CN102181419A

CN102181419A - Trichoderma reesie protease as well as preparation method and application thereof

Info

Publication number: CN102181419A
Application number: CN 201110020041
Authority: CN
Inventors: ***; 张珊嘉; 唐鹏
Original assignee: SHANGHAI YOUTELL BIOCHEMICAL CO Ltd; HUNAN YOUTEER BIOCHEMICAL CO Ltd
Current assignee: SHANGHAI YOUTELL BIOCHEMICAL CO Ltd; HUNAN YOUTEER BIOCHEMICAL CO Ltd
Priority date: 2011-01-18
Filing date: 2011-01-18
Publication date: 2011-09-14
Also published as: WO2012097668A1

Abstract

The invention discloses a protease the amino acid sequence of which is shown as SEQ ID No.1 or the protease has an amino acid sequence over 80% of homology shown in the sequence of SEQ ID No.1; and in addition, the invention also discloses a primer used for constructing the protease expression plasmid, the sequence of the primer is shown as SEQ ID No.2 and SEQ ID No.3; and the invention also discloses a preparation method of the protease, comprising the following steps: 1) magnifying a trichoderma reesei gene; 2) constructing the trichoderma reesei prolease expression plasmid; and 3) transforming the trichoderma reesei, and screening high-yield strain of the protease. The invention also discloses application of the protease in beer saccharification.

Description

A kind of Trichodermareesei proteolytic enzyme and its production and application

Technical field

The present invention relates to biological technical field, be specifically related to a kind of proteolytic enzyme, relate in particular to a kind of Trichodermareesei proteolytic enzyme; In addition, the invention still further relates to the preparation method and the application of this Trichodermareesei proteolytic enzyme.

Background technology

The existing in the world enzyme of finding has 2138 kinds, wherein has 185 kinds to be used for actual production, and annual sales volume has reached more than 3,000,000,000 dollars.Proteolytic enzyme accounts for 59% in the industrial enzymes, and amylases accounts for 28%, and lipase accounts for 3%.

Proteolytic enzyme is most important a kind of industrial enzyme preparation, can catalytic proteins and polypeptide peptide bond hydrolysis.It extensively is present in pluck, plant stem-leaf, fruit and the microorganism.Various organisms can both be synthesized it, and microbial protease has productive value most.The commodity production of proteolytic enzyme started from for 20 beginnings of the century, and the thirties, microbial protease began to be used in food and tanning industry.Early 1950s, Japanese scholar at first finds to exist in the mould proteolytic enzyme of several types, particularly aspartic protease.Early 1960s, Holland begins to produce the washing composition that adds Sumizyme MP.Up to the present, on the world market more than the commercially available protein enzyme 80-100 kind.

The classification of 1 proteolytic enzyme

Mode by proteolytic enzyme protolysate matter can be divided into following several: (1) cuts the inner peptide bond of protein molecule, generates relative molecular mass smaller polypeptides class, and this fermentoid generally is endopeptidase; (2) peptide bond of incision protein or peptide molecule amino or C-terminal, and the amino acid that dissociates, this fermentoid is exopeptidase; Act on the aminoterminal aminopeptidase that is called, act on the carboxypeptidase that is called of C-terminal; (3) the fat key of protein hydrolysate or polypeptide; (4) amido bond of protein hydrolysate or polypeptide.

Source by enzyme can be divided into animal protease, plant protease, microbial protease.Microbial protease can be divided into bacteria protease, mold protease, endotrypsin and actinomycetes proteolytic enzyme again.

Can be divided into the aspartic protease of pH2.5-5.0, the Sumizyme MP of pH9.5-10.5, the neutral protease of pH6-8 by the optimal pH of proteolytic enzyme effect.

The purposes of 2 proteolytic enzyme

2.1 be used for foodstuffs industry

2.1.1 fermentation industry

Brewageing of soy sauce is exactly the protein that utilizes in the proteolytic enzyme decomposition raw material, makes it be degraded to peptone, polypeptide, amino acid, generates color in the product of one.In the brewage, when relatively poor or Fructus Hordei Germinatus consumption reduces auxiliary material and increases when malt quality, often need to replenish proteolytic enzyme, protein is fully degraded, improve the content of alpha-amino nitrogen in the wheat juice.Aspartic protease can also reduce the cold muddiness of beer as the clarify beer agent, increases the stability of beer.In the processing of fish sauce, add proteolytic enzyme and can shorten fermentation time, improve local flavor.

2.1.2 the production of cheese

Cheese is earlier milk to be added lactobacillus-fermented to become sour milk, adds proteolytic enzyme again, and the specific peptide bond Phe105-Met106 incision with dairy protein generates guarantee of k-junket and polypeptide, at Ca ²⁺Under the situation about existing, and solidify, through stripping and slicing, squeeze and the slaking of fermenting forms with salt.

2.1.3 the debitterize of protein hydrolystate

Protein enzymic hydrolysate bitter taste is caused with the peptide that the centre contains proline residue by hydrophobic amino acid residues wherein.Enzymatic protein initial stage hydrolyzed solution bitter taste is obvious, and this is because protein under the effect of enzyme, is decomposed into amino acid and peptide class, wherein the hydrophobic peptide of short chain shows bitter taste, and at protein not during hydrolysis, hydrophobic group is overshadowed in the molecule, comes out in the hydrolytic process and presents bitter taste.The proteolytic enzyme of peptase is rich in use, by acting on the amino acid hydrophobic amino acid of peptide chain, makes its hydrolysis become free amino acid, and it is discharged, and goes bitter purpose thereby reach debitterize.

2.1.4 the production of flavoring

Flavor seasoning is that a class contains material such as amino acid, peptide class, Nucleotide and sugar and has the food flavouring of the flesh of fish and vegetable flavor.Its method for making has two kinds on decomposition type and extracting type, and decomposition type is to be that raw material is made into the hydrolysate of animal and plant albumen such as the flesh of fish, gelatin, gluten, soybean protein by proteolytic enzyme.

2.1.5 flour processing

To the limited hydrolysis of mucedin, reduce gluten strength, improve the processing property and the mechanical property of dough, make the dough softness, the dough kneading time shortens, and improves the extensibility of dough.

2.1.6 the tenderization of meat

Hydrolysis meat reticular tissue easily boils tenderization.

2.1.7 fruit and vegetable juice processing

Cooperate with other enzyme, quicken the disintegration of vegetable cell, solve the cold turbid phenomenon that causes by protein, prolong the quality guaranteed period of product, and can increase the local flavor of beverage.

2.2 be used for washing composition

The output of whole world washing composition has reached 1.3 hundred million tons, reaches more than 80% in the ratio of European enzymatic laundry powder, and the U.S. has more than 45%, and Japan also reaches about 60%, and the sales volume of whole world Sumizyme MP accounts for 25% of zymin market, the world.Albumen spot (bloodstain, the spot of difficulty such as egg stain and human body sweat stain removal) can finish the scale removal purpose by protease hydrolysis, to be the Decomposition of utilizing enzyme with albumen such as insoluble egg, milk, blood steep the principle of scale removal becomes water miscible peptide or amino acid and remove from the fiber that adheres to.

2.3 be used for tanning production

Scleroproein is the useful component of leather in the raw material skin of process hides, also has many non-fibrous albumen to be present in fibre gap and the epidermis in addition, though these protein contents are few, if do not remove, the finished product leather is stiff and crisp.Proteolytic enzyme can not decompose celloglobulin collagen, and can decompose the albumen that is connected between fur and epidermis, makes collegen filament loose, tanned leather softness.

2.4 make gelatin and soluble collagen fiber

Traditional technology is with grease in the raw materials such as lime water logging peeling, bone and foreign protein etc., and this technology is consuming time to reach the several months, and labour intensity is big, and rate of gum output is low and energy consumption is big.With the foreign protein outside the protease digestion collagen, then collagen is cleaned post-heating and take out glue, obtain gelatin purity height, quality is good, and relative molecular mass is even, and is with short production cycle, gelatin yield height.

2.5 silk scouring

The raw silk fabric must come unstuck, silk gum is a kind of protein, and China always comes unstuck with alkali soap method high temperature refining silk, and shortcoming is a lot, alkali matter invasion and attack fibroin, easily cause the scared gloss that influences, after coming unstuck with proteolytic enzyme, the finished product feel is lubricated soft, gloss is bright-coloured, and usually time is short, and service temperature is low, and labour productivity improves.

2.6 be used for fodder industry

Add proteolytic enzyme or be blended directly in the feed animal and plant albumen of can degrading, raising protein and energy utilization ratio, reduction feed cost at feed formulation.

2.7 be used for pharmaceutical industries

Enzymotherapy is with the method for enzyme as medicine treatment disease.With in the enzyme, proteolytic enzyme accounts for more than 50% in treatment.Proteolytic enzyme can be used as the debridement agent of digestive pharmaceutical, antiphlogistic, surgery and thrombolytic agent etc. on clinical treatment.

Trichodermareesei is a kind of mycelium fungi that zymin is produced that is widely used in, and it is strong that this fungi has the protein excretion ability, is fit to the characteristics of extensive liquid submerged fermentation.

Summary of the invention

One of the technical problem to be solved in the present invention provides a kind of Trichodermareesei proteolytic enzyme.

Two of the technical problem to be solved in the present invention provides the primer that is used to make up above-mentioned Trichodermareesei proteolytic enzyme expression plasmid.

Three of the technical problem to be solved in the present invention provides the preparation method of this Trichodermareesei proteolytic enzyme.

Four of the technical problem to be solved in the present invention provides the application of this Trichodermareesei proteolytic enzyme.

For solving the problems of the technologies described above, the present invention adopts following technical scheme:

In one aspect of the invention, provide a kind of Trichodermareesei proteolytic enzyme, its aminoacid sequence perhaps has the above aminoacid sequence of sequence 80% homology shown in the SEQ ID NO.1 shown in SEQ IDNO.1.

In another aspect of this invention, provide a kind of primer that is used to make up above-mentioned Trichodermareesei proteolytic enzyme expression plasmid, its sequence is:

Upstream: 5 '-CAACCGCGGACTGCGCATCATGGCCAGCCTTCGCCT-3 ' (shown in SEQ ID NO:2) or its complementary strand;

Downstream: 5 '-ATGCTCGAGTTAA GCACTGTTCCCGTTGAAG-3 ' (shown in SEQ ID NO:3) or its complementary strand.

In another aspect of this invention, provide a kind of preparation method of above-mentioned Trichodermareesei proteolytic enzyme, this method comprises the steps:

1) Trichodermareesei gene amplification;

2) structure of Trichodermareesei proteolytic enzyme expression plasmid;

3) screening of the conversion of Trichodermareesei and proteinase high-yield bacterial strain.

Step 1) is specially: nucleotide sequence or its complementary strand shown in design SEQ ID NO:2 and the SEQ ID NO:3 are primer, are that template is carried out pcr amplification with the Trichodermareesei genomic dna.The reaction conditions of described pcr amplification is 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 59 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations; Last 72 ℃ are extended 5min.

Step 2) be specially: with the PCR product of step 1) gained by Sac II and Xho I restriction enzyme enzyme liberating after, separate with sepharose, purifying reclaims and connects, connect by the T4DNA ligase enzyme then, the penetrating cell of transformed into escherichia coli DH5 α, extract plasmid by Analysis and Screening, finish the structure of Trichodermareesei proteolytic enzyme expression plasmid.

Step 3) is specially: at first, with EcoRI degradation step 2) plasmid that makes up forms the plasmid fragment, adopts chemical conversion process to change this plasmid fragment over to Trichodermareesei RUT C-30 host cell; Cultivate containing on the PDA substratum of hygromycin B then, again eugonic colony inoculation is put on the PDA substratum, by growth and germination, green spores is transferred on the fresh PDA substratum again, change stable transformant of generation with separation and purification, (the pH value of this trichoderma cellulase inducing culture liquid is 4.8 at trichoderma cellulase inducing culture liquid with the spore inoculating of these transformants, with lactose as inductor), after in triangular flask, cultivating, it is centrifugal to sample, get supernatant liquor and carry out the SDS-PAGE electrophoretic analysis, supernatant liquor is made substrate with azo-casein and is measured proteinase activity simultaneously.

In another aspect of this invention, provide the application of a kind of above-mentioned Trichodermareesei proteolytic enzyme in beer saccharification.

The present invention designs the nucleotide sequence shown in SEQ ID NO:2 and the SEQ ID NO:3 or its complementary strand is a primer, with the Trichodermareesei genomic dna is that template is carried out pcr amplification, carry out the structure of Trichodermareesei proteolytic enzyme expression plasmid then, carry out the conversion of Trichodermareesei and the screening of proteinase high-yield bacterial strain again, the Trichodermareesei protease P roB that obtains thus is obvious through experimental verification effect in beer saccharification.

Description of drawings

Fig. 1 is the synoptic diagram of proteolytic enzyme in the embodiment of the invention 1 (ProB) efficient expression plasmid pTrProB.

Fig. 2 is the synoptic diagram of SDS-PAGE analysing protein band in the embodiment of the invention 1.

Fig. 3 is the PH specificity analysis synoptic diagram of protease P roB in the embodiment of the invention 1.

Embodiment

Following examples only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The preparation of embodiment 1 Trichodermareesei protease P roB

One. microorganism and plasmid material:

The bacillus coli DH 5 alpha that is used to clone is provided by Invitrogen company.Being used to provide the proteinase gene resource and efficiently expressing the host is Trichodermareesei RUT C-30, buys from American Type CultureCollection (ATCC), and initial plasmid is criticized pBluescipt KS () and bought from Stratagene company.

Two. gene proB finds:

Carry out homology analysis by proteinase gene at the Trichodermareesei genome sequence, find to be a proteolytic enzyme by the protein 121495 of Trichodermareesei genome encoding.This proteinic sequence is (SEQ ID NO.1 represents the aminoacid sequence of Trichodermareesei protease P roB) shown in SEQ ID NO.1.

Three. the structure of Trichodermareesei proteinase gene ProB expression plasmid:

Obtain two kinds of deoxyribonucleotide chain TrAlpF1 (CAACCGCGGACTGCGCATCATGGCCAGCCTTCGCCT with synthetic method, shown in SEQ ID NO.2) and TrAlpR1 (ATGCTCGAGTTAA GCACTGTTCCCGTTGAAG, shown in SEQ ID NO.3), above primer entrusts Sigma Chemical company synthetic, be primer with these two kinds of deoxyribonucleotide chains then, with Trichodermareesei RUT C-30 genomic dna is template, and (reaction conditions of pcr amplification is 95 ℃ of pre-sex change 5min to the dna molecular of a 1.4kb of PCR amplification; 94 ℃ of sex change 45s, 59 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations; Last 72 ℃ are extended 5min), because TrAlpF1 has Sac II restriction enzyme site, and TrAlpR1 contains the XhoI restriction enzyme site, this DNA and plasmid are criticized pT3C (accession number, EF127523, Li etal. 2007.Appl.Microbiol.Biotechnol.74:1264-1275) by behind Sac II and the XhoI restriction enzyme enzyme liberating, separates the band of PCR amplification and degraded and two bands of 10.0 and 0.7kb after the pT3C degraded with sepharose.10.0kb band cuts from glue and purifies, be connected by the T4DNA ligase enzyme with PCR 1.4kb band then, the penetrating cell of transformed into escherichia coli DH5 α, by analyzing eight plasmids in the transformant, obtain the gene that two plasmids have required coded prediction proteolytic enzyme, primary pT3C 3 ' end PstI site converts the XhoI site to by sudden change.The plasmid called after pTrProbB that makes up, proteolytic enzyme (ProB) efficient expression plasmid pTrProbB as shown in Figure 1.

Four, the screening of the conversion of Trichodermareesei and proteinase high-yield bacterial strain:

The present invention has used Trichodermareesei RUT C-30 to be host cell, the present invention adopts method (the Penttila et al. of chemical conversion, 1987 Gene 61:155-164) will change host cell over to the plasmid fragment that EcoRI degrades behind the pTrProbB, cultivate containing on the PDA substratum of hygromycin B then, after 5 days eugonic colony inoculation is put on the PDA substratum, by growth in 6 days and germination, green spores is transferred on the fresh PDA substratum again, changeed stable transformant of generation with separation and purification.By this method, obtain 15 commentaries on classics stable transformants of generation, with the spore inoculating of these transformants at trichoderma cellulase inducing culture liquid, this nutrient solution pH 4.8, with lactose as inductor.In triangular flask, cultivate after 6 days, sample centrifugal after, get supernatant liquor, (SDS-PAGE) adds Coomassie blue stain with polyacrylamide gel electrophoresis, analyze by this, see in 15 transformants the 8th and the 10 two transformant produced the protein band (see figure 2) that a molecular weight is approximately 30kDa.Supernatant liquor is made substrate with azo-casein and is measured proteinase activity (seeing Table 1) simultaneously.As seen, the 8th and the 10th transform the vigor of this substrate of degraded apparently higher than other transformants or initial host.

Table 1 Trichodermareesei RUT C-30 transformant supernatant liquor protein content and proteinase activity

Transformant	Protein content (mg/ml)	Degraded azo-casein vigor
			RUT-C30	2.22	0.107
1	3.21	0.085
			2	3.56	0.056
3	3.27	0.089
			4	3.17	0.056
5	3.98	0.120
			6	3.10	0.113
7	3.20	0.091
			8	3.39	1.115
9	3.96	0.098
			10	3.02	0.782
11	2.96	0.072
			12	2.56	0.781
13	2.31	0.056
			14	2.67	0.052

15

2.01

0.081

Five. Trichodermareesei protease P roB specificity analysis

1. various enzyme biopsies are surveyed as shown in table 2, and analytical procedure is as follows:

A. the cellulase detection method is a reducing sugar method, 1g solid enzyme powder, at 50 ℃, pH is under 4.80 conditions, per minute from concentration be the carboxymethylcellulose sodium solution of 10mg/mL degraded to discharge the needed enzyme amount of 1 μ mol reducing sugar be a unit of activity (IU), reducing sugar is with glucose equivalent;

B. the detection method of zytase is a reducing sugar method, 1g solid enzyme powder, at 50 ℃, pH is under 5.50 conditions, per minute from concentration be the oat xylan solution of 10mg/ml degraded to discharge the needed enzyme amount of 1 μ mol reducing sugar be a unit of activity (IU), reducing sugar is with wood sugar equivalent;

C. the method that diastatic enzyme biopsy is surveyed according to GB8725-2009 detects;

D. the detection method of proteolytic enzyme is: 1g solid enzyme powder, and at 30 ℃, pH is under 4.80 conditions, it is 1 enzyme activity unit that the 1min hydrolyzed casein produces 1 μ g tyrosine, represents with u/g.

Table 2

Enzyme is formed	Enzyme is lived
		Cellulase IU/g	5.0(pH?4.8，50℃)
Xylanase I U/g	22.0(pH?5.5，50℃)
		Amylase (U/g)	0
Proteolytic enzyme U/g	102(pH?4.8，30℃)

2. the pH specificity analysis of protease P roB (30 ℃) is as table 3 and shown in Figure 3:

Table 3

pH	3.5	4.0	4.5	5.0	5.5	6.0	6.5	7.0	7.5	8.0	10.5
												Enzyme U/g alive	0	53	105	104	87	42	0	0	0	0	0

From last table 3 and Fig. 3 as can be seen protease P roB be aspartic protease, enzyme is lived the highest between pH4.5 to 5.0.

Embodiment 2. Trichodermareesei protease P roB are the effect comparison experiment in beer saccharification:

One, protease P roB application experiment (Fructus Hordei Germinatus 80g in Fructus Hordei Germinatus of making of the embodiment of the invention 1, add water 320ml, transferring pH is 5.3, enzyme-added, the beginning saccharification, Mashing process: 37 ℃ (insulation 20min) → 50 ℃ (insulation 30min) → 65 ℃ (insulation 20min) → 70 ℃ of (insulation 20min) → 76 ℃ (filtrations).Experimental result is as shown in table 4:

Table 4

Enzyme concentration	0	1g	2g	3g
					5min filters the amount ml of wheat juice	89	112	118	115
Turbidity EBC	59.2	51.7	51.7	49.6
					Pol %	15.5	15.6	15.6	15.6
α-NHan Liang mg/l	270	279	290	295

As shown in table 4, add protease P roB 2g after, compare with blank, filtration velocity is fast 20%, turbidity is low by 12%, α-NHan Liang increases 20mg/l.

Two, with beta-glucanase, fungi neutral xylanase and outsourcing neutral protease (available from Tianjin Li Hua company) are in specific enzyme composite (the protease 3 0000U/g wherein of ratio that lives, beta-glucanase 1500IU/g, zytase 6500IU/g) becomes prozyme (i.e. prozyme in the table 5), the protease P roB (i.e. protease P roB in the table 5) that makes with the embodiment of the invention 1 is contrast experiment (Fructus Hordei Germinatus 80g, add water 320ml, transferring pH is 5.3, add the experiment enzyme, the beginning saccharification, Mashing process: 37 ℃ (insulation 20min) → 50 ℃ (insulation 30min) → 65 ℃ (insulation 20min) → 70 ℃ of (insulation 20min) → 76 ℃ (filtrations).Experimental result is as shown in table 5:

Table 5

As shown in table 5, protease P roB of the present invention is with the prozyme contrast experiment after composite, and filtration velocity is slow slightly 2.5%, and turbidity hangs down 6.7EBC, the α-NHan Liang height 9mg/l.

Three, with prozyme (the protease P roB 100U/g that the embodiment of the invention 1 makes, beta-glucanase 17IU/g, zytase 37IU/g) with prozyme UTC-500 (outsourcing neutral protease (available from Tianjin Li Hua company) 35000U/g, beta-glucanase 6000IU/g, zytase 13000IU/g) is contrast experiment (Fructus Hordei Germinatus 80g, add water 320ml, transferring pH is 5.3, enzyme-added, the beginning saccharification, Mashing process: 37 ℃ (insulation 20min) → 50 ℃ (insulation 30min) → 65 ℃ (insulation 20min) → 70 ℃ of (insulation 20min) → 76 ℃ (filtrations).Experimental result is as shown in table 6:

Table 6

Shown in table 6 result, the compound beta-glucanase of protease P roB, after the fungi neutral xylanase, effect is than UTC-500, and filtration is more or less the same, turbidity is low 4.6EBC, the high 7mg/l of α-NHan Liang.

Sum up: from 3 contrast experiments' of embodiment 2 experimental result, protease P roB of the present invention in beer saccharification effect obviously than outsourcing neutral protease good (α-NHan Liang is higher).

Sequence table

＜110〉Hunan Youteer Biochemical Co., Ltd., the biochemical company limited of Shanghai You Teer

＜120〉a kind of Trichodermareesei proteolytic enzyme and its production and application

<130>CPC-NP-10-14891

<160>3

<170>PatentIn?version?3.4

<210>1

<211>409

<212>PRT

＜213〉artificial sequence

MASLRRLALY?LGALLPAVLA?APAVNYKLPE?AVPNKFIVTL?KDGASVDTDS?HLTWVKDLHR 60

RSLGKRSTAG?VEKTYNIDSW?NAYAGEFDEE?TVKQIKANPD?VASVEPDYIM?WLSDIVEDKR 120

ALTTQTGAPW?GLGTVSHRTP?GSTSYIYDTS?AGSGTFAYVV?DSGINIAHQQ?FGGRASLGYN 180

AAGGDHVDTL?GHGTHVSGTI?GGSTYGVAKQ?ASLISVKVFQ?GNSASTSVIL?DGYNWAVNDI 240

VSRNRASKSA?INMSLGGPAS?STWATAINAA?FNKGVLTIVA?AGNGDALGNP?QPVSSTSPAN 300

VPNAITVAAL?DINWRTASFT?NYGAGVDVFA?PGVNILSSWI?GSNTATNTIS?GTSMATPHVV 360

GLALYLQALE?GLSTPTAVTN?RIKALATTGR?VTGSLNGSPN?TLIFNGNSA 409

<210>2

<211>36

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>2

caaccgcgga?ctgcgcatca?tggccagcct?tcgcct 36

<210>3

<211>31

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

atgctcgagt?taagcactgt?tcccgttgaa?g 31

Claims

1. a Trichodermareesei proteolytic enzyme is characterized in that, its aminoacid sequence perhaps has the above aminoacid sequence of sequence 80% homology shown in the SEQ ID NO.1 shown in SEQ ID NO.1.

2. primer that is used to make up the described Trichodermareesei proteolytic enzyme of claim 1 expression plasmid is characterized in that its sequence is:

3. the preparation method of a Trichodermareesei proteolytic enzyme as claimed in claim 1 is characterized in that, this method comprises the steps:

1) Trichodermareesei gene amplification;

2) structure of Trichodermareesei proteolytic enzyme expression plasmid;

4. the preparation method of Trichodermareesei proteolytic enzyme as claimed in claim 3, it is characterized in that, step 1) is specially: nucleotide sequence or its complementary strand shown in design SEQ ID NO:2 and the SEQ ID NO:3 are primer, are that template is carried out pcr amplification with the Trichodermareesei genomic dna.

5. the preparation method of Trichodermareesei proteolytic enzyme as claimed in claim 4 is characterized in that, the reaction conditions of described pcr amplification is 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 59 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations; Last 72 ℃ are extended 5min.

6. the preparation method of Trichodermareesei proteolytic enzyme as claimed in claim 3, it is characterized in that, step 2) be specially: with the PCR product of step 1) gained by Sac II and XhoI restriction enzyme enzyme liberating after, separate with sepharose, purifying reclaims and connects, and connects the penetrating cell of transformed into escherichia coli DH5 α then by the T4 dna ligase, extract plasmid by Analysis and Screening, finish the structure of Trichodermareesei proteolytic enzyme expression plasmid.

7. the preparation method of Trichodermareesei proteolytic enzyme as claimed in claim 3, it is characterized in that, step 3) is specially: at first, with EcoRI degradation step 2) plasmid that makes up forms the plasmid fragment, adopts chemical conversion process to change this plasmid fragment over to Trichodermareesei RUT C-30 host cell; Cultivate containing on the PDA substratum of hygromycin B then, again eugonic colony inoculation is put on the PDA substratum, by growth and germination, green spores is transferred on the fresh PDA substratum again, is changeed stable transformant of generation with separation and purification, with the spore inoculating of these transformants at trichoderma cellulase inducing culture liquid, after in triangular flask, cultivating, it is centrifugal to sample, and gets supernatant liquor and carries out the SDS-PAGE electrophoretic analysis, and supernatant liquor is made substrate with azo-casein and measured proteinase activity simultaneously.

8. the preparation method of Trichodermareesei proteolytic enzyme as claimed in claim 7 is characterized in that, the pH value of described trichoderma cellulase inducing culture liquid is 4.8, uses lactose as inductor.

9. the application of a Trichodermareesei proteolytic enzyme as claimed in claim 1 in beer saccharification.