Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-11-27
2004-07-27
Meller, Michael (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S219000, C435S183000, C435S221000, C435S252500
Reexamination Certificate
active
06767729
ABSTRACT:
TECHNICAL FIELD
The present invention relates to an enzyme solution and a method for producing the same, an enzyme preparation obtained from the enzyme solution, a proteolytic enzyme preparation containing the enzyme solution or the enzyme preparation as an active component, and a strain producing the proteolytic enzyme.
BACKGROUND OF THE INVENTION
In extremely diversified industrial fields, techniques of hydrolyzing various proteins into peptides or amino acids have been utilized widely. The techniques are applied to the preparation of, for example, medical enteric formulas and nutritious supplements as food materials. The techniques are also applied to increase the food utilization efficiency by enhancing the efficiency of hydrolyzing proteins hard to be hydrolyzed, which are contained in soybean protein and the like. Further, the techniques are utilized for the preparation of amino acid seasoning from proteins as raw materials. Still further, the techniques are utilized for the preparation of bread with enhanced rise.
Although chemical degradation methods using hydrochloric acid and the like so as to hydrolyze protein are efficient, the methods may result in the formation of undesirable by-products because of severe hydrolyzing conditions. Particularly in industrial fields of foods, seasonings, materials for nutrition and the like relating to human bodies, methods capable of hydrolyzing protein under mild conditions are prefered (for example, Japanese Patent Publication No. Hei-7-53106, Japanese Patent Laid-open No. Hei-11-75765, and the like).
Herein, proteolytic enzymes for industrial use for the purposes described above have been produced so far, mainly by means of various bacteria and fungi. However, it is generally said that proteolytic enzymes obtained from bacteria and those obtained from fungi have both advantages and disadvantages. Actually, proteolytic enzymes for industrial use having sufficient activity and stability have not been available.
In other words, bacteria-derived proteolytic enzymes generally have good heat resistance but have low peptidase activity for hydrolyzing protein into amino acids, or low potency of hydrolyzing proteins into low molecules. Therefore, the degradation products obtained by using bacteria-derived proteolytic enzymes contain great amounts of high-molecular peptides. Hence, such products have a disadvantage in that the bitterness thereof as food materials, seasonings and the like is strong, and in that the intestinal absorption thereof as materials for nutrition is slow. Although bacteria-derived proteolytic enzymes have good heat resistance, there has hardly been reported such proteolytic enzyme with high heat resistance that can retain peptidase activity, for example, in a high temperature range of about 60° C.
In contrast, fungi-derived proteolytic enzymes are generally excellent in peptidase activity, wide cleavage specificity of peptide bond, potency of hydrolyzing proteins into low molecules and the like. However, the heat resistance, for example, the potency of protein hydrolysis in a moderate to high temperature range of about 50° C. or higher, is poor. Therefore, such proteolytic enzymes essentially require protein hydrolyzing steps in a relatively low temperature range, readily allowing the propagation of contaminated microorganisms.
As described above, there have not been provided proteolytic enzymes with high thermal stability such that the enzyme is never inactivated in moderate to high temperature ranges and also with great potency of hydrolyzing proteins into low molecules.
Further, it is desired to effectively hydrolyze proteins hard to be hydrolyzed, such as those contained in soybeans and the like, as useful protein materials. It is thus important that proteolytic enzymes have wide cleavage specificity for peptide bonds in protein. However, no proteolytic enzyme has been provided, which has both the thermal stability and the great potency of hydrolyzing proteins into low molecules and which additionally has high potency of hydrolyzing proteins hard to be hydrolyzed.
DISCLOSURE OF THE INVENTION
The present inventors cultivated a bacterium of genus Bacillus, which was isolated from the dough of “Mantou (a kind of Asian steamed buns)” as one of traditional foods in the Mongol District. It was then found that the enzyme solution consequently obtained had the enzyme characteristics as described above.
Based on the findings, the invention provides a proteolytic enzyme solution with thermal stability in moderate to high temperature ranges and with excellent potency of hydrolyzing proteins into low molecules as well. Further, the invention provides a proteolytic enzyme solution additionally having the potency of effectively hydrolyzing protein hard to be hydrolyzed. Still further, the invention provides a method for producing such enzyme solution. Further, the invention provides an enzyme preparation obtained by separating the enzyme protein from the enzyme solution. And yet further, the invention provides a proteolytic enzyme preparation containing the enzyme preparation as the active component, which can be used for given uses. Still further, the invention provides a strain producing proteolytic enzyme to prepare the enzyme solution and the enzyme preparation.
The enzyme solution of the invention is an enzyme solution with proteolytic activity, which can be obtained by cultivating a bacterium of the genus Bacillus, and has such highly heat-resistant peptidase activity that the residual activity of the enzyme after 1-hour heat treatment at 60 to 65° C. at pH 7 is substantially 100%. The use of the enzyme solution enables the protein hydrolyzing process under temperature conditions never permitting the propagation of contaminated microorganisms, for example at 50° C. or higher, or in moderate to high temperature ranges up to 60 to 65° C. Further, the peptidase activity can allow sufficient potency of hydrolyzing proteins into low molecules. The peptidase activity is more preferably an aminopeptidase activity.
More preferably, the enzyme solution of the invention additionally has protease activity and collagenase activity in combination. The protease activity includes neutral protease activity with favorable pH within the neutral region and alkaline protease activity with favorable pH within the alkaline region. Such enzyme solution can be expected of more excellent potency of hydrolyzing proteins into low molecules and the large initial reaction velocity as a general characteristic of protease activity. Therefore, proteolytic reaction can be speedily promoted. For meat tenderization, for example, meat tenderization by the peptidase activity and the protease activity can progress, simultaneously with the cleavage of collagen in the connective tissues by the collagenase activity, so that very tasty meat can be prepared.
Still more preferably, the enzyme solution of the invention has at least one of the following characteristics 1) to 4) so as to provide additional effects described below.
1) Enzyme solution with peptide cleavage site specificity capable of cleaving sites of at least ten amino acid types bonded in the peptide chain of protein. Regarding such wide range of peptide cleavage site specificity, peptide cleavage site specificity capable of cleaving 10 or more, preferably 12 amino acid types bonded at the carboxyl termini can be observed. Such amino acid types specifically include leucine, isoleucine, phenylalanine, lysine, valine, alanine, threonine, glycine, serine, glutamine, asparagine and arginine.
Typically, general proteolytic enzymes have peptide cleavage site specificity capable of substantially cleaving 5 to 6 or less, at most less than 10 amino acid types bonded. Therefore, the peptide cleavage site specificity of the enzyme solution covers a very wide range. Thus, the enzyme solution can retain great potency of hydrolyzing proteins into low molecules. Probably owing to the wide range of peptide cleavage site specificity, the enzyme solution has effective proteolytic activity over proteins, such as thos
Ando Keiichi
Hirano Kenichi
Nagano Hiroko
Shoji Zenya
Amano Enzyme Inc.
Meller Michael
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