Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1996-06-07
1998-03-17
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
424 9467, 435135, 435212, 435219, 435220, 435221, C12P 2106, C12P 762, C12N 948, C12N 950, C12N 952, C12N 954, A61K 3846
Patent
active
057285446
DESCRIPTION:
BRIEF SUMMARY
This application claims benefit of International application PCT/JP94/02050, filed Dec. 6, 1994.
FIELD OF THE INVENTION
This invention relates to novel thermolysin-like neutral metallo-proteases and to use thereof, more specifically in the production of benzyloxycarbonyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester.
Thermolysin is a useful enzyme which is commercially available and used in a wide variety of fields, for example in detergent compositions, in food processing and in cosmetic formulations. It is further used in the synthesis of benzyloxycarbonyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester (hereinafter referred to briefly as Z-APM), which is a precursor of aspartame, an artificial sweetener.
BACKGROUND OF THE INVENTION
Thermolysin was first found in the culture broth of Bacillus thermoproteolyticus (Endo, s. (1962) J. Fermentation Tech., 40, 346-353) and a number of investigations have been conducted thereon. Thus, for instance, its amino acid sequence (Titani, K., et al., (1972) Nature New Biol., 238, 35-37) and the three-dimensional structure of the enzyme (Holmes, M. A. and Matthews, B. W., (1982) J. Mol. Biol. 160., 623-639) have been elucidated. Meanwhile, the protease gene was cloned from Bacillus thermoproteolyticus (EP-A-0418625) and the amino acid sequence of the mature enzyme as deduced from the nucleotide sequence of said gene was found to be different from the original primary structure as shown by Titani in two positions. Thus, it was reported that the 37th (from the amino terminal) amino acid residue of the mature enzyme is not aspartic acid but asparagine and the 119th one is not glutamic acid but glutamine. This amino acid sequence is identical with that coded by nprM, one of the protease genes cloned from Bacillus thermoproteolyticus (Kubo, M., et al., (1988) Journal of General Microbiology 134, 1883-1892).
Therefore, in the present specification, the protease coded by this nprM gene or the gene from Bacillus thermoproteolyticus is referred to as "wild type thermolysin-like neutral metallo-protease".
Alteration of specific activity and stability of thermolysin-like neutral metallo-protease has been reported (Kubo M., et al., (1992) Applied and Environmental Microbiology, 58, 3779-3783). In this article various mutants have been described which differ in one or more amino acid residues in the primary structure, especially at positions 93, 110, 114, 115, 136, 137, 143, 151, 157, 193, 211, 217 and 221. But in this reference, the activity was measured only by casein digestion method. None of these mutants, however, did show any substantially improved activity in relation to Z-APM synthesis or digestion. It now (as described further in the examples of previous European patent application of the applicant, application No. 93200773.5) also has been established that the activity for casein digestion does not correlate to that for Z-APM synthesis: it appears that even if the specific activity for casein digestion increases, the specific activity for Z-APM synthesis does not always increase.
In addition, the applicant previously found that useful novel proteases could be derived from thermolysin-like neutral metallo-protease having the (wild type) amino acid sequence of SEQ ID NO:1 shown below, by replacing one or more amino acid residues at certain positions with other amino acid residues than original ones. ##STR1##
Specifically, applicant already filed a European patent application (Application No. 93200773.5) for such novel modified proteases obtained from said wild type by replacement of at least one of the following amino acid residues with an amino acid different therefrom: 144th (leucine), 150th (aspartic acid), 187th (glutamic acid) and 227th (asparagine) amino acid residues.
The specific activity of the modified enzymes mentioned in said earlier patent application (not yet laid open at the date of filing of the present application) and having the single amino acid replacement at one of the positions 144th, 150th, 187th and 227th was not larger than 2 times of that o
REFERENCES:
patent: 5496710 (1996-03-01), Nagao et al.
Kubo et al. (Jul. 1988) Cloning and Nucleotide Sequence of the Highly Thermostable Neutral Protease Gene from Bacillus stearothermophilus, J. General Microbiology 134: 1883-1892.
Imanaka et al: "A new way of enhancing the thermostability of proteases", Nature, vol. 324, No. 6098, Dec. 18, 1986, pp. 695-697.
Kubo et al: "Alteration of specific activity and stability of thermostable neutral protease by site directed mutagenesis", Appl Environ Microbiol, vol. 58, No. 11, 1992, pp. 3779-3783.
Kostrov et al:"Structure-finction relationship for Bacilli metalloproteases", oage 884; Biological Abstracts, vol. 87, No. 12, 1989, abstract No. 130218, & Mol. Biol., vol. 23, No. 1, 1989, pp. 255-265.
Database WPI, Section Ch, Week 9303, Derwent Publications Ltd., London, GB; AN 93-022706 & JP,A,04 349 883, Dec. 4, 1992.
Endo Kimiko
Hanzawa Satoshi
Kidokoro Shun-ichi
Miki Yoichiro
Miyake Toshio
Holland Sweetner Company V.o.F.
Sagami Chemical Research Center
Stole Einar
Wax Robert A.
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