Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1997-08-14
2000-10-03
Achutamurthy, Ponnathapu
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
435 691, 435223, 426 40, 426 42, C12P 2106, C12N 958, A23C 912
Patent
active
06127142&
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to at least partially deglycosylated microbially derived milk clotting enzymes and in particular to aspartic proteases derived from Rhizomucor miehei and having improved milk clotting activity.
TECHNICAL BACKGROUND FOR THE INVENTION AND THE PRIOR ART
Milk clotting enzymes are widely used in the cheese manufacturing industry to provide a curd of the major milk proteins. Commercially available milk clotting enzymes include native (or homologous) enzymes derived from microbial species or animal tissue sources such as calf stomachs, or such enzymes can be provided as gene products of recombinant cells expressing a heterologous milk clotting enzyme of animal or microbial origin.
Milk clotting enzymes of microbial origin are in commercial use in the dairy industry. In the following, such enzymes are also referred to as microbial milk clotting enzymes, microbial rennets or microbial coagulants. Examples of such enzymes include proteases natively produced by the zygomycete filamentous fungal species Rhizomucor miehei and Rhizomucor pusillus and protease produced by the fungal species Endothia parasitica. Enzymes having milk clotting activity can also be produced by other microbial species, including Rhizopus species, Physarum species, Penicillium species and Bacillus species.
The major milk clotting enzyme derived from Rhizomucor miehei is an aspartic protease (EC 3.4.23) being produced extracellularly by this fungal species and having a relatively high milk clotting activity and a relatively low proteolytic activity, i.e. with a desirable low ratio between proteolytic activity and milk clotting activity. A commercial coagulant containing Rhizomucor miehei aspartic protease is also referred to in the industry as a Mucor rennin.
Microbial coagulant compositions may be based on enzymes produced by microbial strains naturally producing milk clotting enzymes (homologous enzymes), or they can be based on enzymes produced by a heterologous strain. Thus, as an example Aikawa et al. 1990, J. Biol. Chem., 265, 13955-13959 have disclosed the expression of the Rhizomucor pusillus rennin in a strain of Saccharomyces cerevisiae.
One advantage of using a heterologous production strain in the industrial manufacturing of microbial coagulants such as aspartic proteases is that undesirable contamination with other proteases generally occurring in homologously produced enzyme preparations can be at least partially eliminated.
It is well-known that microbially produced enzymes may be glycosylated when expressed, the degree of glycosylation depending on the type of enzyme and the microbial species expressing the enzyme. Thus, Aikawa et al., supra found that the mature Rhizomucor pusillus aspartic protease as produced homologously in that species only contained a few glycosidic moieties in its molecules whereas the enzyme, when it was expressed in Saccharomyces cerevisiae, was highly glycosylated (about 37 residues/mole). These investigators found that deglycosylation of the heterologous protease which was achieved by treating this milk clotting enzyme with endo-.beta.-N-acetylgalactosaminidase resulted in enhancement of its milk clotting activity, whereas treating the homologously produced protease similarly did not affect the milk clotting activity hereof.
Gray et al., 1986, Gene, 48, 41-53 disclosed expression of Rhizomucor miehei aspartic protease in Aspergillus nidulans and found that this heterologously produced protease had the same specific activity as determined in a milk turbidity assay as had the corresponding homologously produced protease.
Recently, a commercial Rhizomucor miehei derived aspartic protease produced in Aspergillus oryzae has been disclosed (Novo Nordisk, Biotimes, June 1994). The milk clotting activity of this heterologous protease was studied by Christensen et al., 1988, Biotechnology, 6, 1419-1422. These authors found that the protease was overglycosylated which, however, according to these authors did not alter the specific activity of the enzyme. I
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P.A. McBride-Warren and W.S. Rickert--"Structural and Functional Determinaants of Mucor Mieheiprotease"--Biochimica Et Biophysica Acta, vol. 328 (1973) pp. 52-60.
Tove Christensen, et al. "High Level Expression of Recombinant--Genes in Aspergillus Oryzae" Bio/Technology,--vol. 6. (Dec. 1988) pp. 1419-1422.
Gene, vol. 48, 1986, pp. 41-53, G. Gray et al Primary Structure of Mucor Miehie Aspartyl Protease: Evidence for a Zymogen Intermediate.
Molecular & General Genetics 241 (3-4). 1993. 312-318. Murakami et al Characterization of an Aspartic Proteinase of Mucor Pusillus Expressed in Aspergillus Oryzae.
J. Bacteriol (1994), 176(9), 2635-9 Murakami, Kohji et al "A Mucor Pusillus Mutant Defective in Asparagine-Linked Glycosylation".
J. Biol. Chem. (1990), 265(23), 13955-9, Aikawa, Junichi et al "Effects of Glycosylation on the Secretion and Enzyme Activity of Mucor Rennin, an Aspartic Proteinase of Mucor Pusillus, Produced by Recombinant Yeast".
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Harboe Marianne Kirsten
Kristensen Pia Bach
Achutamurthy Ponnathapu
Chr. Hansen A/S
Tung Peter P.
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