcium binding site (Ca
1,2
), that is composed of ligands that are conserved in all TLPs. The other, single binding sites (Ca
3
and Ca
4
) are composed of ligands that are conserved only in the more stable TLPs such as thermolysin and the TLP produced by
B. stearothermophilus
(TLP-ste). At elevated temperatures, TLPs are irreversibly inactivated as a consequence of autolysis. Autolysis follows first-order kinetics because its rate is determined by local unfolding processes that render the protease susceptible to autoproteolytic cleavage (Eijsink, V. G. H., Van den Burg. B., Vriend, G., Berendsen, H. J. C. and Venema, G. (1991) Biochem. Internatl. 24, 517-525). In their studies on the contribution of calcium ions to thermolysin stability, Dahlquist et al. (Dahlquist, F. W., Long, J. W, and Bigbee, W. L. (1976) Biochemistry 15, 1103-1111. ) and Roche and Voordouw (Roche, R. S. and Voordouw, G. (1978) CRC Crit. Rev. Biochem. 5, 1-23) concluded that the initial steps in thermal inactivation are accompanied by the release of one calcium ion (Ca
3
or Ca
4
). Extensive mutagenesis studies of the TLP-ste have shown that a region near the Ca
3
site is crucial for thermal stability (Veltman, O. R., Vriend, G., Middelhoven, P. J., Van den Burg, B., Venema, G. and Eijsink, V. G. H. (1996) Protein Engng. 9, 1181-1189. ). Thus, thermal inactivation seems to be dominated by one single ‘weak’ region, near Ca
3
. Considering the expected high structural similarity between thermolysin and TLP-ste (85 percent sequence identity) the studies on TLP-ste suggest that the critical calcium ion is Ca
3
rather than Ca
4
.
Metallo endopeptidases can be applied in several industrial processes, for instance in the preparation of the artificial sweetener aspartame, but also in the leather industry, for dehairing or dewooling, in breweries and in the production of (poly)peptide or protein hydrolysates. These processes may need to be performed at high temperatures to accelarate the processes. However, the enzymes used normally are not resistant to elevated temperatures. Proteases that are more stable at higher temperatures can for example be obtained via mutations in the 55-69 area, however, they still do not withstand boiling for periods lasting longer than several minutes. On the other hand, boiling is a simple method to denature the enzyme and thus a method to stop the enzymatic process. Thus, there is a need to develop enzymes that are more resistant to the temperatures found at boiling for prolonged periods in a watery environment, with an half-life >0.5 hour at 100° C. but that on the other hand, if needed, may be controlled by other methods to stop the enzymatic processes.
The invention provides strategies to design and construct genetically engineered thermolysine-like proteases that are far more resistant to boiling than those known before. The invention also provides the proteases resulting from such strategies as well as the use of said products in industrial processes and intermediates in making the products. The invention provides mutations leading to the introduction of disulfide bridges stabilizing the area in metallo-endopeptidases that is most susceptible to unfolding after heating which area is hereinafter referred to as the “functional part of the sequence”. In a first embodiment, the invention provides a recombinant DNA molecule comprising a least a functional part of the sequence of FIG.
7
and coding functional part of the sequence of
FIG. 7A
[SEQUENCE ID No. 1] or
FIG. 7B
[SEQUENCE ID No. 2] or
FIG. 7
(which combines the SEQUENCE ID No. 1 and the SEQUENCE ID No. 2 as more fully explained, infra) coding wherein at least one codon is mutated to code for cysteine to generate a stabilizing disulfide bridge, and a polypeptide derived of said DNA molecule. For example, an important mutation concerns the introduction of a disulfide bridge cross-linking residue 60 [in SEQUENCE ID No. 2] or residue 63 [in SEQUENCE ID No. 1] in the critical region with residue 8 (common to each of SEQUENCE ID No. 1 and SEQUENCE ID No. 2) in the underlying &bgr;-hairpin. For example, the invention provides a mutated TLP-ste variant with a half life at 100° C. of almost 3 hours which is more than 1000 times that of the wild-type while it maintained its specific activity at 37° C. Furthermore, the invention provides mutants that are active and stable in the presence of high concentrations of denaturing agents and which cleavage specificity at both moderate and high temperatures is largely unaffected by the stabilizing mutations.
The invention also provides calcium-dependent and—independent variants of t
Van den Burg Lambertus
Veltman Oene Robert
Venema Gerard
Liberman Arthur L.
Michaelson Peter L.
Michaelson & Wallace
Prouty Rebecca E.
Rao Manjunath N.
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