PRODUCTION BY YEASTS OF ASPARTIC PROTEINASES FROM PLANT ORIGIN WITH SHEEP'S, COW'S, GOAT'S MILK, ETC. CLOTTING AND PROTEOLYTIC ACTIVITY

Abstract: This invention is valid for recombinant enzymes produced from transformed yeast with coding genes for plant-origin aspartic acid proteinases. These proteinases have considerable sheep's, cow's and goat's milk clotting activity. They can be produced in large quantities by cultivating transformed yeast in a liquid medium. They are secreted into the culture medium and can be supplied in liquid or lyophilised form. The activity of these enzymes is similar to that of chymosin (an animal-origin enzyme) used in the production of cheese on an industrial scale. Recombinant aspartic acid proteinases differ from chymosin in their casein cleavage capacity. Recombinant plant enzymes cleave alpha , beta and kappa caseins. Chymosin only cleaves kappa casein. The ability of plant-origin recombinant aspartic acid proteinases to cleave alpha , beta and kappa caseins is responsible for the special flavour, smell and consistency of the cheese produced.

Description: DESCRIPTION Production by yeasts of aspartic proteinases from plant origin with sheep's, cow's, goat's milk, etc. clotting and proteolytic activity. Introduction The use of a yeast expression system has become a way of producing large quantities of different types of compounds on an industrial scale. Regarding the production of plant-origin aspartic acid proteinases with industrial applications, there has not been any news of yeast expression with regard to production for use on an industrial scale. The object of this invention patent, described below, refers to the construction of plasmids, the transformations of yeast strains and the production of plant-origin aspartic acid proteinases. Construction of Plasmids. Transformation of Yeast Strains and Production of Plant-origin Proteinases The insertion of coding gene CYPRO11 into a plant-origin proteinase constitutes the experimental model for controlling the yeast expression of plant-origin aspartic acid enzymes. Two Escherichia co/-yeast expression system vectors were constructed, using a type 2μ multi-copy plasmid and a centromeric plasmid having a low number of copies. The choice of gene used was the leucine deficient one (LEU2). The expression cassette contained developer G7 and four different leader sequences upstream from the heterologous gene. Transcription of the heterologous gene was stopped by a PGK1 terminator. From the different leader sequences tested (native prosequence, preSUC2-proCYPRO11 , preMFα-proCYPRO11 and preproMFα), we concluded that preMFα-proCYPRO11 was the best leader sequence for the production of plant-origin aspartic acid proteinases, whether cyprosins corresponding to the plant-origin model proteins coded by gene CYPRO11 , or other commercially interesting plant-origin acidic aspartic proteinases. The MFα yeast presequence is sufficient to develop secretion of the aspartic acid proteinase into the culture medium, and the use of a prosequence of the gene is not necessary. The native prosequence was essential to the active protein's production. The use of centromeric plasmids having a low number of copies gave better results than type 2μ multi-copy plasmids. Different yeast strains were tested, including Saccharomyces cerevisae BJ1991 (MATα Ieu2 trpl ura3-52 prbl-1122 pep4-3), BJ2168 (MATα Ieu2 trpl ura3-52 prcl-1122 pep4-3), MT302/1c-a {arg5-6 Ieu2-12 his3-11 his3- 15 peb4-3 adel), W303-1 a (MATα Ieu2-3, 112 ura3-1 trpl-1 his3-11, 15 ade2-1 can1-100 GAL SUC2). These strains were kept on YPD agar plates containing 1 % yeast extract, 2% bacto-peptone, 2% glucose and 1.5% agar. The transformed yeast was grown in an SD medium (0.67% yeast nitrogen base without aminoacids, DIFCO, 2% (w/v) glucose), supplemented with aminoacids suited to the auxotrophy needs of each strain, except for the leucine one. The cultures were collected and washed once with sterile distilled water. The cells were resuspended in a YPGal medium (1 % yeast extract, 2% bacto-peptone, 4% galactose) and used to inoculate the same medium at a density of A 6 oo = 0.2. The cultures were incubated in the same culture conditions until they reached densities of A 60 o = 2, 6 or 10. Of the yeast strains tested, protease deficient strain BJ1991 produced and secreted into the culture medium the largest quantities of aspartic acid proteinase with considerable milk clotting and proteolytic activity. The secretion of proteolytic enzymes was therefore dependent on culture growth. The recombinant proteinase with the highest degree of clotting and proteolytic activity was obtained in the stationary phase of the YPGal medium's growth (A 6 oo = 10). In the exponential phase (A 6 oo = 2), the yeast cells secreted an inactive recombinant proteinase having a high molecular weight. It was considered to be an unprocessed form of the proteinase in which a specific region of the genes of plant-origin acidic aspartic proteinases called a specific plant insert had not been removed. The largest sub-unit of the recombinant proteinases secreted by the yeast was glycosilated, in the only site possible for glycosilation, and contained a considerable number of manose type glycan chains. Preparation of Polyclonal Antibodies The total proteic extract used to produce polyclonal antibodies against plant-origin acidic aspartic proteinase with considerable coagulation and proteolytic activity was obtained from the dry flowers of Cynara cardunculus by maceration in a mortar in liquid nitrogen and extraction with 50mM of Tris HCI buffer at a pH of 8.3 (Heimgartner et al., 1990). The proteins were fractionated in 12% SDS-PAGE using 100μg of total protein extract per well. The gel was tinted with a 0.02% Commassie Blue solution in distilled water. The bands corresponding to the largest sub-unit of the plant enzyme (31-32.5kDa in the SDS-PAGE gel) were isolated and the content of each well was sent to EUROGENTEC (Belgium) for the production of antibodies. Isolation of the Plant-origin Proteinase and Western Blotting Analysis Isolation of the recombinant plant-origin proteinase from the cell extracts was done using 30ml of yeast cells grown to densities of A 6 oo = 2, 6 or 10. After collection, the cells were washed with distilled water, resuspended in 500μl of buffer and exploded by shaking them with glass balls. Isolation of the recombinant proteinase from the culture medium was done after collecting the medium and concentrating it almost 10 times by ultracentrifugation. The proteinase concentration was ascertained using the Bio-Rad protein analysis kit in accordance with the manufacturer's instructions. 50μg of total proteic extract from the yeast cells or 1.125g of the concentrated culture medium was analysed in 12% SDS-PAGE. The proteins were transferred to a nitro-cellulose membrane (Bio-Rad) using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) equipment in accordance with the manufacturer's instructions. Proteins were detected using polyclonal antibody CCMPI prepared in accordance with the description in the previous section and Boehnger Mannheim's Chemiluminescence Western Blotting Kit in accordance with the manufacturer's instructions. The results obtained showed that the transformed yeast produces plant- origin aspartic acid proteinase and that the inactive form is found in cells in the exponential growth phase while the active form is secreted into the culture medium. This peculiarity is decisive when it comes to getting good performance for the extraction and purification of plant-origin acidic aspartic proteinases produced from yeast. Analysis of the Plant-origin Recombinant Enzyme's Clotting and Proteolytic Activity Proteolytic activity was analysed in accordance with the Twinning method (1984). The casein preparation marked with isothiocyanate (casein-FTC) was made in accordance with the author's instructions. The reactive mixture contained 30μl of 0.2M sodium citrate buffer, pH 5.1 , 20I of casein- FTC and 20μl of enzyme solution (3μg/μl in the case of total proteic extract from the yeast cells or 150ng/μl in the case of concentrated culture medium). Two control tests were done by replacing the enzymatic solution with the reactive buffer. The samples were incubated at 37°C for 30 minutes. Reaction was stopped by adding 120μl of 5% t chloracetate acid (TCA) in all but one of the controls. In the latter case, the same amount of 0.5M Tris HCI buffer at a pH of 8.0 (positive control) was added. The samples were centrifuged and a 150μl aliquot of the supernatant fraction was diluted to 3ml with 0.5M Tris HCI buffer at a pH of 8.5. The control (without enzymes), whose reaction was stopped with the TCA solution, was used to ascertain the formation of soluble fluorescent compounds in TCA with enzyme intervention. Relative fluorescence of the samples was ascertained using wavelengths of 490nm for excitation and 525nm for emission in a Shimadzu RF-1501 (Shimadzu Corporation, Kyoto, Japan) spectrofluorimeter. The percentage of relative fluorescence (%RF) was calculated by subtracting the negative control values from the values, and considering the positive control values as 100%RF. For statistical analysis of the results, each sample had three replicas and three independent readings were taken. The data obtained were analysed with the Student's t test (α=0.05). Greatest proteolytic activity, obtained for the best combination/yeast strain, was 15% RF/μ of protein. This figure refers to standard culture conditions, and can be increased under conditions optimised for industrial purposes and using Sec- strains, that is, strains chosen for their maximum recombinant proteinase secretion into the culture medium. Ascertaining Clloting Activity Clotting activity was ascertained in test tubes, using unconcentrated culture medium in accordance with the following method: 10ml of the culture medium of the transformed YPGal yeast cells was added to 3ml 12% of skimmed milk (bacto-Difco) and 100ml mM CaCI 2 . The pH of the culture medium for the culture grown to either A 6 oo = 6 or 10 was approximately 5.0. For the culture medium of the culture grown to A 6 oo = 2, the pH was adjusted to 5.0 using HCI. The samples were kept at 37°C until the onset of coagulation. The coagulation was evident.

Claims: CLAIMS 1. Transformed yeast cultures are characterised by their content in coding genes for plant-origin aspartic acid proteinases and their production of plant-origin aspartic acid proteinases with milk clottting activity, secreted in active form into the culture medium. They can be extracted from the culture medium, purified and supplied in liquid or lyophilised form on a domestic or industrial scale as milk clotting enzymes. 2. The transformed yeast cultures described in claim 1 are characterised by the stable integration of coding genes for plant- origin aspartic acid proteinases. 3. The transformed yeast cultures described in claims 1 and 2 are characterised by their ability to produce aspartic acid proteinases, confirmed by the use of produced antibodies against cardoon aspartic acid proteinases, and their milk clotting activity, confirmed by milk clotting tests. 4. The transformed yeast cultures described in claims 1 , 2 and 3 are characterised by their ability to secrete recombinant aspartic acid proteinases into the culture medium. 5. The transformed yeast cultures described in claims 1 , 2, 3 and 4 are characterised by their ability to produce recombinant aspartic acid proteinases capable of effectively coagulating milk from different origins, especially sheep's, cow's and goat's milk. 6. The transformed yeast cultures described in claims 1 , 2, 3, 4 and 5 are characterised by their ability to produce aspartic acid proteinases which cleave α, β and K caseins from milk from different origins. 7. The transformed yeast cultures described in claims 1 , 2, 3, 4 are characterised by their production of recombinant aspartic acid proteinases, including cyprosins and cardosins, capable of giving cheese a special flavour, smell and consistency.

Inventors: CALIXTO FILOMENA DA CONCEICAO, PLANTA RUDY J, SOARES PAIS MARIA SALOME, CALIXTO, FILOMENA DA CONCEICAO, S., S., PLANTA, RUDY, J., SOARES PAIS, MARIA, SALOME
Applicants: CALIXTO FILOMENA DA CONCEICAO, INST DE CIENCIA APLIC E TECNOL, PLANTA RUDY J, SOARES PAIS MARIA SALOME, CALIXTO, FILOMENA DA CONCEICAO, S., S., INSTITUTO DE CIENCIA APLICADA E TECNOLOGIA (ICAT), PLANTA, RUDY, J., SOARES PAIS, MARIA, SALOME
Application year: 2000
Main classification: C12N1
IPC codes: C12N1, C12N9, C12N15