Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the invention relates to a DNA fragment which contains at least one mutant protease gene.
• Lactic acid bacteria are gram-positive bacteria which are used in the production of a series of fermented foodstuffs, including dairy products. These bacteria, in particular those which belong to the genus Lactococcus lactis (L.lactis) , have a complex proteolytic system for degrading milk proteins to small peptides and free amino acids which are then used for the cell growth: see Thomas, T.D. et al., FEMS Microbiol. Rev. »6 (1987), pages 2 ⁇ 5-268.
• a key enzyme in this proteolytic system is a large serine-protease which is localized on the cell membrane and which is involved in the initial cleavage of caseins which are the most important milk proteins.
• This protease is essential for the cell growth and it produces peptides which contribute to the development of the flavour of a number of dairy products: see Visser, S. et al., Appl. Microbiol. Biotechnol. 2j (1988), pages 61-66.
• proteases localized on the cell membrane can be divided into two main groups on the basis of their caseinolytic properties, viz. type I and type III proteases; see Visser, S. et al., Appl. Environ. Microbiol. 52 (1986), pages 1162-1166.
• Type III proteases degrade ⁇ sl-, ⁇ - and k-casein, while type I proteases mainly degrade ⁇ -casein.
• type I proteases On acting on casein as a whole, type I proteases produce much more bitter-tasting peptides than type III proteases (see Visser, S. et al., Neth. Milk Dairy J. 3_ (1983), pages I69- l8 ⁇ ) .
• proteases are immunologically related proteins having similar molecular weights.
• the protease genes of a representative of each group i.e. the type I protease of L.lactis Wg2 and the type III protease of L.lactis SK11 have been cloned and expressed in L.lactis; see Kok, J., et al., Appl. Environ. Microbiol. 52 (1 85) pages 94-101 and De Vos, .M., Bimolecular Engineering in the European Community (1986), pages 465-472, Martinus Nijhoff Publishers, The Netherlands.
• the complete nucleotide sequence of the L.lactis g2 protease gene has been determined by Kok, J. et al., Appl. Environ. Microbiol 5_4 (1988), pages 231-238 and the Applicant has determined the complete nucleotide sequence of the L.lactis SK11 protease gene (EP-A-0,307,011) ; these sequences are shown in Fig. 1.
• the protease genes code for very large pre-pro-proteins (>200 kDa) which are converted at the N- and C-terminus to obtain the mature active enzyme (Haandrikman, A. et al., J. Bacteriol.
• mutant proteases have been developed in which "mutant protease gene" is understood to mean: - a gene made up of sections of parent protease genes of several lactococcal strains, inter alia L.lactis SK11 and L.lactis g2, as well as - a parent protease gene of a lactococcal strain, the DNA sequence of which has been altered in a manner such that, in the mutant protease for which the gene codes:
• the invention therefore relates to DNA fragments containing at least one mutant protease gene, the encoded protease having modified properties compared to the properties of the parent protease(s). These properties include the thermostability, alkaline/acid pH stability, oxidative stability, autoproteolysis, calcium ion binding, cleavage and substrate specificity, catalytic activity and pH activity profile of the proteases according to the invention.
• a particular embodiment of a mutant protease gene is a hybrid protease gene which is made up exclusively of sections of the Wg2 protease gene and the SK11 protease gene.
• the invention relates to cloning vectors, preferably as described in EP-A-0,157,441 or EP-A-0,228,726, which contain such a DNA fragment according to the invention, to host cells, advantageously lactococcal strains, in particular the L.lactis strain MGI363, deposited at the Centraal Bureau voor Schimmelcultures under number CBS 364.89. which have been trans ⁇ formed with a cloning vector specified above, to mutant proteases preparable with such host cells, and also to the products, ' such as foodstuffs and flavourings, which have been obtained with the aid of the host cells or proteases specified above.
• host cells advantageously lactococcal strains, in particular the L.lactis strain MGI363, deposited at the Centraal Bureau voor Schimmelcultures under number CBS 364.89. which have been trans ⁇ formed with a cloning vector specified above, to mutant proteases preparable with such host cells, and also to the products, ' such
• E.coli TGI K12, (_/ ⁇ lac-pro), supE, thi, hsdD5/ F*traD36, proA + B + , lad*, lacZ ⁇ M15.
• the strain L.lactis subsp. lactis MGI363 is a plasmid-free derivative of L.lactis subsp. lactis 712 and is described by Gasson, M.J., J. Bacteriol. 154 (1983) , pages 1-9- This strain was used as host for all the plasmid transformations.
• the plasmid pGKV552 contained the complete protease gene (prtP gene) and a functional maturation protein gene (prtM gene) of L.lactis subsp. cremoris Wg2, introduced into the lactococcal cloning vector pGKl4, as described by Haandrikman, A. et al., J. Bacteriol.
• the prtM gene codes for a trans-active, maturation protein which, according to the Applicant, is regarded as essential for proteolytic activity.
• the section of the plasmid which contains both the prtM gene and the prtP gene and also promoter regions for each is termed the protease region.
• the plasmid pNZ521 contained the complete protease gene (prtP gene) and a functional maturation protein gene (prtM gene) of L.lactis subsp. cremoris SKll introduced into the lactococcal cloning vector pNZ122, as described by Vos, P. et al. J. Bacteriol.
• Plasmid pNZ521 appears to have a reasonably high copy number in the strain L.lactis subsp. lactis MGI363 and, in addition, to be stably maintained without antibiotic selection pressure.
• the promoter region for the prtM and prtP genes of strain SKll and Wg2 are not completely identical.
• the prtM genes of strains SKll and Wg2 code for identical maturation proteins prtM (Haandrikman A. c.s. and Vos P. c.s., J. Bacteriol. 121, (1989), pages 2789-2802).
• the cloning method was carried out in accordance with generally known cloning techniques, such as those described by Maniatis, T. , et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1982, with only slight modifications. All the necessary enzymes were obtained from Bethesda Research Laboratories and New England Biolabs Corp. and were used in accordance with the supplier's instructions.
• the plasmids were introduced into the strain L.lactis MGI363 by means of electro- poration (see Chassy, B.M. et al. , FEMS Microbiol. Lett.
• fragment (a) is a 1055 bp Clal-BamHI fragment with the coding sequence of a section of the pre-pro-sequence and the first 173 amino acids of the mature protease.
• Fragment (b) is a 970 bp BamHI- SacII fragment which codes for the amino acids 174-496 and fragment (c) is a 1778 bp SacII-BstEII fragment which codes for the amino acids 497-1089 of the protease (see Fig. 2).
• a restriction site is present which is unique to one of the two proteases, viz Pstl, Kpnl and Bglll (see Fig. 2) .
• the hybrid protease plasmids were therefore initially analysed with the aid of restriction enzyme analysis and then verified by a nucleotide sequence analysis at the relevant section of the plasmid DNA.
• the protease plasmids designated pNZ521a, pNZ521b and ⁇ NZ521c and also pGKV552a, pGKV552b and pGKV552c were constructed. Furthermore the fragment (c) of the SKll protease was introduced into the plasmid pGKV552a, and this resulted in the plasmid pGKV552ac, and fragments (b) and (c) of the SKll protease were introduced and this resulted in plasmid pGKV552abc.
• fragment (c) of the g2 protease was introduced in plasmid pNZ521a and this resulted in plasmid pNZ521ac.
• DNA fragments containing only sections of the fragment (c) of the Wg2 protease were exchanged in plasmid pNZ521; see Fig. 2.
• Fragment (cl) is a 1.45 kb EcoRI-BstEII fragment which corresponds to the amino acids 605-1089
• fragment (c2) is a 1.11 kb MStl-BstEII fragment which corresponds to the amino acids 721-1089
• fragment (c3) is a 0.75 kb Styl-BstEII fragment which corresponds to the amino acids 840-1089.
• the plasmids obtained were designated pNZ521cl, pNZ521c2 and pNZ521c3.
• DNA fragments containing only sections of fragment (a) of SKll protease were introduced in plasmid pGKV552c; see Fig.2.
• Fragment (al) is a 235 bp Sphl-BamHI fragment which corresponds to to the amino acids 97-173 and fragment (a2) is a 33 bp SauI-BamHI fragment which corresponds to amino acids 163-173•
• the plasmids obtained were designated pGKV552alc and pGKV552a2c.
• hybrid proteases The used nomen ⁇ clature for the hybrid proteases is as follows: wild-type SKll and Wg2 proteases are designated ABCD and abed respectively and hybrid proteases are indicated with the corresponding fragments of SKll and g2 protease, which are present therein; see Fig.3- The production of the functional protease, which was specified by each of these plasmids, was verified by analysis of the growth characteristics of L.lactis MGI363 strain containing these plasmids in milk.
• the obtained plasmids were designated (between the brackets the resulting mutation) : pNZ57 ⁇ (S443A), pNZ567 (K138N), pNZ573 (N166D), pNZ570 (R747 ), PNZ571 (K748T) and pNZ590 (V1759R) .
• the result of one of the mutations made in the protease gene was a deletion of 9 nucleotides coding for amino acids A137. KI38 and T139 of the L. lactis SKll protease.
• the obtained plasmid was designated pNZ560.
• This restriction site was then used to insert additional nucleotides at this position making use of synthetically-made 9 base pair double- strand adapter fragments.
• the result of some of these constructions is shown in fig. 5b.
• the obtained plasmids were designated (between brackets the resulting inserted amino acids) : pNZ562 (GDT) , PNZ563 (GLA), pNZ564 (GPP), pNZ565 (GSAGTTGTT) and pNZ566 (GKTGLAGKTGKTGKT) .
• pNZ562 GDT
• PNZ563 GLA
• pNZ564 GPP
• pNZ565 GSAGTTGTT
• pNZ566 GKTGLAGKTGKTGKT
• the isolation of the obtained mutant proteases The protease was isolated by releasing it from the cells, in principle specifically in the manner described by Exterkate, F.A. et al., Appl. Environ. Microbiol. _9_ (1985), pages 328-332.
• the L.lactis cells were cultured for 12-20 hours in a medium based on whey. Then the cells were collected by centrifuging and quickly washed once with "release" buffer (50 mM sodium acetate/ phosphate pH: 6.5) « The cells were suspended in l/50th of the original volume of the "release” buffer and incubated at 30°C while being shaken gently.
• the cells were again separated by centrifuging and the supernatant, which contained the protease released, was stored at 0°C.
• the cell pellet obtained on centrifuging was again suspended in "release” buffer and incubated at 30°C. Then the cells were again centrifuged, after which the two "release” fractions were combined and the pH was adjusted to a value of 5-4 with concentrated acetic acid.
• the protease isolated was placed in sample bottles and stored at -80 * C.
• the plasmids ⁇ NZ521 and pGKV552 which both contain the structural protease gene (prtP gene) and also the essential part of the prtM gene of L.lactis SKll or L.lactis Wg2 were used.
• prtP gene structural protease gene
• Fig. 2 three different DNA fragments designated (a), (b) and (c) as shown in Fig. 2 were exchanged between the two protease plasmids and this resulted in the plasmids pNZ521a, pNZ521b, pNZ521c, pGKV552a, pGKV552b and pGKV552c.
• pGKV552a was constructed viz. pGKV552ac and pGKV552abc which contain fragment (c) and both fragments (b) and (c) of the SKll protease gene respectively.
• a derivative of pNZ521a was constructed which also contained fragment (c) of the Wg2 protease gene and was designated pNZ521ac.
• derivatives of the plasmid pNZ521c were constructed which contained only a section of the fragment (c) of the Wg2 protease gene, and this resulted in the plasmids pNZ521cl, pNZ521c2 and pNZ521c3 and derivatives of plasmid ⁇ GKV552c were constructed which only contained a section of fragment (a) of the SKll protease gene and resulted in the plasmids pGKV552alc and pGKV552a2c (see Fig. 2) .
• a diagrammatic representation of the proteases which have been synthesized by the L.lactis strains containing the plasmids concerned is shown in Fig.3.
• the protease was isolated from the L.lactis strains by releasing the enzyme from the cell membrane by incubation in a Ca 2+ -free buffer. To determine the cleavage specificity of the proteases concerned, various quantities of all the separate protease preparations were incubated with ⁇ sl-, ⁇ - and k-casein and the casein degradation products obtained were separated on SDS-PAGE gels.
• An exchange of the fragment (b) and/or fragment (d) does not affect the cleavage specificity for ⁇ sl-casein. More particularly it is supposed that the protease which is specified by the plasmid pGKV552ac is the only hybrid derived from the Wg2 protease which is capable of efficiently cleaving the ⁇ sl-casein, comparable to the wild-type SKll protease.
• the abovementioned results indicate that at least one of the fragments (a) and (c) of the SKll protease is necessary to degrade the ⁇ sl casein and both fragments are necessary to efficiently degrade the ⁇ sl-casein.
• fragment (c) of the SKll protease is necessary to degrade ⁇ sl-casein since fragment (c) does not have any homology with subtilisin or other serine proteases which are in fact capable of degrading ⁇ sl-casein.
• the L.lactis SKll and Wg2 proteases are both capable of degrading ⁇ -casein, but with a markedly different specificity (see Visser, S. et al., Appl. Environ. Microbiol. 2 (1986), pages 1162-1166).
• the various hybrid proteases according to the invention were also capable of degrading ⁇ -casein.
• the fragments (a) and (c) both altered the cleavage specificity for ⁇ - casein (Figs. 4c and 4d) .
• Fragments (a) and (c) both affect the cleavage specificity of Lactococcus protease for ⁇ -casein, and fragment (b) has no effect.
• L.lactis MGI363 transformed with either pNZ521a or with pGKV552c yields hybrid proteases which both appear to have one and the same cleavage specificity.
• L.lactis MG1363 transformed with either pNZ521c or with pGKV552a Fig. 4c
• L.lactis MGI363 bacteria transformed with either pGKV552 or pGKV552b have an identical cleavage specificity.
• fragment (a) and fragment (c) are essential for determining the specificity and that, in the case of the hybrid proteases investigated, it is not important whether the other sections of the protein originate from strain SKll or Wg2.
• a combination of fragment (a) of the one protease with fragment (c) of the other protease results in a hybrid protease having a specificity which differs from both SKll and Wg2 proteases.
• fragment (a) corresponds to a sequence of 173 amino acids in which only 7 amino acid substitutions between the SKll protease and Wg2 protease have been found (see Table A) .
• fragment (c) corresponds to a region of 593 amino acids in which only 9 amino acid substitutions have been found between the SKll and Wg2 proteases (see Table A) .
• the ⁇ -casein degradation by the proteases specified by the plasmids pNZ521cl, c2 and c3 (see Fig. 3) was investigated in order to determine which of these substitutions affects the cleavage specificity.
• fragment (a) amino acid sequences which have a homology with the substrate binding region of serine proteases of the subtilisin family In this connection, the Applicant assumes that a similar role occurs for this region in the lactococcal proteases.
• fragment (c) also affects the cleavage specificity is surprising and makes it probable that, compared with the subtilisins, the lactococcal proteases contain additional elements for determining the cleavage specificity.
• the proteases described here therefore have two regions which are involved in the binding and cleavage of substrate molecules.
• One region is situated in fragment (a) after residue 130 and is homologous with the substrate binding region of serine proteases of the subtilisin family (Wells., J. et al., Proc. Natl. Acad. Sci. USA 84 (1987), pages 5167-5171; McPhalen, CA. , et al., Proc. Natl. Acad. Sci. USA 82 (1985), pages 7242-7246; Betzel, C. et al., Eur. J. Biochem. 128 (1988), pages 155-171) « The other region is situated at residues 77 and 7 ⁇ 8 in fragment (c); a corresponding (homologous) region is not, however, present in subtilisins or other serine proteases. Mutant proteases obtained by site-directed mutagenesis
• mutant proteases having new cleavage specificities can also be obtained by carrying out single amino acid substitutions. These substitutions then have to be carried out, in particular, in the regions which are essential for the cleavage specificity.
• amino acid serine 433 of L.lactis SKll protease was replaced by alanine, as a result of which the so- called S433A mutant SKll protease was obtained (Fig. 5a) .
• S433A mutant proved to be completely inactive, which bears out the hypothesis that S433 represents the serine residue of the active site triad.
• mutant proteases having altered properties can be obtained by omitting (so-called “deletion”) or introducing (so- called "insertion") one or more amino acids. If this deletion or insertion takes place in a region which is essential for tne cleavage specificity, mutant proteases can be obtained having novel catalytic properties.
• a mutant protease derived from L.lactis SKll protease was made in which amino acids A137.
• K138 and T139 are missing (Fig. 5b) .
• This / ⁇ 137 ⁇ 139 mutant SKll protease was also found to cleave ⁇ sl- and ⁇ -casein in a different manner from "wild-type" SKll or Wg2 protease (Fig. 6a and 6b).
• a new AccIII restriction enzyme cleavage site was also introduced into the DNA fragment, as a result of which it became possible to insert one or more codons at the site where the codons for residues 137" 139 have been omitted.
• mutant proteases derived from SKll proteases were made, the residues 137-139 (i.e. AKT) being replaced by the three residues GDT, GPP and GLA, resulting in the mutant proteases A137G/K138D, A137G/K138P/T139P and A137G/K138L/T139A respectively.
• mutant proteases termed ⁇ 137-139/ins9 and _ ⁇ 137-139/insl5, were made, the residues 137-139 (i.e. AKT) being replaced by 9 residues (i.e. GSAGTTGTT) and fifteen residues (i.e. GKTGLAGKTGKTGKT) , respectively (Fig. 5b) .
• Amino acid deletions or insertions can also be made in regions which determine properties of the protease other than cleavage specificity.
• a mutant protease derived from SKll protease termed / ⁇ 205 ⁇ 219/S, was made in which the segment containing amino acids E205-M219 was replaced by a single serine residue (Fig. 5b).
• This omitted segment contains, in particular, peptide bonds which were found to be sensitive to autoproteolysis, as a result of which the "wild-type" SKll protease may inactivate itself.
• these cleavage-sensitive peptide bonds are no longer present.
• the C-terminal section of L.lactis serine protease is responsible for binding to the cell membrane.
• the effect is of a mutation in the hydrophobic area of the C-terminal section which is completely or partially responsible for the anchoring in the cell membrane.
• the mutation V1759R was introduced in the L.lactis SKll protease, as a result of which the mutant V1759R protease was secreted almost completely into the medium.
• curtailment to 1272 amino acids still in fact yields an active and stable secreted protease
• curtailment to 1090 amino acids (BstEII site in Fig. 2) or 937 amino acids (Bglll site in Fig. 2) no longer yielded an active and/or stable protease.
• the invention relates, in particular, to DNA fragments which contain at least one mutant protease gene, which gene is made up of sections of protease genes of several lactococcal strains or of a protease gene of one lacto ⁇ coccal strain, the DNA sequence of which has been altered at one or more sites.
• a mutant protease gene is made up of at least sections of a protease gene, in particular a type I protease gene of L.lactis, more particularly of L.lactis subsp. cremoris Wg2 and a protease gene, in particular a type III protease gene of L.lactis, more particularly of L.lactis subsp. cremoris SKll.
• the invention also relates to DNA fragments which contain at least one hybrid protease gene which is made up of the type I protease gene or L.lactis subsp. cremoris Wg2, of which one or more codons coding for amino acids as shown in Table A, in particular the amino acids A62, T89, T131, TI38, Sl42, Ll44, D166, L747 and T748, have been altered into codons which code for the amino acids specified in Table A at corresponding sites in the type III protease gene of L.lactis subsp. cremoris SKll.
• the invention also relates to DNA fragments containing at least one hybrid protease gene which is made up of the type III protease gene of L.lactis subsp. cremoris SKll, of which one or more codons coding for amino acids as shown in Table A, in particular the amino acids V62, K89, S131, K138, A142, V144, N166, R747 and K748, have been altered into codons which code for the amino acids specified in Table A, at corresponding sites in the type I protease gene of L.lactis subsp cremoris Wg2.
• the invention relates to DNA fragments which contain at least one mutant protease gene which is made up of the type I protease gene of L.lactis subsp. cremoris Wg2, of which one or more codons coding for the amino acids as shown in Table A, in particular the amino acids A62, T89, T131, TI38, Sl42, Ll44, Dl66, L747 and/or T748, have been altered into codons which do not code for the amino acids specified in Table A at corresponding sites in the type III protease gene L.lactis subsp. cremoris SKll.
• the invention relates to DNA fragments which contain at least one mutant protease gene which is made up of the type III protease gene of L.lactis subsp. cremoris SKll, of which one or more codons coding for amino acids as shown in Table A, in particular the amino acids, V62, K89, S13L KI38, Al42, Vl44, N166, R747 and K748, have been changed into codons which do not code for the amino acids specified in Table A at corresponding sites in the type I protease gene of L.lactis subsp. cremoris Wg2.
• the invention also relates to DNA fragments which contain at least one mutant protease gene which is made up of the type I protease gene of L.lactis subsp. cremoris Wg2, of which one or more codons are missing, including, preferably, at least one codon coding for the amino acids A62, T89, T131, TI38, Sl42, Ll44, D166, L747. and/or T748, and/or to which one or more codons have been added.
• the invention also relates to DNA fragments which contain at least one mutant protease gene which is made up of the type III protease gene of L.lactis subsp cremoris SKll, of which, one or more codons are missing, including, preferably, at least one codon coding for the amino acids V ⁇ 2 , K89, S131, K138, Al42, Vl44, N166, R77 and/or K748, and/or to which one or more codons have been added.
• the invention also relates to the cloning vectors, including plasmids, which contain a DNA fragment according to the invention, to host cells, preferably lactococcal strains, in particular the L.lactis strain MGI363, which have been transformed with a cloning vector specified above, to the proteases obtained with such host cells and also to the foodstuffs, flavourings etc., which are obtained with the aid of said host cells or proteases.
• host cells preferably lactococcal strains, in particular the L.lactis strain MGI363
• the proteases obtained with such host cells and also to the foodstuffs, flavourings etc. which are obtained with the aid of said host cells or proteases.
• the invention is therefore based on providing new proteases and cultures contain ⁇ ing these proteases for preparing all kinds of products which can be prepared with the aid of lactic acid bacteria.
• the protein cleavage products formed in this manner are either used as such or further reacted.
• examples of such products are butter, margarines and other spreads for bread which contain fermented milk or skimmed milk in the aqueous phase.
• other fermented milk products such as butter milk and fermented skimmed milk, quark and other fresh and ripened types of cheeses and yoghurt and also
• flavouring mixtures are obtained by making use of the modified proteases according to the invention, while human or animal foodstuffs based on proteins other than milk proteins can also be prepared with the aid of the proteases according to the invention.
• An important embodiment of the invention is therefore the use of one or more of the mutant proteases described above or of cultures which contain one or more of said mutant proteases for preparing the products just specified which contain protein cleavage products.
• Fig. 1 Nucleotide sequence of the L. lactis SKll protease gene and the amino acid sequence of the protease derived there ⁇ from. The numberings of the nucleotides and amino acids are indicated: nucleotide position 1 is the first base of the start codon of the protease gene, while amino acid position 1 is the first amino acid (D) of the mature protease. In addition, the first 10 amino acids of the mature protease are underlined. The first 17 amino acids of the prtM protein which is coded upstream of the prtP gene in reverse order, starting with M, K, K, K, M, R etc. , are also indicated.
• Fig. 2 Structure of hybrid protease plasmids.
• the upper and lower diagrams from the top show the protease regions on plasmids pSKlll and pWV05 of, respectively, strain SKll and Wg2, with the prtM gene on the left and the prtP gene on the right.
• the blank bars indicate the coding regions of the prtP gene and the black bars indicate the coding regions of the prtM gene.
• the hatched bar indicates the duplication of l8 ⁇ bp which is present in the SKll prtP gen.
• the lengths are indicated in kb, position 0 being the initiation codon of the protease gene.
• each relevant restriction enzyme is indicated by: B, BamHI; Bg, Bglll; Bs, BstEII; C, Clal; E, EcoRI. H, Hindlll; K, Kpnl; M, Mstl; P, Pstl; RV, EcoRV; S, Saul; Sc; SacII; Sp, Sphl; St, Styl (not all Styl-sites are indicated).
• B BamHI
• Bg Bglll
• Bs, BstEII C
• Clal Clal
• E EcoRI. H, Hindlll
• K Kpnl
• M Mstl
• P Pstl
• RV EcoRV
• S Saul
• Sc SacII
• Sp Sphl
• St Styl
• protease gene which is present in the various plasmids is shown, with the plasmid code indicated on the left and the protease code indicated on the right, the sequences derived from SKll being shown as blank bars and the sequences derived from Wg2 as hatched bars.
• the "wild-type" SKll and Wg2 protease genes are present on plasmids pNZ521 and pGKV552, respectively.
• the DNA fragments are shown which were ex ⁇ changed between both protease genes (see Fig.2).
• the sizes of the mature protease coded by these plasmids are indicated in units of 100 amino acids.
• proteases and subtilisin are shown dia- grammatically at the bottom of the figure, with the pre-peptide (on the left) and the membrane anchor (on the right) dotted, the pro-peptide cross-hatched and the most homologous regions in black, which are around the active site amino acid residues aspartic acid (D) , histidine (H) and serine (S) .
• the horizontal hatched regions, further indicated with B, represent the substrate binding region in subtilisin and the corresponding area in the L.lactis protease.
• the vertical strokes above the lactococcal protease indicate approximately the position of the amino acid differences between the proteases of strain SKll and strain Wg2 (see Table A).
• the blank square indicates the position of the deletion of 60 amino acids in Wg2 protease in comparison with SKll protease.
• Fig. 4a SDS polyacrylamide gel patterns of ⁇ sl-casein incubated with release fractions of "wild-type” and hybrid protease- containing L. Lactis strains
• Lanes 1 and 10 contain marker proteins of ovalbumin, carbonic anhydrase, ⁇ -lactoglobulin, lysozyme and bovine trypsin inhibitor having molecular weights of, respectively, 43, 29, 18.4, 14.3 and 6.2 kilodaltons.
• Lane 9 contains ⁇ sl-casein not incubated with release fraction.
• Lanes 2 to 8 inclusive contain ⁇ sl-casein incubated with release fractions of L.lactis MGI363 containing, respectively, plasmids pGKV552c, pGKV552a, pNZ521c, pNZ521b, ⁇ NZ521a, pGKV552 and pNZ521.
• Fig. 4b SDS-polyacrylamide gel patterns of ⁇ sl-casein incubated with release fractions of "wild-type" and hybrid protease containing L.lactis strains.
• Lanes 1 to 9 contain ⁇ sl-casein incubated with release fractions of L.lactis MG1363 containing, respectively, plasmids pNZ521, pGKV552, pGKV552a, pGKV552b, pGKV552c, pGKV552ac, pGKV552abc, pGKV552alc and pGKV552a2c.
• Fig. 4c SDS-polyacrylamide gel patterns of ⁇ -casein incubated with release fractions of "wild-type” and hybrid protease- containing L.lactis strains.
• Lane 1 contains ⁇ -casein not incubated with a release fraction.
• Lanes 2 to 7 contain ⁇ -casein incubated with release fractions of L.lactis MGI363 containing, respectively, plasmids pNZ521, pNZ521a, pNZ521c, pGKV552, pGKV552a and pGKV552c.
• Fig. 4d SDS-polyacrylamide gel patterns of ⁇ -casein incubated with release fractions of "wild-type” and hybrid protease- containing L.lactis strains.
• Lane 1 contains ⁇ -casein incubated with release fraction of the plasmid-free, protease-deficient strain L.lactis MGI363.
• Lanes 2 to 9 inclusive contain ⁇ -casein incubated with the release fractions of L.lactis MGI363 containing, respectively, the plasmids pNZ521, pGKV552, pNZ521a, pNZ521b, pNZ521c, pNZ521cl, pNZ521c2 and pNZ521c3-
• Figs. 5a and 5b Overview of the oligonucleotides used in the site- directed mutagenesis of the L.lactis SKll protease gene.
• the "wild-type" L.lactis SKll nucleotide sequence is in each case indicated in the top line with the amino sequence above it and its position in the protease.
• the nucleotide sequence of the oligonucleotide used and the amino acid sequence resulting therefrom is in each case shown underneath.
• the altered nucleotides are indicated by *.
• the two nucleotides on either side of the deletion concerned are connected by a line.
• the inserted double-strand oligonucleot ⁇ ide concerned and the altered amino acid sequence resulting there ⁇ from are shown. The nomenclature of mutants is given at the left.
• Fig. 6a SDS-polyacrylamide gel patterns of ⁇ sl-casein incubated with release fractions of "wild-type” and mutant protease containing L.lactis strains.
• Lanes 1 to 10 contain ⁇ sl-casein incubated with release fractions of L.lactis MGI363 containing respectively plasmids
• PNZ567 K138N
• pNZ573 N166D
• pNZ521 wild type
• pNZ562 GDT
• pNZ564 GPP
• pNZ563 GLA
• pNZ56 ⁇ / ⁇ 137--39
• PNZ571 K748T
• PNZ565 ins9
• pNZ566 insl5
• Fig. 6b SDS-polyacrylamide gel patterns of ⁇ -casein incubated with release fractions of "wild-type” and mutant protease containing L.lactis strains.
• Lanes 1 to 10 contain ⁇ -casein incubated with release fractions of L.lactis MGI363 containing respectively plasmids pNZ567 (K138N), PNZ573 (N166D), pNZ521 (wild type), pNZ562 (GDT), PNZ564 (GPP), pNZ563 (GLA), P NZ56 ⁇ (/ ⁇ 137-_39) .
• PNZ571 (K748T) PNZ565 (ins9) and pNZ566 (insl5).

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