Protease for wound conditioning and skin care

Abstract

We have identified by molecular cloning a protease which originates from the larvae of Lucilia sericata and which was termed debrilase due to its activities useful for debridement of wounds. Described is a nucleic acid molecule encoding a serine protease having the ability to cleave fibrin and casein which is (a) a nucleic acid molecule encoding the serine protease comprising or consisting of the amino acid sequence of SEQ ID NO: 4 as well as to nucleic acid molecules encoding precursors or fragments of said serine protease; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 3; (c) a nucleic acid molecule encoding a serine protease the amino acid sequence of which is at least 80% identical to the amino acid sequence of (a), preferably at least 85% identical, more preferably at least 90% identical, and most preferred 95% identical; (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 80% identical to the nucleotide sequence of (b), preferably at least 85% identical, more preferably at least 90% identical, and most preferred 95% identical; (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (b) or (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U.

Claims

1. An isolated nucleic acid molecule encoding (i) a serine protease having the ability to cleave fibrin and casein, said serine protease encoded by (a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 4; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 3; (c) a nucleic acid molecule encoding the amino acid sequence which is at least 90% identical to the amino acid sequence of (a); (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence at least 90% identical to the nucleotide sequence of (b); (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U.Iadd., wherein the coding sequence of the isolated nucleic acid molecule is operably linked to a heterologous promoter.Iaddend..

2. A vector encoding the nucleic acid molecule of claim 1.

3. An isolated host cell transformed, transduced or transfected with the vector of claim 2.

4. A method of producing a serine protease comprising culturing the host cell of claim 3 and isolating the serine protease, the propeptide or the pre-propeptide produced.

5. A serine protease, propeptide, or pre-propeptide encoded by the nucleic acid molecule of claim 1 or produced by the method of claim 4.Iadd., wherein the serine protease, propeptide, or pre-propeptide is provided in a pharmaceutical composition with a pharmaceutically acceptable sterile carrier.Iaddend..

6. A fusion protein comprising the serine protease, the propeptide, or the pre-propeptide of claim 5.Iadd., wherein the fusion protein is a pre-propeptide having a heterologous signal sequence.Iaddend..

7. A composition comprising the nucleic acid of claim 1, the vector of claim 2, or the host cell of claim 3 or combinations thereof.

8. The composition of claim 7 which is a cosmetic composition.

9. The composition of claim 7 which is a pharmaceutical composition.

10. A method for treatment of skin peeling, skin smoothening or the intervention with scar formation, comprising contacting skin with the nucleic acid of claim 1, the vector of claim 2, or the host cell of claim 3.

11. A method for treatment of wounds, comprising contacting a wound with the nucleic acid of claim 1, the vector of claim 2, or the host cell of claim 3.

12. The method of claim 11 wherein the wounds are chronic or slow healing wounds.

13. A method for treatment of skin diseases accompanied by impaired wound healing comprising contacting a skin disease with the nucleic acid of claim 1, the vector of claim 2, or the host cell of claim 3.

14. The method claim 10, additionally comprising administering at least one component selected from the group of a further protease, nuclease, excipient, anti-microbial agent and pain-relieving agent.

15. The nucleic acid molecule of claim 1, wherein the nucleic acid encodes a propeptide of the serine protease of (i), and the propeptide is encoded by a nucleic acid molecule selected from: (a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 6; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 5; (c) a nucleic acid molecule encoding the amino acid sequence of which is at least 90% identical to the amino acid sequence of (a); (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 90% identical to the nucleotide sequence of (b); (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U.

16. The nucleic acid molecule of claim 1, wherein the nucleic acid encodes a pre-propeptide of the serine protease of (i), wherein the pre-propeptide is encoded by a nucleic acid molecule selected from (a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 1; (c) a nucleic acid molecule encoding the amino acid sequence which is at least 90% identical to the amino acid sequence of (a); (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence at least 90% identical to the nucleotide sequence of (b); (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U.

17. The method of claim 11, additionally comprising administering at least one component selected from the group of a further protease, nuclease, excipient, anti-microbial agent and pain-relieving agent.

18. The method of claim 12, additionally comprising administering at least one component selected from the group of a further protease, nuclease, excipient, anti-microbial agent and pain-relieving agent.

.Iadd.19. The isolated nucleic acid molecule of claim 1, wherein the heterologous promoter is a yeast promoter..Iaddend.

.Iadd.20. The isolated nucleic acid molecule of claim 19, wherein the heterologous promoter is a methanol inducible promoter..Iaddend.

.Iadd.21. The isolated nucleic acid molecule of claim 19, wherein the heterologous promoter is a yeast promoter selected from an AOX1 promoter and a GAL1 promoter..Iaddend.

.Iadd.22. The isolated nucleic acid molecule of claim 1, wherein the serine protease is in the form of a propeptide or pre-propeptide..Iaddend.

.Iadd.23. The isolated host cell of claim 3, wherein the isolated host cell is a yeast cell..Iaddend.

.Iadd.24. The isolated host cell of claim 23, wherein the yeast cell is of a species selected from Saccharomyces cerevisiae, Hansenula polymorpha and Pichia sp..Iaddend.

.Iadd.25. The isolated host cell of claim 24, wherein the yeast cell is a Pichia pastoris cell..Iaddend.

.Iadd.26. The serine protease, propeptide, or pre-propeptide of claim 5 which is a propeptide..Iaddend.

.Iadd.27. The serine protease, propeptide, or pre-propeptide of claim 5 which is a propeptide of SEQ ID NO. 6..Iaddend.

.Iadd.28. The propeptide of claim 27, wherein the pharmaceutical composition is suitable for topical administration..Iaddend.

.Iadd.29. The propeptide of claim 27, wherein the pharmaceutical composition is a gel..Iaddend.

.Iadd.30. The propeptide of claim 29 wherein the pharmaceutical composition comprises at least one gel-forming agent selected from cellulose derivatives, vinyl polymers, and carboxypoly-methylene derivatives..Iaddend.

.Iadd.31. The propeptide of claim 30 wherein the pharmaceutical composition comprises at least one gel-forming agent selected from as methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, polyvinyl alcohols, polyvinyl pyrrolidone, and carbopoline..Iaddend.

.Iadd.32. The propeptide of claim 29 wherein the pharmaceutical composition comprises at least one gel-forming agent selected from pectins, gums, alginates, agar and gelatin..Iaddend.

.Iadd.33. The propeptide of claim 29 wherein the pharmaceutical composition comprises one or more auxiliary agents selected from preservatives, antioxidants, stabilizers, colorants and perfumes..Iaddend.

.Iadd.34. The fusion protein of claim 6, wherein the heterologous sequence is a yeast signal sequence..Iaddend.

.Iadd.35. The fusion protein of claim 34, wherein the pre-propeptide comprises the debrilase propeptide sequence of SEQ ID No. 6 and wherein the native signal peptide of debrilase (MFRFVALFAFVSCALA) (SEQ. ID. NO 7) is substituted by the vector encoded-factor signal sequence from Saccharomyces cerevisiae..Iaddend.

Description

(1) The figures show:

(2) FIG. 1: Diffusion assay on agar containing casein showing a clearing zone around a debrilase secreting clone of E. coli.

(3) FIG. 2: Activity assay in agar containing fibrin showing a clearing zone around debrilase active sample A) debrilase positive clone growing on fibrin containing substrate, B) agar diffusion assay with crude extracts from a debrilase-positive recombinant E. coli clone: (a) undiluted crude extract b) 1:10 dilution in PBS c) 1:50 dilution in PBS.

(4) FIG. 3: A) enzyme assay for identifying the activity profile of recombinant debrilase by use of known serine protease (PMSF) and trypsin like serine protease (APMSF) inhibitors; B) activity assay with debrilase in puffers of different pH-values to identify the pH-profile of recombinant debrilase.

(5) FIG. 4: Activation of PAR 2 in a cell based receptor assay with HEK232 cells with Ca.sup.2+-flux as read-out.

(6) FIG. 5: Alignment of the pre-propeptide amino acid of debrilase (A) (SEQ. ID. NO:2) and cDNA nucleotide sequence (B) (SEQ. ID. NO:1) with the nearest neighbour sequence being the trypsin like protein from Sarcophaga bullata (SEQ. ID. NOs:9 and 10) showing 76% identity for nucleotide and protein sequence using NCBI BLAST algorithm. (A) The signal peptide of debrilase consists of amino acids 1 to 16 (the signal peptide is shown bold), the propeptide consists of amino acids 17 to 254 (propeptidic amino acids 17 to 26 are underlined) and the proteolitically processed mature polypeptide of debrilase consists of amino acids 27 to 254.

(7) FIG. 6: (A) SDS PAGE of culture supernatant: 1) after centrifugation; 2) pre-filtration [0.45 m]; 3) sterile filtration [0.22 m], MW of the propeptide 25.7 kDa. (B) Visualisation of activated mature Debrilase; 5) after dialysis and subsequent pH shift from 5.5 to 8; 6) affinity chromatography column flow-through (unbound protein); 7) wash flow through, (8 to 12) fractionated elution 1 to 5, respectively (molecular weight of the mature protein 24.6 kDa).

(8) The examples illustrate the invention.

EXAMPLES

General Methods and Materials

(9) Isolation of mRNA of Different Larval Stases from Lucilia sericata

(10) 2 days old maggots from the green bottlefly (Lucilia sericata) were purchased from BioMonde (BioMonde, 22885 Barsbttel, Germany). Isolation of total RNA was done after feeding for 24 hours (second instar stage) on blood-agar (1.5% (w/v) agar containing 10% bovine blood) to enhance production of proteases and after 3 days when the production of enzymes is markedly decreased in the third instar stage (Chambers et al. 2000, Degradation of extracellular matrix components by defined proteinases from the greenbottle larva Lucilia sericata used for the clinical debridement of non-healing wounds, Brit J Dermatol. 148: 14-23). For isolation of total-RNA 3 larvae (ca. 30 mg) were shock frozen with liquid nitrogen and pulverised mechanically. RNA was isolated using the kit Total RNA Isolation, NucleoSpin RNA II (purchased from Macherey-Nagel, 52313 Duren, Germany) yielding 47 g total RNA.

(11) Generation of an E. coli cDNA Library from Total RNA Isolated from Larvae of Lucilia sericata

(12) The generation of a cDNA-library from said Lucilia sericata RNA was accomplished according to the SMART cDNA Library Construction Kit User Manual (CLONTECH Laboratories, Inc.). In vitro packaging of the final ligation reaction and subsequent transformation in E. coli XL-1 Blue resulted in 1.510E6 primary plaque forming units.

(13) Primary phages were harvested and stored in phage stabilization buffer containing 7% DMSO at 80 C. for subsequent infection and mass excision in E. coli.

(14) Conversion of the lambda phage library into the corresponding plasmid library was performed by Cre recombinase-mediated mass excision of the phage embedded plasmids according to the SMART cDNA Library Construction Kit User Manual (CLONTECH Laboratories, Inc.).

(15) Heterogeneity and Quality of the Library was Tested by Restriction Analysis

(16) Identification of Proteases from Lydiai Sericata cDNA-Library

(17) Colony forming units resulted from mass excision described above were screened on agar media containing 2% skim milk under selective conditions. Expression of heterologous proteases was driven by the vector comprising inducible P.sub.lac promoter. Colonies expressing heterologous proteases were detected by the formation of clearing zones in the turbid Skim Milk medium around the colonies.

(18) Isolation and Purification of Debrilase Protein from E. Coli Culture Expression Culture

(19) In order to obtain enzyme samples of the debrilase containing sufficient enzymatic activity for a characterisation of the enzyme either the cDNA clone expressing the corresponding protease or a more suited expression construct set up in a typical expression vector like e.g the pET26b-vector (Novagen) and a suitable expression host like e.g. E. coli Rosetta (DE3) (Novagen) were used. For the construction of the expression constructs, the corresponding debrilase genes were PCR amplified to introduce unique restriction enzyme recognition sequences upstream and downstream of the open reading frame (ORF) which allowed to ligate the genes encoding the protease with the expression vector e.g. pET26b in a definite way. The restriction enzyme recognition sequences were chosen on the basis of their absence in the coding region of the debrilase gene and could be e.g. NdeI, HindIII, EcoRI, XhoI. The absence of unwanted second site mutations due to erroneous amplification by the polymerase was confirmed by sequencing of the cloned amplicon.

(20) The cDNA clones or the expression constructs were used to inoculate e.g. 200 ml of culture medium complemented with the appropriate antibiotic in a 1 l Erlenmayer flask. LB-medium and antibiotics in the following concentration were used: 100 g/ml ampicillin, 25 g/ml kanamycin, chloroamphenicol 12.5 g/ml. The initial optical density (OD.sub.580) was adjusted to 0.05 and the cells grown at a temperature of 28 C. on a gyratory shaker. When the optical density reached the value of about 1 the expression from the lac-promoter of pTriplEx2 (Clontech) or from the T7-promoter of vectors from the pET-vector series e.g. pET26 was induced by addition of IPTG in the concentration of 20 M-500 M. Cells were harvested 4 to 20 h after induction by centrifugation. The cell sediment was resuspended in 5 ml 1PBS, pH 7.0 and the cells disrupted by ultrasonication.

(21) The molecular weight of debrilase is 27.4 kD for the complete protein including signal sequence, 25.7 for the hypothetical pro-peptide and 24.6 for the mature protein.

(22) Solid Phase Activity Assay for Fibrinolytic Activity

(23) To identify fibrinolytic activity in extracts of casein degrading clones a solid agar assay was used. For this, a solution A containing 200 mg agarose, 87 mg NaCl and 3 mg CaCl.sub.2 were added to 10 ml 0.1 M Tris pH7.4 and incubated at 50 C. To a second solution B containing 10 ml 0.9% NaCl and 100 mg Fibrin (Sigma F3879), 20 l Plasminogen (2 units/mg, Sigma P7397) e added and pre-incubated at 50 C. Both solutions were combined with 12 l Thrombin (175-350 NIH units, Sigma T4265) and spread on agar plates using micro-well forming devices. In case of a fibrinolytic activity a clear zone appears in the opaque medium around extract containing cavities.

(24) To identify fibrinolytic activity in casein degrading clones a solid agar assay was used. Therefore a medium A consisting of 10 ml 2 Luria Bertani containing 200 mg agarose, 87 mg NaCl and 3 mg CaCl.sub.2 was incubated at 50 C. To a solution B containing 10 ml 0.9% NaCl and 100 mg Fibrin (Sigma F3879), 20 l plasminogen (2 units/mg, Sigma P7397) was added and pre-incubated at 50 C. Both solutions were combined with 12 l thrombin (175-350 NIH units, Sigma T4265) and appropriate antibiotics for selection and poured on agar plates under sterile conditions forming a opaque agar medium. In case of fibrinolytic activity expressed by heterologous clones a clear zone appears around the colony after over night incubation.

(25) Liquid Phase Activity Assay and Inhibitory Profiling

(26) Quantification of proteolytic activity was conducted by a fluorescence assay in 96-well plates with BODIPY-FL-Casein (Molecular Probes) used as substrate. Hereby, on the undigested substrate casein the labelling molecules are in close contact to each other and therefore fluorescence is suppressed by a quenching mechanism. In case of hydrolysis these molecules separate from each other and fluorescence can be excited at 485 nm and measured at 520 nm.

(27) A standard assay contained 5 g/ml BODIPY-FL-Casein in 100 l PBS buffer and a series of dilutions of protease extract. Samples were incubated for 60 min at 37 C. and fluorescence was measured using a spectrophotometer (NovoStar, BMG LABTECH, Offenburg, Germany), applying the following parameters: excitation 485 nm, emission 520 nm, gain 5, one cycle, 10 flashes. Serine protease inhibitors APMSF (4-amidinophenylmethanesulfonyl fluoride) and PMSF (phenylmethanesulfonyl fluoride) were used for testing the specificity of the isolated protease. The following assay concentration were used:

(28) TABLE-US-00001 PMSF = serine protease inhibitor 5 mmol/l APMSF = trypsin like serine protease inhibitor 1 mmol/l

(29) The Sequence Listing submitted herewith electronically is incorporated herein by reference.

(30) The following examples are illustrative, but not limiting the scope of the present invention. Reasonable variations, such as those occur to the person skilled in the art, can be made herein without departing from the scope of the present invention.

(31) Pichia pastoris Expression Culture

(32) Heterologous expression of the serine protease of the invention, i.e. debrilase was carried out in the methylotrophic yeast Pichia pastoris, which is classified as a GRAS-organism by the Food and Drug Administration and is therefore established for pharmaceutical production processes. For expression an integrative vector system was used, providing a methanol inducible promoter. The native signal peptide of debrilase (MFRFVALFAFVSCALA) (SEQ. ID. NO:7) was substituted by the vector encoded-factor signal sequence from Saccharomyces cerevisiae to facilitate efficient secretion in Pichia.

(33) Fermentation was carried out in mineral medium with glycerol as sole carbon source in the first batch phase, fed batch mode was started after consumption of initial batch glycerol. Induction of heterologous expression was initiated by supplementation of methanol as inducer/carbon source and sorbitol as additional non-inhibiting feed during gene expression.

(34) Stationary growth of the culture was reached after batch/fed-batch (FB) phase leading to an Optical Density of about 600. Induction was initiated by adding MeOH. To prevent heterologous enzyme from being activated autocatalytically at this point, process temperature was decreased from 30 C. to 20 C. Expression of debrilase was supported in the end phase of the process by feeding. Autocatalytic activation of the propeptide was achieved by increasing the pH of the culture medium from 5.5 to 6.8, during the purification procedure (see below).

(35) Purification of Debrilase Protein from Pichia pastoris Culture Expression Culture

(36) Purification was carried out by affinity chromatography using serine protease inhibitor benzamidine coupled to sepharose carrier material (GE Healthcare). After removal of unbound protein purified the serine protease of the invention was harvested by competitive elution using buffer with addition of free benzamidine. SDS PAGE of the downstream procedure is visualized in FIG. 6. After subsequent dialysis against citrate storage buffer the purified protein was lyophilized and displayed as white dry powder.

(37) N-Terminal Sequencing of Recombinantly Expressed Debrilase

(38) For Edman sequencing the blot was fitted into the sample preparation cartridge. For determination of the amino acid sequence the protein sequencer Procise 492 (Applied Biosystems) was used. Reagents and protocols were applied as advised by the manufacturer. The resulting chromatograms were analysed using appropriate software (Applied Biosystems). Prior to each sample a standard sample and a blank were run.

Example 1

Screening for Proteases in an Expression Library Generated from Lucilia sericata

(39) A phage library of 810E6 primary clones was screened for the expression of proteases. For this end, the phage library was transferred into a plasmid library by co-infection of E. coli with a f1 type helper phage according to the SMART cDNA Library Construction Kit User Manual (CLONTECH Laboratories, Inc.). The resulting colonies harbour the intrinsic plasmids excised from the phage vector.

(40) The heterogeneity of the plasmid library was tested by isolation of forty plasmids from representative clones. In fact, every plasmid harboured an insert and these inserts showed complete diversity in size. In total, 310E5 cfu were screened on solid media containing 2% skim milk under selective conditions.

(41) By isolation and sequencing of plasmid DNA from sixteen of these halo-forming colonies a pre-propeptide sequence of a protease with 76% identity on the amino acid level to a trypsin-like enzyme from Sarcophaga bullata was identified (SEQ ID NO: 2).

Example 2

Characterisation of a Fibrinolytic Protease

(42) The identification of fibrinolytic activity was performed in agar plate assays described above. Colonies expressing fibrinolytic activity were identified by streaking out recombinant cells on nutrient agar containing turbid fibrin substrate. Cells harbouring plasmids with the identified protease showed clear zones around the colony after over night incubation at 37 C.

(43) Fibrinolytic activity in cell free crude extracts was determined using buffered agar medium containing fibrin. Plates contained wells with a capacity of up to 200 l which were formed using microplate devices during solification of the agar medium. Recombinant cells were sonicated and the resulting extract was centrifuged, places into the agar wells and incubated over night at 37 C. Crude extract with fibrinolytic activity was detected by a clear zone around microwells of the recombinant clone harbouring the protease gene of SEQ ID NO. 1.

Example 3

Profiling of a Fibrinolytic Protease

(44) The recombinant protease type was identified in a liquid assay in a 96-well plate using inhibitors specific for different types of proteases. Herein, specific inhibitors for trypsin (4-amidinophenylmethyl-sulphonyl fluoride, APMSF), serine (phenylmethylsulphonyl fluoride, PMSF), aspartyl-(pepstatin A) and metallo-type proteases (1,10-phenanthroline) were measured. As shown in FIG. 3 A, the inhibitor-profile of the recombinant clone suggests a trypsin type activity, which corresponds to comparative amino acid sequence alignment data of SEQ ID NO: 2 with the BLAST data base, showing the closest homology being 76% to an enzyme from Sarcophaga bullata (FIG. 5).

Example 4

Stability at Different pH-Values

(45) Activity of the debrilase was measured in liquid assay using appropriate buffer systems in the range of 3-10 (0.1 to 0.2 M citric acid buffer, pH 3-7; 0.05 M tris buffer, pH 8; 0.1 M carbonate buffer, pH 9-10) and the fluorogenic substrate Z-Gly-Gly-Arg-AMC. The release of 7-amino-4-methylcoumarin (AMC) was measured with a BMC Novostar Fluorometer (.sub.excitation 365 nm, .sub.emission 440 nm). The results are shown in FIG. 3 B. Debrilase exhibits enzymatic activity in a pH-range of 5-10 which corresponds to the similarly wide pH-range of the wound milieu.

Example 5

Activation of PAR 2 (Calcium Imaging)

(46) The activity of debrilase as agonist of PAR 2 was monitored over time in a cell based fluorescence assay by use of a wild type HEK293 cell line endogenously expressing human PAR2. In brief, 1 day prior to performing the assay, HEK293 cells expressing human PAR2 were plated onto 96-well, black-walled, assay plates, at a density of 45,000 cells per well. Using a 96-well microplate reader (FlexStation, Molecular Devices, Sunnyvale, Calif.), the change of the cellular calcium concentration was monitored by use of the calcium sensitive fluorescent dye fluo-4 (excitation 494 nm, emission 516 nm). The PAR 2 agonist trypsin (8 nM) was used as positive control. The dye-loaded cells in plates were placed into the fluorescence microplate reader to monitor fluorescence (excitation 488 nm, emission 520 nm) change after the addition of 50 l assay buffer (118 mM NaCl; 4.7 mM KCl; 1.2 mM MgSO.sub.4; 1.2 mM KH.sub.2PO.sub.4 4.2 mM NaHCO.sub.2; 1.3 mM CaCl.sub.2; 10 mM HEPES (pH: 7.4)) supplemented with an agonist. Calcium mobilization was quantified as the change of peak fluorescence (F) over the baseline level (F). Data were expressed as the mean S.E. of the F/F value (=RFU, relative fluorescence units) of replicated independent samples. The analysis was done with the software of the FlexStation.

Example 6

The Sequence of the First 10 Amino Acids of the Mature Debrilase

(47) The aim of the example is to determine the sequence of the first 10 amino acids by N-terminal Edman sequencing. The sample (protein from FIG. 6 B, lane 5) was successfully sequenced from the N-terminus. The following major sequence was detected:

(48) IVNGVDTTIQ (SEQ. ID. NO:8)

(49) which corresponds to the mature protein proposed by analysis of the primary sequence (FIG. 5A).

Example 7

Molecular and Enzymatic Properties of Debrilase

(50) TABLE-US-00002 TABLE 1 Summary of molecular and enzymatic properties of Debrilase molecular weight: 27.4 kDa (pre-pro-peptide), 25.7 kDa (pro-peptide), 24.6 kDa (mature protein) K.sub.m: 0.07 mM* K.sub.cat: 37.5 .Math. s.sup.1 specific activity: 90 mol* .Math. min.sup.1 .Math. mg.sup.1 enzyme pH.sub.opt: 8 T.sub.opt: 37 C. *measured in fluorometric assay using Z-Gly-Gly-Arg-AMC as substrate