Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Utility Patent
1996-03-06
2001-01-02
Lankford, Jr., Leon B. (Department: 1808)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S070100
Utility Patent
active
06168939
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a novel endopeptidase produced from the culture of
Lactobacillus helveticus.
The endopeptidase of the present invention does not degrade proteins but degrades peptides, and thus can be used together with other proteolytic enzymes such as proteinases and aminopeptidases to effectively produce protein hydrolysate used for foods and drinks, and medicines. In addition, the endopeptidase of the present invention can be used solely to selectively produce specific peptides.
BACKGROUND OF THE INVENTION
Heretofore, a number of proteolytic enzymes derived from various sources are known, and they have been utilized in various fields. Among them, proteolytic enzymes derived from lactococci have been used in food processing industries because of their safety. In particularly, endopeptidases derived from lactococci hydrolyze large peptides generated by the action of proteinases on proteins and are deemed to be very important enzymes for the effective degradation of proteins. For example, an endopeptidase produced by
Lactococcus lactis
ssp, cremoris was purified and its properties are well elucidated [Japanese Laid-open (KOKAI) Patent Application No. 268955 (1993)].
However, purified endopeptidase has heretofor not been obtained from
Lactobacillus helveticus
, a lactobacillus which is used to ferment dairy products such as fermented milk and cheese and plays an important role in industrial fields, particularly the food industry.
The inventors of the present invention have found a novel endopeptidase produced by
Lactobacillus helveticus
which specifically hydrolyzes peptides by attacking peptide bonds within peptide without hydrolyzing proteins such as various caseins.
SUMMARY OF THE INVENTION
One object of the present invention is to provide an endopeptidase which produced by
Lactobacillus helveticus
, specifically hydrolyzes peptides having the following physicochemical properties:
(1) a molecular weight of about 70 kDa, as determined by SDS polyacrylamide gel electrophoresis;
(2) an isoelectric point of about 4.8;
(3) an optimal temperature of about 30° C.;
(4) an optimal pH of about 7.0; a Michaelis constant (K
m
value) of about 0.20 mM and a maximum velocity (V
max
value) of about 56 &mgr;mol/min./mg by hydrolysis of the substrate Tyr-Gly-Gly-Phe-Met;
(6) substrate specificity whereby the endopeptidase attacks peptide bonds inside peptides to hydrolyze the peptides without hydrolyzing proteins;
(7) it is inhibited by the enzyme inhibitors 1,10-phenanthroline, ethylenediaminetetra-acetic acid (EDTA), and p-chloromercuribenzenesulfonic acid (p-CMBS).
(8) the endopeptidase's activity is inhibited by Cu
2+
, Zn
2+
, and Fe
2+
.
The endopeptidase of the present invention does not react with a polyclonal antibody raised against an endopeptidase produced by
Lactococcus lactis
ssp. cremoris Wg2 strain of the lactic acid-forming coccus. Thus the endopeptidase of the present invention is immunologically different from that produced by the Wg2 strain.
Furthermore, the amino acid sequence of the N-terminus of the endopeptidase of the present invention is Val-Arg-Gly-Gly-Ala-Gly-Asp-Ile-Thr-Glu-Ala-Asp-Leu-Ser-Ala-Arg-Pro-Gln-Asp-Asn-Leu-Tyr-Leu-Ala-Val-Asn- (SEQ ID NO:2). The homology of the sequence to data bases of all proteins and DNAs was searched in the literature and using a BLAST program, but no homologous or nearly homologous enzyme was found. Thus the enzyme of the present invention is considered as a novel enzyme.
REFERENCES:
patent: 4515891 (1985-05-01), Yokogawa et al.
patent: 0 406 598 B1 (1991-01-01), None
patent: 0522203 A1 (1993-01-01), None
patent: 0 633 316 A1 (1995-01-01), None
patent: WO 94/16082 (1994-07-01), None
patent: WO 94/26882 (1994-11-01), None
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C.M. Nowakowski et al., “Cloning of Peptidase Genes fromLactobacillus HelveticusCNRZ32”Applied Microbiology and Biotechnology39:204-210 (1993).
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H. Kataoka et al. “Determination of Glutathrone and Related Aminothiols by Gas Chromatography with Flames Photometric Detection”Biomed. Chromatography9(2):85-89 (1995).
H. Miyakawa et al., “Purification and Characterization of an Aminopeptidase fromLactobacillus helveticusLHE-511”,J. Dairy Sci. 75(1):27-35 (1992).
H. Miyakawa et al., “Purification and Characterization of a Prolyl Aminopeptidase fromLactobacillus helveticusLHE-511”,Milk Science Intl. 49(11):615-619 (1994).
H. Miyakawa et al., “Purification and Characterization of an X-Prolyl Dipeptidyl Aminopeptidase fromLactobacillus helveticusLHE-511”Milk Science Intl. 49(12):670-673 (1994).
Bosman Boukje
Iwasaki Taisuke
Sasaki Masahiro
Takafuji Shin'ichi
Tan Paris S. T.
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