CN1405305A - Efficient, broad-spectrum keratinase and its gene - Google Patents

Abstract

The invention provides a keratinase and its coding gene. It has advantages of high activity, good heat stability, wide PH range and efficient degradation of alpha & beta-keratinase. It has also the character of common protease and can degrade many kinds of protein derived from plants and animals. It can be widely used in the trades such as feed, foods, washing, wool spinning and pharmacy.

Description

A kind of efficient, broad-spectrum keratinase and gene thereof

Technical field

The present invention relates to a kind of M-Zyme and gene thereof.

Technical background

M-Zyme is the enzyme of class energy hydrolysis of keratin, and some M-Zyme has hydrolytic action to the multiple proteins except that Keratin sulfate simultaneously.Keratic feature is highly interconnected and form with the halfcystine disulfide linkage, water insoluble, salt, diluted acid and diluted alkaline, can not be degraded by general proteolytic enzyme.Keratin sulfate is the major ingredient of animal feather, hair, hoof, extensively exists in the skin of humans and animals, scar, scab.

M-Zyme can be applicable to fodder industry, and feather, pig hair the like waste are converted into high nutrient fodder albumen.Keratin sulfate is the main ingredient in the waste-feather that produces in the bird course of processing, protein content is up to more than 90% in the feather meal, essential amino acids content is high, it is the good protein source in the animal-feed, but it is highly interconnected and form with the halfcystine disulfide linkage, water insoluble, can not be degraded by general proteolytic enzyme, thereby it is difficult in animal body by digestibility and utilization.How the feather fermented product being converted into the available protein source of animal is present problem demanding prompt solution.Handled with the method for boiling or chemistry in the past, and made nutritive substance by considerable damage on the one hand, also caused environmental pollution on the other hand.Utilize M-Zyme to address this problem effectively, its advantage is more economical, and the product nutritive value is good simultaneously, and pollution-free.The feather waste material is very abundant, only at the feather waste in 1 year in Corolla state, U.S. north just above 100,000 tons, China is simple stomach livestock and poultry cultivation big country, annual feather waste is above 600,000 tons, this part good protein source fails to be utilized effectively at present, the wasting of resources and contaminate environment have been caused, and China is the country of the serious scarcity of feed protein resource, according to China's feed industrial development planning, breach to feedstuff protein in 2010 will reach 8,000,000 tons, and effective development and use of feather waste have great significance to China this protein resource country that there is a serious shortage in the supply.

M-Zyme also can be applicable to foodstuffs industry, is amino acid and small peptide as making keratin degrading, as the trophology medicine; Be used for producing high-nutrition foods such as fish sauce, meat peptone, or as the tenderization agent.In fur processing industry, its alternative common proteolytic enzyme or be used with common proteolytic enzyme improves treatment effect and also reduces processing cost.In detergent industry, can be used for enzyme-containing detergent.In beauty and health care, M-Zyme solubilized skin surface scurf and blackspot make skin softness, smooth, and can remove dandruff, softening hair and hair etc.Pharmaceutically, M-Zyme can impel wound healing, remove scab and epithelium regeneration, is the specifics of surgical operation and skin burn, wound, also is the specifics of treatment homocystinuria.

The research work of M-Zyme has the history of decades, and the many microorganisms of occurring in nature all can secrete M-Zyme, and the fungi of existing multiple product M-Zyme is separated with bacterium both at home and abroad at present, domestic as aspergillus (Zhang Daohai, biotechnology, 4:11-14,1994; Luo Wen etc., microbiology circular, 20:209～212,1993), streptomycete (Tu Guoquan, Agricultural University Of Jiangxi's journal, 16:399～403,1994; Ding Zhengmin etc., microorganism journal, 33:227～232,1993) etc.The M-Zyme that abroad is separated to produces bacterium and also mainly concentrates on streptomycete and the fungi, as streptomyces fradiae (Noval, J Bacteriol, 77:251,1959), (Malviya, Indian J Experiment Biol.30:103,1992) such as fungi Cylindrocarpon, Graphium and Microsporum.The M-Zyme that produces has also been carried out preliminary zymologic property research, but because the enzyme major part that these bacterial strains produce is not carried out purifying, can not determine these bacterial strains decompose keratic ability be realize by a kind of enzyme or finish by the mixing protease that comprises M-Zyme.

(Appl Environ Microbiol such as U.S. Lin in 1992,58:3271,1992) be separated to the genus bacillus PWD-1 that M-Zyme is produced in a strain, and the M-Zyme that is produced separation and purification and zymologic property research have been carried out, find that this M-Zyme need not other proteolytic enzyme and cooperates the Keratin sulfate of just degrading, its molecular weight is about 30kD, optimal pH is 7.5, specific activity is the 5990U/mg zymoprotein, enzyme has self-catalytic pyrolysis effect, less stable, the transformation period at room temperature only has 4～5 days.It goes back the multiple animal/vegetable protein of degradable except the Keratin sulfate of degrading.

Up to the present, the commercialization production and the practical application widely of M-Zyme are not arranged also both at home and abroad, its most important reason is: 1. the output of M-Zyme is too low in the natural bacterial strain, makes production cost high, is difficult to commercialization production; 2. the validity of the M-Zyme that is separated to is good inadequately, effect slowly, as need a couple of days and even tens of days feather bar complete hydrolysis could have been limited its practical application.The screening of efficient M-Zyme with separates, the validity of the various M-Zymes that are separated to both at home and abroad at present is all unsatisfactory, is abroad also attempting carrying out the validity of molecular modification with the raising enzyme by protein engineering, but is not also making a breakthrough.

Recognized that both at home and abroad engineered method is expected to the unit output that first mate's degree improves M-Zyme.Abroad attempt in recent years, but up to the present, only be isolated and cloned into a M-Zyme encoding gene (Shih, US Patent 5712147,1998 abroad; Wo Patent95/33056), this gene source is in Bacillus licheniformis PWD-1, structure gene total length 1140bp, 379 amino acid of encoding.Successfully this gene was expressed in recombined bacillus subtilis in 1997, the M-Zyme of expressing has normal biologic activity, its M-Zyme expression amount of recon the highest on the highest shaking table level has improved 3 times of (Lin than original strain, J IndMicrobiol Biotechnol, 19:134,1997), laboratory ferment to recombined bacillus subtilis studies show that, the expression amount of M-Zyme reaches 300～500U/mL fermented liquid (Wang, JInd Microbiol Biotechnol, 22:608～616,1999).Because the bacillus subtilis genetic engineering bacterial strain that makes up is also lower than original natural bacterial strain unit output, there is autolysis in this M-Zyme simultaneously, and is extremely unstable, therefore also do not reach the requirement of scale operation.Though domesticly carry out the M-Zyme research work early, all concentrate on the strains separation, also do not isolate a M-Zyme encoding gene at present, let alone make up the efficient bacterial strain of genetically engineered.

Summary of the invention

The purpose of this invention is to provide good M-Zyme of a kind of character and encoding gene thereof, by engineered method, utilize the reorganization bio-reactor to come the cheap M-Zyme of producing of industrialization that genetic resources is provided for further.

The inventor screens a kind of natural bacterial strain, and the M-Zyme that it produced has good character.The M-Zyme that the present invention screens derives from fungi Trichophyton Trichophyton sp.95, has high specific activity.Its specific activity is 7000U/mg, be higher than the various M-Zymes in the domestic and international report, as derive from M-Zyme (6000U/mg) (the Applied Environ.Microbio.58:3271-3275 of Bacillus licheniformis PWD-1,1992) and derive from M-Zyme (124.8U/mg) (Agricultural University Of Jiangxi's journal, 19 (4): 60-65) of streptomycete.Inventor's screening and separating to this M-Zyme simultaneously alpha-keratin (as wool and hair) and beta-keratin (as feather) are had the efficient degradation effect, and the present both at home and abroad M-Zyme in the various sources of the report beta-keratin of generally can only degrading; The optimal pH of this M-Zyme is 7.0, and useful effect pH value broad is 4～10, have good thermostability, handled 1 hour down at 70 ℃, its enzymic activity also maintains about 90%, and the no self-catalytic pyrolysis effect of enzyme itself, the various M-Zymes in being better than reporting.As the optimum pH that derives from the M-Zyme of Bacillus licheniformis PWD-1 is 7.9, poor heat resistance, and can self-catalytic pyrolysis (Applied Environ.Microbio.58:3271-3275,1992).The M-Zyme useful effect pH value that derives from streptomycete is 7～10, handles 0.5 hour down at 70 ℃, and its enzymic activity only maintains about 70% (Agricultural University Of Jiangxi's journal, 19 (4): 60-65).

The present invention is isolated and cloned into the encoding gene of this M-Zyme, and it has the nucleotide sequence shown in the SEQ ID NO:1, for this reason the transformation of gene and efficiently express the genetic material that provides good in various heterologous gene expression systems.Method separating clone by PCR this M-Zyme gene kerB, the DNA complete sequence analysis is the result show, these M-Zyme total length 1434 Nucleotide, 477 amino acid of encoding, there is not homology with the aminoacid sequence of external unique M-Zyme gene kerA (deriving from Bacilluslicheniformis PWD-1) of being cloned into, be a new M-Zyme gene, its aminoacid sequence is shown in SEQ ID NO:2.

The SDS-PAGE of the M-Zyme behind Brief Description Of Drawings Fig. 1 purifying analyzes

1. medium supernatant; 2. ion exchange chromatography; 3. gel chromatography;

4. standard protein molecular weight; Optimal reactive temperature Fig. 4 of optimal pH Fig. 3 M-Zyme of Fig. 2 M-Zyme. thermostability Fig. 5 of M-Zyme. M-Zyme coding gene sequence Fig. 6. the M-Zyme aminoacid sequence of deriving

Embodiment

Experiment one

This description of test produces the screening procedure of the natural bacterial strain of M-Zyme.

Adopting feather keratin is unique N source, and the mode of enrichment culture is screened M-Zyme and produced bacterium continuously.Rot to be inoculated into screening and culturing liquid (1.5g/LKH after feather soil sample sample dilutes routinely ₂PO ₄, 0.025g/L MgSO ₄.7H ₂O, 0.025g/L CaCl, 0.015g/L FeSO ₄.7H ₂O, 0.005g/L ZnSO ₄, the 0.5g/L feather meal, pH6.0) in, cultivate 10～15d for 30 ℃, the bacterium liquid of picking breeding growth is cultivated 3 continuously and is taken turns in this nutrient solution, coating separates on the screening culture medium flat board again, the renewed vaccination of picking list bacterium colony is in screening and culturing liquid, and well-grown bacterial strain repeats plate isolation again and liquid screening is cultivated, and makes the bacterial strain purifying.Screen bacterial strain 24 strains of secretion M-Zyme by this method.

Can secrete M-Zyme for the further bacterial strain that screens that confirms, strain cultured solution is carried out enzyme assay.Identical (Appl.Environ.Microbiol, 58:3271～3275) of enzymatic determination method and Lin report.The nitrine Keratin sulfate of 5mg is joined in the 0.8mL 0.1mol/L Tris-HCl damping fluid (pH7.5), add 0.2mL enzyme diluent, 50 ℃ of water-baths vibrations 15 minutes add 0.2mL 10%TCA termination reaction, behind the reacting liquid filtering in 450nm place survey OD value.Contrast adds substrate again after adding enzyme diluent and TCA earlier.Enzymic activity be defined as enzyme reaction after 15 minutes the OD value increase by 0.01 and be 1U.By enzyme assay, selected the active bacterial strain of M-Zyme, a wherein the highest strain enzymic activity reaches the 64U/mL nutrient solution, and preliminary evaluation is Trichophyton sp.95, and as the material of further research.

Experiment two

The purifying procedure of this description of test M-Zyme.

At first carry out the ammonium sulfate precipitation of M-Zyme, bacterial strain shaking table in screening culture medium was cultivated after 6～7 days, clear enzyme solution on the centrifuging and taking, crude enzyme liquid is placed ice bath, slowly add ammonium sulfate to 50% saturation ratio while stirring, put 2 hours on ice, the centrifugal 30min of 13000rpm then, get precipitation, precipitation is heavy molten with 0.1mol/L Tris-HCl damping fluid (pH7.5), becomes to concentrate enzyme liquid.Then carry out the ion exchange chromatography purifying, will concentrate HiTrap_Q_Sepharose_XL on the enzyme liquid (amersham pharmacia biotech prepacked column) anion column.Application of sample 0.5ml with the Tris-HCl buffered soln wash-out balance pillar of pH8.5,0.02mol/L, uses 0～1mol/L NaCl gradient elution of same buffer preparation instead earlier, and flow velocity is 2ml/min, fraction collection, every pipe 1ml.Then the solution in the collection tube is surveyed enzyme work and carried out the protein electrophoresis analysis.Carrying out gel chromatography at last separates, detection behind ion-exchange chromatography there is enzyme collection sample alive again through a molecular sieve Superdex_75_HR_10/30 (amersham pharmacia biotech prepacked column), application of sample 0.5ml, with pH5.5 acetate buffer solution wash-out, flow velocity is 0.4ml/min, fraction collection, every pipe 1ml obtains pure M-Zyme KERB.

Respectively go on foot enzyme activity, the protein content of sample more than having measured respectively and carried out SDS-PAGE.Purification result sees Table 1, and crude enzyme liquid is behind a few step purifying, and specific activity is brought up to 7020.6/mg from 88.4/mg, and the purifying multiple is 79.4 times.Protein electrophoresis result (Fig. 1) shows that the M-Zyme maturation protein behind the purifying is single band on electrophoresis, molecular weight is about 43kD.

Table 1: M-Zyme KERB purification step and result

Specific activity (U/mg) purifying multiple

Crude enzyme liquid 88.4 1

Concentrated solution 548.1 6.2

Cross ion column 4172.5 47.2

Cross molecular sieve 7020.6 79.4

Annotate: protein concn is measured with the Coomassie brilliant blue method

Experiment three

The measuring method of the zymologic property of this description of test M-Zyme.

The optimal pH measuring method of M-Zyme is as follows:

Purified M-Zyme carries out enzymatic reaction to measure its optimal pH under different pH values.The substrate Keratin sulfate carries out the M-Zyme vitality test under 50 ℃ in the damping fluid of different pH.Result (Fig. 2) shows that the optimal pH of KERB is 7.0, and in the scope of pH4～10, enzymic activity all maintains more than 60% of maximum enzyme activity.

The optimum temperuture of M-Zyme and thermal stability determination method are as follows:

Enzymatic reaction is carried out in being determined as under Tris-HCl damping fluid (pH7.0) system and differing temps of the optimum temperuture of M-Zyme.Temperature tolerance is determined as M-Zyme and handles different time down at 70 ℃, carries out enzyme assay again under 50 ℃.Enzyme reaction optimum temperuture measurement result (Fig. 3) shows that its optimum temperuture is 60 ℃.The heat stability test of enzyme shows (Fig. 4), and behind 70 ℃ of processing 30min, residual enzyme is lived and also had about 95%, and behind the processing 60min, enzymic activity still maintains more than 90%.

The chicken feather and the human hair that in the Tris-HCl of pH7.0 damping fluid, add wash clean respectively, the M-Zyme that adds 300U again, 50 ℃ of following water-baths were vibrated 3 hours, can be observed all dissolvings fully of feather and hair, this illustrates this M-Zyme beta-keratin (as feather) of not only degrading, the alpha-keratin (hair) of can also degrading.

The common protease activity of KERB is measured by the industry standard QB/T1803-93 of the People's Republic of China (PRC).The result shows, the KERB casein of degrading efficiently, and the M-Zyme activity of 1U is equivalent to the protease activity of 1.2U.Simultaneously, KERB is not to the bovine serum albumin (BSA) of solubility, the collagen protein etc. of capacitive all has Degradation.

KERB maturation protein N behind purifying end has been carried out determined amino acid sequence, and the result shows that preceding 8 aminoacid sequences of its N-end are: A-E-S-T-L-G-A-A.

Experiment four

The separating clone program of this description of test M-Zyme encoding gene kerB

Neutral cracking process is adopted in the extraction of the total DNA of bacterial strain, strain culturing was collected thalline after 5 days, with liquid nitrogen freezing and grind to form powdery, add 10mL SDS-TE damping fluid (4%SDS, 10mmol/LTris, 0.1mmol/LEDTA in the 10mg thalline, pH8.0) cracking 5min at room temperature, with isopyknic phenol, phenol-chloroform, chloroform extracting successively, ethanol sedimentation.

According to the synthetic degenerated primer of this M-Zyme N terminal amino acid sequence of measuring, respectively with the encoding sequence of 20 random primer groups to pcr amplification M-Zyme maturation protein.Be cloned into respectively on the PGEM-T carrier after the PCR fragment electrophoresis that contains the maturing enzyme encoding sequence reclaims, carry out sequential analysis.According to the result of sequential analysis, resynthesis inverse PCR primer, respectively with the random primer group to back amplification gene 5 ' end signal peptide-coding sequence.

According to the synthetic degenerated primer of this maturing enzyme N terminal amino acid sequence, the degenerated primer of design is: P1 5 ' GC (C/T/A/G) GA (G/A) AG (T/C) AC (G/A) CT (G/A/T/C) GG 3 '

The primer of the other end adopts 20 random primers, and is right with primer P1 group respectively, is template with total DNA, carries out pcr amplification.The PCR reaction parameter is: 94 ℃ of sex change 1min, 50 ℃ of annealing 45sec, 72 ℃ of extension 1min; Back 72 ℃ of insulation 10min are taken turns in circulation 30.Wherein only one group of primer amplification has arrived the PCR fragment of about 1.6kb.According to this proteic molecular weight of measuring, infer that the encoding gene length of this zymoprotein is no more than 1.4kb, if this PCR fragment is the M-Zyme encoding gene, should comprise complete structure gene 3 ' end.The PCR fragment of this 1.6kb of picking is cloned into it on the pGEM-T carrier, carries out sequencing.The result shows, in this fragment the reading frame of a long 1359bp arranged, and 6 amino acid of 5 ' end coding are consistent with the determined amino acid sequence result, tentatively are judged as the goal gene that we intend cloning.

In order to clone the signal coding sequence of zymoprotein N-end, we are according to the sequence results of said determination, the synthetic one section inverse PCR primer P2:5 ' GTTGAACTCACGGCTCGC 3 ' of design, same and the random primer group is right, carry out pcr amplification, obtain the PCR fragment of an about 600bp, it is cloned on the pGEM-T plasmid, carry out sequential analysis.

The result of comprehensive these two sections sequences, obtain the structured coding gene nphy (Fig. 5) of complete M-Zyme, 1434 Nucleotide of this full length gene, 477 amino acid of encoding, tactical rule according to signal peptide sequence is inferred, 25 amino acid of N-end are signal peptide, and the cleavage site of signal peptide is after+25 Gly, and this result is consistent with the N terminal amino acid sequence measurement result of maturing enzyme.The aminoacid sequence of the M-Zyme gene kerA of the gene order of this M-Zyme and present unique report (deriving from Bacillus licheniformis PWD-1) does not have homology, is a new M-Zyme gene.

＜110〉＜120〉、＜130〉I2001258＜160〉3＜170〉PatentIn version 3.1＜210〉1＜211〉1434＜212〉DNA＜213〉Trichophyton＜220〉＜221〉CDS＜222〉 ( 1 ) .. ( 1431 )＜223〉＜220〉＜221〉sig_peptide＜222〉 ( 1 ) .. ( 75 )＜223〉＜220〉＜221〉mat_peptide＜222〉 ( 76 ) .. ( )＜223〉＜400〉1atg ggc tcg tac gcc ctt ccc aga tcc ggt atc cgc cgg aag atc cac 48Met Gly Ser Tyr Ala Leu Pro Arg Ser Gly Ile Arg Arg Lys Ile His-25-20-15-10ggc ctg ctg ctg acg ctg gtc gtc ggc gtc ctc ggc acg gtc acc gca 96Gly Leu Leu Leu Thr Leu Val Val Gly Val Leu Gly Thr Val Thr Ala

-5 -1 1 5ctg?gtg?gcg?ccg?ccg?acc?gca?cac?gcc?gcc?gag?agc?acg?ctc?ggc?gcc 144Leu?Val?Ala?Pro?Pro?Thr?Ala?His?Ala?Ala?Glu?Ser?Thr?Leu?Gly?Ala

10 15 20gcg?gcg?gca?cag?agc?ggc?cgc?tac?ttc?ggc?acc?gcc?atc?gcc?tcg?ggc 192Ala?Ala?Ala?Gln?Ser?Gly?Arg?Tyr?Phe?Gly?Thr?Ala?Ile?Ala?Ser?Gly

25 30 35aag?ctg?ggc?gac?tcg?gcg?tac?acg?acg?atc?gcg?agc?cgt?gag?ttc?aac 240Lys?Leu?Gly?Asp?Ser?Ala?Tyr?Thr?Thr?Ile?Ala?Ser?Arg?Glu?Phe?Asn40 45 50 55atg?gtg?acg?gcc?gag?aac?gag?atg?aag?atc?gac?gcc?acc?gaa?ccg?cag 288Met?Val?Thr?Ala?Glu?Asn?Glu?Met?Lys?Ile?Asp?Ala?Thr?Glu?Pro?Gln

60 65 70cgg?ggc?cag?ttc?aac?ttc?tcc?gcc?ggt?gac?cgc?gtc?tac?aac?tgg?gcg 336Arg?Gly?Gln?Phe?Asn?Phe?Ser?Ala?Gly?Asp?Arg?Val?Tyr?Asn?Trp?Ala

75 80 85gtg?cag?aac?ggc?aag?cag?gtg?cgc?ggc?cac?acc?ctg?gcc?tgg?cac?tcc 384Val?Gln?Asn?Gly?Lys?Gln?Val?Arg?Gly?His?Thr?Leu?Ala?Trp?His?Ser

90 95 100cag?cag?ccc?ggc?tgg?atg?cag?agc?ctc?agc?ggc?agc?aca?ctg?cgc?cag 432Gln?Gln?Pro?Gly?Trp?Met?Gln?Ser?Leu?Ser?Gly?Ser?Thr?Leu?Arg?Gln

105 110 115gcg?atg?atc?gac?cac?atc?aac?ggc?gtg?atg?ggc?cac?tac?aag?ggc?aag 480Ala?Met?Ile?Asp?His?Ile?Asn?Gly?Val?Met?Gly?His?Tyr?Lys?Gly?Lys120 125 130 135atc?gcc?cag?tgg?gac?gtc?gtg?aac?gag?gcc?ttc?tcg?gac?gac?ggt?tcg 528Ile?Ala?Gln?Trp?Asp?Val?Val?Asn?Glu?Ala?Phe?Ser?Asp?Asp?Gly?Ser

140 145 150ggc?ggc?cgc?cgg?gat?tcc?aac?ctg?cag?cgc?acc?ggc?aac?gac?tgg?atc 576Gly?Gly?Arg?Arg?Asp?Ser?Asn?Leu?Gln?Arg?Thr?Gly?Asn?Asp?Trp?Ile

155 160 165gag?gtc?gcc?ttc?cgc?acc?gcg?cgc?gcc?gcc?gac?ccg?gcg?gcc?aag?ctc 624Glu?Val?Ala?Phe?Arg?Thr?Ala?Arg?Ala?Ala?Asp?Pro?Ala?Ala?Lys?Leu

170 175 180tgc?tac?aac?gac?tac?aac?atc?gag?aac?tgg?acc?tgg?gcg?aag?acc?cag 672Cys?Tyr?Asn?Asp?Tyr?Asn?Ile?Glu?Asn?Trp?Thr?Trp?Ala?Lys?Thr?Gln

185 190 195ggc?gtg?tac?aac?atg?gtc?cgc?gac?ttc?aag?cag?cgc?ggc?gtg?ccg?atc 720Gly?Val?Tyr?Asn?Met?Val?Arg?Asp?Phe?Lys?Gln?Arg?Gly?Val?Pro?Ile200 205 210 215gac?tgc?gtc?ggc?ttc?cag?tcg?cac?ttc?aac?agc?ggc?agc?ccc?tac?aac 768Asp?Cys?Val?Gly?Phe?Gln?Ser?His?Phe?Asn?Ser?Gly?Ser?Pro?Tyr?Asn

220 225 230agc?aac?ttc?cgc?acc?acc?ctc?cag?aac?ttc?gcc?gcc?ctc?ggc?gtc?gac 816Ser?Asn?Phe?Arg?Thr?Thr?Leu?Gln?Asn?Phe?Ala?Ala?Leu?Gly?Val?Asp

235 240 245gtg?gcc?atc?acc?gaa?ctc?gac?atc?cag?ggc?gcg?tcg?tcc?tcg?acc?tac 864Val?Ala?Ile?Thr?Glu?Leu?Asp?Ile?Gln?Gly?Ala?Ser?Ser?Ser?Thr?Tyr

250 255 260gcc?gcc?gtg?acc?aac?gac?tgc?ctg?gcc?gtc?tcg?cgc?tgc?ctg?ggc?atc 912Ala?Ala?Val?Thr?Asn?Asp?Cys?Leu?Ala?Val?Ser?Arg?Cys?Leu?Gly?Ile

265 270 275acc?gtc?tgg?ggc?gtg?cgc?gac?acc?gac?tcc?tgg?cga?tcg?ggg?gac?acg 960Thr?Val?Trp?Gly?Val?Arg?Asp?Thr?Asp?Ser?Trp?Arg?Ser?Gly?Asp?Thr280 285 290 295ccg?ctg?ctg?ttc?aac?ggc?gac?ggc?agc?aag?aag?gct?gcc?tac?acc?gcc 1008Pro?Leu?Leu?Phe?Asn?Gly?Asp?Gly?Ser?Lys?Lys?Ala?Ala?Tyr?Thr?Ala

300 305 310gtc?ctc?aac?gca?ctc?aac?ggc?ggc?tcc?tcc?acg?ccg?ccg?cct?tcg?ggt 1056Val?Leu?Asn?Ala?Leu?Asn?Gly?Gly?Ser?Ser?Thr?Pro?Pro?Pro?Ser?Gly

315 320 325ggc?gga?cag?atc?aag?ggc?gtc?ggt?tcg?ggc?cgt?tgc?ctg?gac?gtg?ccc 1104Gly?Gly?Gln?Ile?Lys?Gly?Val?Gly?Ser?Gly?Arg?Cys?Leu?Asp?Val?Pro

330 335 340aac?gcc?agc?acc?acc?gac?ggc?acc?cag?gtt?cag?ttg?tac?gac?tgc?cac 1152Asn?Ala?Ser?Thr?Thr?Asp?Gly?Thr?Gln?Val?Gln?Leu?Tyr?Asp?Cys?His

345 350 355agc?gcc?acc?aac?cag?cag?tgg?acg?tac?acc?gac?gcc?ggc?gag?ctc?agg 1200Ser?Ala?Thr?Asn?Gln?Gln?Trp?Thr?Tyr?Thr?Asp?Ala?Gly?Glu?Leu?Arg360 365 370 375gtc?tac?ggc?gac?aag?tgc?ctg?gac?gcc?gcc?ggc?acc?ggc?aac?ggc?acc 1248Val?Tyr?Gly?Asp?Lys?Cys?Leu?Asp?Ala?Ala?Gly?Thr?Gly?Asn?Gly?Thr

380 385 390aag?gtc?cag?atc?tac?age?tgc?tgg?ggc?ggc?gac?aac?cag?aag?tgg?cgc 1296Lys?Val?Gln?Ile?Tyr?Ser?Cys?Trp?Gly?Gly?Asp?Asn?Gln?Lys?Trp?Arg

395 400 405ctc?aac?tcc?gac?gga?tcc?atc?gtc?ggc?gtc?cag?tcg?ggc?ctc?tgc?ctc 1344Leu?Asn?Ser?Asp?Gly?Ser?Ile?Val?Gly?Val?Gln?Ser?Gly?Leu?Cys?Leu

410 415 420gac?gcc?gtc?gga?ggc?gga?acc?gcc?aac?ggc?acc?ctg?atc?cag?ctc?tac 1392Asp?Ala?Val?Gly?Gly?Gly?Thr?Ala?Asn?Gly?Thr?Leu?Ile?Gln?Leu?Tyr

425 430 435tcc?tgc?tcc?aac?ggc?agc?aac?cag?cgc?tgg?acc?cgc?acc?tga 1434Ser?Cys?Ser?Asn?Gly?Ser?Asn?Gln?Arg?Trp?Thr?Arg?Thr440 445 450<210>2<211>477<212>PRT<213>Trichophyton<400>2Met?Gly?Ser?Tyr?Ala?Leu?Pro?Arg?Ser?Gly?Ile?Arg?Arg?Lys?Ile?His-25 -20 -15 -10Gly?Leu?Leu?Leu?Thr?Leu?Val?Val?Gly?Val?Leu?Gly?Thr?Val?Thr?Ala

-5 -1 1 5Leu?Val?Ala?Pro?Pro?Thr?Ala?His?Ala?Ala?Glu?Ser?Thr?Leu?Gly?Ala

10 15 20Ala?Ala?Ala?Gln?Ser?Gly?Arg?Tyr?Phe?Gly?Thr?Ala?Ile?Ala?Ser?Gly 25 30 35Lys?Leu?Gly?Asp?Ser?Ala?Tyr?Thr?Thr?Ile?Ala?Ser?Arg?Glu?Phe?Asn40 45 50 55Met?Val?Thr?Ala?Glu?Asn?Glu?Met?Lys?Ile?Asp?Ala?Thr?Glu?Pro?Gln

60 65 70Arg?Gly?Gln?Phe?Asn?Phe?Ser?Ala?Gly?Asp?Arg?Val?Tyr?Asn?Trp?Ala

75 80 85Val?Gln?Asn?Gly?Lys?Gln?Val?Arg?Gly?His?Thr?Leu?Ala?Trp?His?Ser

90 95 100Gln?Gln?Pro?Gly?Trp?Met?Gln?Ser?Leu?Ser?Gly?Ser?Thr?Leu?Arg?Gln

105 110 115Ala?Met?Ile?Asp?His?Ile?Asn?Gly?Val?Met?Gly?His?Tyr?Lys?Gly?Lys120 125 130 135Ile?Ala?Gln?Trp?Asp?Val?Val?Asn?Glu?Ala?Phe?Ser?Asp?Asp?Gly?Ser

140 145 150Gly?Gly?Arg?Arg?Asp?Ser?Asn?Leu?Gln?Arg?Thr?Gly?Asn?Asp?Trp?Ile

155 160 165Glu?Val?Ala?Phe?Arg?Thr?Ala?Arg?Ala?Ala?Asp?Pro?Ala?Ala?Lys?Leu

170 175 180Cys?Tyr?Asn?Asp?Tyr?Asn?Ile?Glu?Asn?Trp?Thr?Trp?Ala?Lys?Thr?Gln

185 190 195Gly?Val?Tyr?Asn?Met?Val?Arg?Asp?Phe?Lys?Gln?Arg?Gly?Val?Pro?Ile200 205 210 215Asp?Cys?Val?Gly?Phe?Gln?Ser?His?Phe?Asn?Ser?Gly?Ser?Pro?Tyr?Asn

220 225 230Ser?Asn?Phe?Arg?Thr?Thr?Leu?Gln?Asn?Phe?Ala?Ala?Leu?Gly?Val?Asp

235 240 245Val?Ala?Ile?Thr?Glu?Leu?Asp?Ile?Gln?Gly?Ala?Ser?Ser?Ser?Thr?Tyr

250 255 260Ala?Ala?Val?Thr?Asn?Asp?Cys?Leu?Ala?Val?Ser?Arg?Cys?Leu?Gly?Ile

265 270 275Thr?Val?Trp?Gly?Val?Arg?Asp?Thr?Asp?Ser?Trp?Arg?Ser?Gly?Asp?Thr280 285 290 295Pro?Leu?Leu?Phe?Asn?Gly?Asp?Gly?Ser?Lys?Lys?Ala?Ala?Tyr?Thr?Ala

300 305 310Val?Leu?Asn?Ala?Leu?Asn?Gly?Gly?Ser?Ser?Thr?Pro?Pro?Pro?Ser?Gly

315 320 325Gly?Gly?Gln?Ile?Lys?Gly?Val?Gly?Ser?Gly?Arg?Cys?Leu?Asp?Val?Pro

330 335 340Asn?Ala?Ser?Thr?Thr?Asp?Gly?Thr?Gln?Val?Gln?Leu?Tyr?Asp?Cys?His

345 350 355Ser?Ala?Thr?Asn?Gln?Gln?Trp?Thr?Tyr?Thr?Asp?Ala?Gly?Glu?Leu?Arg360 365 370 375Val?Tyr?Gly?Asp?Lys?Cys?Leu?Asp?Ala?Ala?Gly?Thr?Gly?Asn?Gly?Thr

380 385 390Lys?Val?Gln?Ile?Tyr?Ser?Cys?Trp?Gly?Gly?Asp?Asn?Gln?Lys?Trp?Arg

395 400 405Leu?Asn?Ser?Asp?Gly?Ser?Ile?Val?Gly?Val?Gln?Ser?Gly?Leu?Cys?Leu

410 415 420Asp?Ala?Val?Gly?Gly?Gly?Thr?Ala?Asn?Gly?Thr?Leu?Ile?Gln?Leu?Tyr

425 430 435Ser?Cys?Ser?Asn?Gly?Ser?Asn?Gln?Arg?Trp?Thr?Arg?Thr440 445 450<210>3<211>18<212>DNA<213>Artificial<400>3gttgaactca?cggctcgc 18

Claims (3)

1. M-Zyme, it has the aminoacid sequence shown in the SEQ ID NO:2.

2. gene of the described M-Zyme of claim 1 of encoding.

3. according to the gene of the described M-Zyme of claim 2, it has the nucleotide sequence shown in the SEQ ID NO:1.

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