CN104672919B - Method for preparing whey protein film from thermally stable recombinant laccase - Google Patents

Abstract

The invention belongs to the field of food industry biotechnology, and relates to a method for preparing whey protein film by using heat-stable recombinant laccase. The whey protein film is formed by adding whey protein, glycerin, recombinant heat-stable laccase and ferulic acid into pure water to form a film-forming liquid. After ultrasonic degassing, heat in a water bath at 80-90°C for 15-25 minutes, ‑55 ℃ drying 36‑48h in the system. The present invention uses whey protein as the base material, glycerin as the plasticizer, and the whey protein film prepared by adding recombined heat-stable laccase as the cross-linking agent is green and edible, and has high tensile strength, light transmittance, and water absorption. The degree and plasticity all show good properties.

Description

A method for preparing whey protein film by using thermostable recombinant laccase

technical field

The invention belongs to the field of food industry biotechnology, and relates to a method for preparing whey protein film by using heat-stable recombinant laccase.

Background technique

While plastic products have brought great convenience to society since their creation, they have also brought great threats to the living environment of human beings. This kind of packaging made of high molecular chemical polymer materials is not only difficult to degrade, but also produces polluting gases due to combustion. With the enhancement of people's awareness of environmental protection, the use of natural green materials to prepare edible packaging films has become the focus of many researchers. Edible packaging materials are made of natural edible substances, such as proteins, polysaccharides (starch, cellulose), etc., and have a porous network structure formed by intermolecular cross-linking. They are edible, easy to degrade, and have a certain Pack protection features.

However, the edible packaging film is not widely used at present and cannot replace the use of plastics. The main reason is that the properties of the prepared edible film cannot be compared with chemical polymer plastics, such as tensile strength, light transmittance, water absorption, Plasticity, soft products (packaging film) are more than hard products (packaging boxes); in addition, cost is also a factor restricting its development. The reason for the above two problems is that one of the core problems of edible packaging materials is that a good cross-linking agent that acts as an intermolecular cross-link has not yet been found. At present, in the early stage of research on crosslinking agents and plasticizers, inorganic reagents are mainly used, such as ammonia water, CMC-Na, polyalcohol epichlorohydrin, dicarboxylic acid, NaH ₂ PO ₄ and so on. Although the above research results have improved the performance of food packaging materials to a certain extent, they are still not comparable to chemical materials. In addition, the inorganic reagent crosslinking agent used also casts a shadow on the "green". As a pure natural biological product, enzyme preparation has the essence of high catalytic efficiency. It is protein itself. After food processing, it will be denatured into non-toxic and harmless food components. Enzyme preparation is a standard green food additive. Therefore, it will be an inevitable trend to use enzymes to catalyze the cross-linking of proteins and polysaccharides, and use enzyme preparations as cross-linking agents for edible packaging materials.

We previously prepared whey protein membranes using recombinant lipoxygenase (LOX), which outperformed control membranes not treated with LOX. It has been reported that the physical treatment method of high temperature heating (80-90°C, treatment for 20-30 minutes) can improve the performance of protein-based membranes. The mechanism is that the heat treatment makes the protein fully denatured, and after the temperature is lowered, new covalent bonds are formed between the polypeptide chains of the protein molecules, which in turn strengthens the intramolecular and intermolecular cross-linking of the protein. However, the recombinant LOX we used earlier was rapidly inactivated under high temperature conditions (half-life of 5-7 minutes at 50°C). Therefore, in the process of preparing the membrane, add recombinant LOX, react at 20-25°C for 2-3h, then spread the membrane, dry at 50-55°C for 36-48h, and peel off the membrane. So far, there have been no reports on adding thermostable enzymes to improve membrane performance during the heating denaturation process of whey protein.

Laccase (Laccase, EC1.10.3.2, Lac), a copper-containing metalloenzyme ubiquitous in nature, can catalyze the oxidation of various phenolic substances such as catechins, tannins, flavonoids, etc. to quinones. One of the main enzyme preparations found to have cross-linking effects on proteins and polysaccharides. Lac can catalyze the cross-linking and polymerization of proteins, probably because Lac can catalyze the oxidation of tyrosine residues in protein molecules and phenolic compounds linked to proteins through non-peptide bonds. In addition, phenolic compounds can be oxidized to quinones, and quinones can also react with amino groups, sulfhydryl groups, indolyl groups and other groups of proteins to cause protein polymerization. A large number of research work on laccase is based on fungi, and the fungi that secrete laccase are mainly concentrated in fungi such as Basidiomycota, Ascomycota and Imperfect fungi, among which the most important one is Basidiomycota White-rot fungi in the phylum Fungi. The biggest problem of fungal laccase is its poor thermal stability, which is not suitable for practical production and processing applications. Bacterial-derived laccase has good thermal stability, and can play a synergistic role with heating and physical denaturation during protein cross-linking to promote the progress of the cross-linking reaction. However, the application of thermally stable recombinant laccase to the preparation of edible packaging films has not been reported yet.

Contents of the invention

The purpose of the present invention is to provide a preparation method of whey protein film aiming at the above-mentioned prior art.

The purpose of the present invention can be achieved through the following technical solutions:

A whey protein film, the whey protein film is obtained by adding whey protein, glycerin, recombinant laccase and ferulic acid into pure water to obtain a film-forming solution, and the film-forming solution is heated in a water bath at 80-90°C for 15-30min , poured into a glass petri dish covered with a plastic film, dried at 50-55°C for 36-48h to remove the film; the amino acid sequence of the recombinant laccase described therein is shown in SEQ ID NO.2.

The coding gene of the recombinant laccase is preferably shown in SEQ ID NO.1.

Described restructuring paint is further preferably prepared by following method:

(1) With Bacillus death valley fmb-103 genomic DNA as a template, the sequence is the upstream primer of SEQ ID NO.3 and the sequence is the downstream primer of SEQ ID NO.4 to synthesize the laccase gene containing SacI and XhoI restriction sites sequence;

(2) The PCR product purified in the previous step was double-digested with SacI and XhoI and inserted between the SacI/XhoI restriction sites of the vector pET-23a to obtain the expression plasmid pET-23a-fmb-L103 containing the laccase gene;

(3) Transform Escherichia coli expression host strain BL21(DE3)pLysS containing fmb-L103 expression plasmid pET-23a-fmb-L103, pick small colonies after culturing at 37°C for 10-11 hours, and insert them into 50ml of ampicillin-containing LB liquid medium, 70-90rpm, culture overnight at 30°C, take the seed solution according to the volume ratio of 1:40 and add it to 100ml LB liquid medium containing ampicillin, shake at 180rpm at 35°C for 2-3 hours until OD600 is about 0.6, add IPTG (final concentration 100 μg/ml) induction, after 5 hours centrifugation collects bacterium;

(4) Resuspend the bacterial cells collected by induced expression, resuspend the bacterial cells with phosphate buffer, break the bacterial cells by ultrasonic wave, centrifuge at 10000×g for 10 min at 4°C, collect the supernatant as the crude enzyme solution, and process the crude enzyme The solution was purified according to the instructions of Ni-NTA His TagKit to obtain the recombinant laccase.

The mass percentage of whey protein in the film-forming liquid is preferably 8%-12%, more preferably 10%; the mass ratio of glycerol to whey protein is preferably 1:4-1:5, more preferably 1:4; The mass percent content of ferulic acid is preferably 1%-1.2% of the mass of the film-forming liquid, more preferably 1%, the amount of recombinant laccase is preferably 10U/g-20U/g film-forming liquid, more preferably 15U/g; the pH of the film-forming liquid is preferably 5.5-6, more preferably 5; the reaction temperature is preferably 85°C, and the reaction time is preferably 20min.

A method for preparing a whey protein film, adding a film-forming solution of whey protein, glycerin, recombinant laccase and ferulic acid into pure water, heating the film-forming solution in a water bath at 80-90°C for 15-25min, pouring it into a In a glass petri dish with a plastic film, dry at 50-55°C for 36-48 hours and remove the film; the amino acid sequence of the recombinant laccase described therein is shown in SEQ ID NO.2.

Described restructuring paint is preferably prepared by the following method:

(1) With Bacillus death valley fmb-103 genomic DNA as a template, the sequence is the upstream primer of SEQ ID NO.3 and the sequence is the downstream primer of SEQ ID NO.4 to synthesize the laccase gene containing SacI and XhoI restriction sites sequence;

(2) The PCR product purified in the previous step was double-digested with SacI and XhoI and inserted between the SacI/XhoI restriction sites of the vector pET-23a to obtain the expression plasmid pET-23a-fmb-L103 containing the laccase gene;

(3) Transform Escherichia coli expression host strain BL21(DE3)pLysS containing fmb-L103 expression plasmid pET-23a-fmb-L103, pick small colonies after culturing at 37°C for 10-11 hours, and insert them into 50ml of ampicillin-containing LB liquid medium, 70-90rpm, culture overnight at 30°C, take the seed solution according to the volume ratio of 1:40 and add it to 100ml LB liquid medium containing ampicillin, shake at 180rpm at 35°C for 2-3 hours until OD600 is about 0.6, add IPTG (final concentration 100 μg/ml) induction, after 5 hours centrifugation collects bacterium;

(4) Resuspend the bacterial cells collected by induced expression, resuspend the bacterial cells with phosphate buffer, break the bacterial cells by ultrasonic wave, centrifuge at 10000×g for 10 min at 4°C, collect the supernatant as the crude enzyme solution, and process the crude enzyme The solution was purified according to the instructions of Ni-NTA His TagKit to obtain the recombinant laccase.

The mass percentage of whey protein in the film-forming liquid is preferably 8%-12%, more preferably 10%; the mass ratio of glycerol to whey protein is preferably 1:4-1:5, more preferably 1:4; The mass percent content of ferulic acid is preferably 1%-1.2% of the mass of the film-forming liquid, more preferably 1%, the amount of recombinant laccase is preferably 10U/g-20U/g film-forming liquid, more preferably 15U/g; the pH of the film-forming liquid is preferably 5.5-6, more preferably 5; the reaction temperature is preferably 85°C, and the reaction time is preferably 20min.

In the method for preparing a whey protein film, it is further preferred to weigh whey protein powder and dissolve it in ultrapure water to prepare a solution with a mass percent content of 10%, stir and add glycerin, ferulic acid and the recombined paint Enzyme, magnetically stirred to make it fully dissolved, then adjusted the pH to 5 with NaOH or HCl solution to form a film-forming liquid, degassed the film-forming liquid with ultrasonic waves, heated it in a constant temperature water area of 85°C for 30 min, coated it into a film or introduced Film formation in flat containers.

Beneficial effect:

In the present invention, by adding heat-stable recombinant laccase oxygenase as a cross-linking agent, and simultaneously adopting a treatment method of high-temperature heat treatment of whey protein matrix, the catalytic effect of the enzyme preparation and the effect of protein thermal denaturation are coordinated, and the whey is prepared. protein film. The prepared whey protein film is green and edible, and has good properties in terms of tensile strength, light transmittance, water absorption and plasticity. application value.

Description of drawings

Fig. 1 Construction process of recombinant fmb-103 laccase gene expression vector

Figure 2 The film-forming effect of whey protein without adding ferulic acid and adding ferulic acid

A is the film-forming effect of whey protein without adding ferulic acid and enzymes

B is the film-forming effect of whey protein with ferulic acid added and no enzyme added

Figure 3 Whey protein film-forming effect without adding thermostable recombinant laccase and adding enzyme

A is the film-forming effect of whey protein without adding thermostable recombinant laccase

B is the film-forming effect of whey protein added with heat-stable recombinant laccase

Figure 4 Adding heat-stable recombinant laccase and ferulic acid to the film-forming effect of whey protein

Biological Material Deposit Information

Death Valley Bacillus fmb-103, classified as Bacillus vallismortis, was preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures. The preservation date was June 08, 2012, and the preservation address was No. 1, No. 1 Beichen West Road, Chaoyang District, Beijing. No. Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No.6198.

detailed description

The present invention is described further below by embodiment.

Embodiment 1: Cloning of Bacillus vallismortis fmb-103 laccase gene

Bacillus vallismortis fmb-103 cells (CGMCCNo.6198) were collected by centrifugation, and the genomic DNA of Bacillus vallismortis fmb-103 was extracted with Shanghai Sangon Genomic DNA Extraction Kit.

Design two primers according to the registered Bacillus laccase gene (No.GU972592.1) in the Genebank database:

Upstream primer F-1: 5'-ATGACACTTGAAAAATTTGTGGATGC-3' (SEQ ID NO.3);

Downstream primer R-1: 5'-TTATTTATGGGGATCAGTTATATC-3' (SEQ ID NO.4);

In a 50 μl system, the final concentration of each primer is 1 μM, the final concentration of dNTPs is 0.2 mM, fmb-103 strain genomic DNA 10 ng, 2 U Pfu DNA polymerase. The amplification program was 94°C for 3min; 30×(94°C for 40s, 53°C for 50s, 72°C for 90s); 72°C for 10min. Agarose gel electrophoresis, gel cutting, recovery by Shanghai Sangon kit, connection of the recovered PCR product with TaKaRapMD19-simple-T vector, transformation of E.coli DH5α, and spreading on LB containing IPTG, X-gal and ampicillin Plate, cultured at 37°C for 13-14 hours, picked white colonies, cultured with shaking, extracted plasmid, confirmed successful connection and sent to Shanghai Sangon for sequencing, analyzed the sequencing results with computer software DNAMAN, and obtained a sequence as shown in SEQ ID NO.1 The ORF with a length of 1542bp, namely the fmb-103 laccase gene (fmb-L103), encodes a protein consisting of 514 amino acids, and its sequence is shown in SEQ ID NO.2.

Embodiment 2: the construction of prokaryotic expression vector of Bacillus vallismortis (Bacillus vallismortis) fmb-103 laccase gene prokaryotic expression vector (accompanying drawing 1)

According to the obtained laccase gene sequence, two primers were designed, the upstream primer plus the SacI recognition sequence, and the downstream primer plus the XhoI recognition sequence (the underlined part is the restriction enzyme recognition sequence):

Upstream primer F-2: 5′-CGC GAGCTC ATGACACTTGAAAAATTTGTGGATGC-3′ (SEQ ID NO.5),

Downstream primer R-2: 5'-CCG CTCGAG TTATTTATGGGGATCAGTTATATC-3' (SEQ ID NO.6),

Add each component according to the following PCR system to amplify the laccase gene:

The PCR program is: 94°C for 3min; 30×(94°C for 40s; 53°C for 50s; 72°C for 90s); 72°C for 10min.

Purify the PCR product with Shanghai Sangon PCR Product Purification Kit, add SacI and XhoI double enzyme digestion, inactivate, ethanol precipitation, redissolve in ddH ₂ O, connect with an appropriate amount of vector pET-23a digested with the same restriction enzyme, and transform the large intestine Bacillus DH5α. A few colonies were randomly picked from the transformation plate, inserted into LB liquid medium, cultured by shaking, a small amount of plasmid was extracted, electrophoresis, PCR verification was carried out using the plasmid lagging behind in electrophoresis as a template, and it was sent to Shanghai Sangon for sequencing after confirming that the connection was successful.

Embodiment 3: the expression of Bacillus vallismortis (Bacillus vallismortis) fmb-103 laccase gene in Escherichia coli

Transform the expression plasmid pET-23a-fmb-L103 containing fmb-L103 into Escherichia coli expression host strain BL21(DE3)pLysS, culture at 37°C for 10-11 hours, pick small colonies, insert into 50ml LB liquid culture containing ampicillin Base, 70-90rpm, 30°C culture overnight, according to the volume ratio of 1:40, take the seed solution and add it to 100ml LB liquid medium containing ampicillin, shake at 180rpm at 35°C for 2-3 hours until the OD600 is about 0.6, add IPTG (final Concentration 100μg/ml) induced. After 1.5 hours, the cells were collected by centrifugation. The bacterium was disrupted, and the supernatant was collected by centrifugation to obtain a crude enzyme solution of recombinant Bacillus vallismortis laccase (fmb-rL103).

The laccase activity was determined using the 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) method with slight modifications: the total volume of the reaction system was 3mL, including 2.45mL 0.2mol/L pH 5.0 citric acid-phosphate buffer solution, 0.5mL 6mmol/LABTS and 50μl enzyme crude extract appropriately diluted with pH 5.0 citric acid-phosphate buffer solution, measure the absorbance value OD of the reaction solution at 420nm within the first 3 minutes of the reaction at 45°C The increasing amount of the enzyme solution was used as a blank control. The amount of enzyme required to generate 1 μmol of reactant per minute was defined as an enzyme activity unit. Calculation formula for laccase activity: laccase activity (U) = V _total × ΔOD/(V _enzyme × ε × Δt × 10 ^-6 ) × dilution factor of total enzyme enzyme solution; where, ε = 3.6 × 10 ⁴ mol/cm ; Δt: 3min; ΔOD: the change value of absorbance OD within 3min; Vtotal: the total volume of the reaction solution in the _enzyme reaction; _Venzyme : the volume of the enzyme solution in the enzyme reaction. The experiment was repeated 3 times and the average value was taken.

The above method was used to measure the recombinant laccase yield of the engineered bacteria transformed with pET-23a-fmb-L103 into BL21(DE3)pLysS was 12000 U/ml fermentation broth.

Embodiment 4: Separation and purification of recombinant Bacillus vallismortis fmb-103 laccase (fmb-rL103)

The fmb-rL103 crude enzyme solution obtained in Example 3 was separated and purified by NTA (nickel column, product of GE Company) affinity chromatography.

Add 5mM imidazole (final concentration) to the sample to enhance the adsorption column.

The column was equilibrated with 20 mM imidazole before loading. The sample passes through the column material three times to achieve the purpose of fully combining fmb-rL103 with the affinity column material.

After loading the sample, it was eluted with 100 mM imidazole (respectively using 10 column bed volumes), and the eluate was collected to measure the enzyme activity. The enzyme activity of the recombinant laccase fmb-rL103 purified and prepared above is 3.6 U/mg protein.

Embodiment 5: the preparation process of whey protein packaging film

Weigh the whey protein powder and dissolve it in ultrapure water to prepare a mass percentage of 10%. The ratio of glycerin and whey protein is 1:4. Slowly add the film-forming liquid during the stirring process, and magnetically stir it to fully dissolve it, then use NaOH Or adjust the pH to 5 with HCl solution, the mass percentage of ferulic acid is 1%, add recombinant fmb-rLac, and the enzyme amount is 15U/g film-forming solution. Heat the film-forming solution in constant temperature water at 85°C for 20 minutes. After the film-forming solution is cooled, degas it with ultrasonic waves, pour it into a glass petri dish covered with a plastic film, react at room temperature for 2 hours at 20°C, and dry at 55°C for 36 hours to remove the film.

Embodiment 6: the mensuration of whey protein membrane property

Determination of film sensory indicators: sensory evaluation of film color, surface smoothness and film viscosity, whether it is easy to peel off the film.

Determination of film thickness (mm): Take five points at random (except the edge of the film) on the film to be tested, measure the thickness with an electronic digital caliper, take the average value and record it.

Film crease (FM) measurement: Fold the film in half, judge the film crease according to the degree of crease, use 0-4 as the index, 0 means no crease, 1 means slight crease, 2 means obvious film crease, 3 means that the film is partially broken after being folded in half, and 4 means that the part of the film folded in half is completely broken.

Measurement of light transmittance: Cut the sample into a rectangle with a certain specification (0.8mm*4.5cm), paste it on the inner smooth surface of the cuvette, and measure its transmittance at a wavelength of 500nm, that is, the light transmittance. Color dish as a control. Make 2 parallels for each sample.

Determination of film water solubility: Dry the film sample (2cm×2cm) at 105°C to constant weight, and calculate the moisture content based on the difference between dry and wet weights after weighing. Put the film with 30mL water The film was dissolved in a beaker at room temperature for 24 hours, and then the film was dried at 105 °C to constant weight, weighed, and the water solubility was calculated according to the difference between the two dry weights.

Water vapor transmission rate measurement: Take a round open plastic cup with a diameter of 3cm and a depth of 4cm, add 3g of dry CaCl ₂ , cut a film sample with a diameter of 7cm, cover the mouth of the cup, and the interface between the film and the cup Seal with paraffin. Each cup was then placed in a desiccator (25°C, 100% relative humidity) with 1000 mL of distilled water added to the bottom. Weighing every 12h for 1 week. The amount of water vapor permeation is determined by the increase in cup weight. Calculate water vapor transfer rate (WVTR) and water vapor transmission rate (WVP) according to ASTM method (E96-93, 1993).

Measurement of mechanical properties: Cut the film into strips of 4cm*2cm, and fix them on a physical property meter for measurement. Set the initial distance between the two fixtures of the physical property meter to 20mm, and the speed to 1mm/sec. After the measurement, record the force (g) at the instant the film breaks and the distance L between the two clamps when the film breaks. Three parallel samples were taken for each sample.

Tensile strength TS (g): The tensile strength is represented by the momentary force when the film is broken.

The elongation at break E (%) is calculated according to the following formula:

E=(L1-L0)/L0

In the formula: E—the elongation at break of the film (%);

L1—the distance between the two clamps when the film is broken (mm);

L0—the initial distance between the two fixtures (mm).

According to the above method, the whey protein film performance indicators prepared by the two methods of recombinant laccase treatment and non-increased recombinant laccase treatment are shown in Table 1:

Table 1 Properties of whey protein film prepared by different treatments

Claims (8)

1. A whey protein film, characterized in that this whey protein film is to obtain a film-forming liquid by adding whey protein, glycerol, recombinant laccase and ferulic acid in pure water, and the film-forming liquid is obtained at 80-90°C Heated in a water bath for 15-30min, poured into a glass petri dish covered with a plastic film, dried at 50-55°C for 36-48h to remove the film; wherein the amino acid sequence of the recombinant laccase is shown in SEQ ID NO.2; wherein , the mass percentage of whey protein in the film-forming liquid is 8%-12%, the mass ratio of glycerin and whey protein is 1:4-1:5, and the mass percentage of ferulic acid is the film-forming liquid 1%-1.2% of the mass, the amount of recombinant laccase is 10U/g-20 U/g film-forming solution. 2. The whey protein film according to claim 1, characterized in that the coding gene of the recombinant laccase is as shown in SEQID NO.1. 3. The whey protein film according to claim 2, characterized in that said recombinant laccase is prepared by the following method: (1) Using the Bacillus death valley fmb-103 genomic DNA as a template, the upstream primer whose sequence is SEQ ID NO.3 and the downstream primer whose sequence is SEQ ID NO.4 synthesize a laccase gene containing SacI and XhoI restriction sites sequence; (2) The PCR product purified in the previous step was double-digested with SacI and XhoI and inserted between the SacI/XhoI restriction sites of the vector pET-23a to obtain the expression plasmid pET-23a-fmb-L103 containing the laccase gene; (3) Transform Escherichia coli expression host strain BL21(DE3)pLysS containing fmb-L103 expression plasmid pET-23a-fmb-L103, pick small colonies after culturing at 37°C for 10-11 hours, and insert them into 50ml ampicillin-containing LB liquid medium, 70-90rpm, culture overnight at 30°C, take the seed solution according to the volume ratio of 1:40 and add it to 100ml LB liquid medium containing ampicillin, shake at 35°C and 180rpm for 2-3 hours until the OD600 is 0.6, add IPTG After induction, the cells were collected by centrifugation after 5 hours; the final concentration of IPTG was 100 μg/ml; (4) Resuspend the cells collected by induced expression, resuspend the cells with phosphate buffer, break up the cells by ultrasonic wave, centrifuge at 10 000 × g for 10 min at 4 °C, collect the supernatant as crude enzyme solution, and process the obtained The crude enzyme solution was purified according to the instructions of the Ni-NTA His Tag Kit to obtain the recombinant laccase. 4. The whey protein film according to claim 1, characterized in that the mass percentage of whey protein in the film-forming liquid is 10%, the ratio of glycerol to whey protein is 1:4, ferulic acid The mass percentage is 1%, the enzyme amount of recombinant laccase is 15U/g, and the pH of the film-forming solution is 5. 5. A method for preparing a whey protein film, characterized in that the film-forming solution of whey protein, glycerol, recombinant laccase and ferulic acid is added to pure water, and the film-forming solution is heated in a water bath at 80-90°C for 15- 25min, pour it into a glass petri dish covered with a plastic film, dry at 50-55°C for 36-48h and uncover the film; the amino acid sequence of the recombinant laccase described therein is shown in SEQ ID NO.2; the film-forming The mass percentage of whey protein in the liquid is 8%-12%, the mass ratio of glycerin and whey protein is 1:4-1:5, the mass percentage of ferulic acid is 1%-1.2%, and the recombinant heat is stable The amount of sex enzyme is 10U/g-20 U/g. 6. according to the method for preparing whey protein film according to claim 5, it is characterized in that described recombinant laccase is prepared by the following method: (1) Using the Bacillus death valley fmb-103 genomic DNA as a template, the upstream primer whose sequence is SEQ ID NO.3 and the downstream primer whose sequence is SEQ ID NO.4 synthesize a laccase gene containing SacI and XhoI restriction sites sequence; (2) The PCR product purified in the previous step was double-digested with SacI and XhoI and inserted between the SacI/XhoI restriction sites of the vector pET-23a to obtain the expression plasmid pET-23a-fmb-L103 containing the laccase gene; (3) Transform Escherichia coli expression host strain BL21(DE3)pLysS containing fmb-L103 expression plasmid pET-23a-fmb-L103, pick small colonies after culturing at 37°C for 10-11 hours, and insert them into 50ml ampicillin-containing LB liquid medium, 70-90rpm, culture overnight at 30°C, take the seed solution according to the volume ratio of 1:40 and add it to 100ml LB liquid medium containing ampicillin, shake at 35°C and 180rpm for 2-3 hours until the OD600 is 0.6, add IPTG After induction, the cells were collected by centrifugation after 5 hours; the final concentration of IPTG was 100 μg/ml; (4) Resuspend the cells collected by induced expression, resuspend the cells with phosphate buffer, break up the cells by ultrasonic wave, centrifuge at 10 000 × g for 10 min at 4 °C, collect the supernatant as crude enzyme solution, and process the obtained The crude enzyme solution was purified according to the instructions of the Ni-NTA His Tag Kit to obtain the recombinant laccase. 7. the method for preparing whey protein film according to claim 5 is characterized in that the mass percentage composition of whey protein is 10% in the described film-forming liquid, and glycerol and whey protein ratio are 1:4, The mass percentage of ferulic acid is 1%, the enzyme amount of recombinant laccase is 15U/g, and the pH of the film-forming solution is 5. 8. the method for preparing whey protein film according to claim 7, is characterized in that taking whey protein powder is dissolved in ultrapure water and is mixed with the solution that mass percentage composition is 10%, stirs and adds glycerin, ferulicum acid and the recombinant laccase, magnetically stirred to make it fully dissolved, and then adjusted the pH to 5 with NaOH or HCl solution to form a film-forming solution. After the film-forming solution was degassed by ultrasonic waves, it was heated in 85°C constant temperature water for 20 minutes, and coated with Cloth to form a film or import it into a flat container to form a film.