CN104672919B - Method for preparing whey protein film from thermally stable recombinant laccase - Google Patents
Method for preparing whey protein film from thermally stable recombinant laccase Download PDFInfo
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Abstract
The invention belongs to the technical field of biology in food industry and relates to a method for preparing a whey protein film from a thermally stable recombinant laccase. The whey protein film is prepared by use of the following steps: adding whey protein, glycerin, the thermally stable recombinant laccase and ferulic acid to pure water to form a film-forming solution, after ultrasonic deaeration, heating in water bath at a temperature ranging from 80 to 90 DEG C for 15-25 minutes, and drying at 50-55 DEG C for 36-48 hours to obtain the film. The whey protein film prepared with the whey protein as the base material, the glycerin as the plasticizer and the added thermally stable recombinant laccase as the cross-linking agent is green and edible, and excellent in such properties as tensile strength, transmission of light, water absorption degree and plasticity.
Description
Technical field
The invention belongs to technical field of food industry biology, is related to a kind of using heat stability restructuring laccase preparation milk surum egg
The method of tunica albuginea.
Background technology
While plastic has brought huge convenience to society since the generation, it is also brought greatly to environment for human survival
Threaten.It is this to be not only difficult to degrade with packing made by polymer chemistry polymeric material, also dusty gass can be produced because of burning.With
Enhancing of the people to environmental consciousness, edible packaging film is prepared using natural green material becomes Jiao of many researcheres
Point.Edible packing material is that such as protein, polysaccharide (starch, cellulose) etc. is raw material, by dividing with edible natural material
The structure with holey that the effect of being cross-linked with each other between son is formed, it is edible, degradable, and there is certain packaging protecting work(
Energy.
But edible packaging film application is not extensive at present, it is impossible to replaces the use of plastics, traces it to its cause, mainly makes
Standby edible film performance cannot be compared with chemical polymer plastic, such as tensile strength, light transmission, absorption of water, plasticity, soft
The more hard product (packing box) of product (packaging film) is few；Cost Problems are also a factor for restricting its development in addition.Cause above-mentioned
Of both problem the reason for exactly one of key problem of edible packing material be still to be not found good to play molecule
Between be cross-linked with each other the cross-linking agent of effect.At present in early days in terms of about cross-linking agent, the research of plasticizer based on inorganic reagent, such as
Ammonia, CMC-Na, polyalcohols epoxychloropropane, dicarboxylic acids, NaH2PO4Deng.The studies above result, although carry to a certain extent
The high performance of packaging material for food, but still can not match in excellence or beauty with chemical material.In addition, the inorganic reagent cross-linking agent for being adopted
Shade has been coverd with to " green "., used as a kind of pure natural biological product, the essence with high catalytic efficiency, itself is just for enzyme preparation
It is protein, Jing after food processing, degeneration is nontoxic food component, and enzyme preparation is the green food additive of standard.
Therefore, using enzyme catalysiss albumen, the crosslinked action of polysaccharide, will be inevitable using enzyme preparation as the cross-linking agent of edible packing material
Trend.
We are prepared for lactalbumin membrane using restructuring lipoxidase (LOX) at early stage, and its performance is better than without LOX process
Control film.Studies have reported that by the physical treatment method of high-temperature heating (80-90 DEG C, process 20-30 minutes), can improve with
Performance of the protein for the film of substrate.Its mechanism is that heat treated causes the abundant degeneration of albumen, reduces protein molecule after temperature
Polypeptide chain between re-form new covalent bond, and then enhance in protein molecule and intermolecular crosslinked action.However,
The restructuring LOX that our early stages are used fast deactivation (50 DEG C of minutes half-life 5-7) under the high temperature conditions.Therefore preparing the mistake of film
Cheng Zhong, adds restructuring LOX, reacts plastic film mulch after 2-3h at 20-25 DEG C, 50-55 DEG C be dried 36-48h after take off film.So far,
During whey protein heat denatured, the enzyme of heat stability is added, and the research for improving film properties has not been reported.
Laccase (Laccase, EC1.10.3.2, Lac), is a kind of copper-containing metal enzyme of nature generally existing, can urge
Change various aldehydes matters such as catechuic acid, tannin, flavone etc. and be oxidized to quinone, be it has now been found that have friendship to protein, polysaccharide
One of main enzyme preparation of connection effect.Lac can crosslink polymerization with catalytic proteins, it may be possible to as Lac can catalytic protein
The oxidation of tyrosine residue and the phenolic compound being connected by non-peptide bond with protein in matter molecule is realizing.In addition,
Phenolic compound can be made to be oxidized to quinone, and quinoness also can occur with the group such as the amino of protein, sulfydryl, indyl it is anti-
Should be so as to causing albumen to be polymerized.A large amount of research work with regard to laccase have drawn from funguses, and the funguses of secretion laccase focus primarily upon
Basidiomycota (Basidiomycota), Ascomycota (Ascomycota) and Fungi Imperfecti (Imperfect fungi) etc. are true
Bacterium, wherein the white rot fungi most importantly in Basidiomycota.The maximum problem of fungal laccase is that its heat stability is poor, is not suitable for
In actual production and processing application.Bacterial origin laccase has good thermal stability, with heating thing during protein cross
Reason degeneration can play synergism, promote the carrying out of cross-linking reaction.And be applied to regard to the restructuring laccase of heat stability at present
The preparation of edible packing membrane has not been reported.
The content of the invention
The purpose of the present invention is for the above-mentioned of prior art, there is provided a kind of preparation method of lactalbumin membrane.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of lactalbumin membrane, the lactalbumin membrane be by pure water add lactalbumin, glycerol, restructuring laccase and
Ferulic acid obtains into film liquid, into film liquid in 80-90 DEG C of heating in water bath 15-30min, pours into and is covered with the glass culture dish of plastic packaging film,
50-55 DEG C is dried 36-48h and takes off obtained by film；The aminoacid sequence of wherein described restructuring laccase is as shown in SEQ ID NO.2.
The encoding gene of described restructuring laccase is preferably as shown in SEQ ID NO.1.
Described restructuring paint is further preferably prepared via a method which：
(1) with dead paddy bacillus cereuss fmb-103 genomic DNAs template, forward primer of the sequence for SEQ ID NO.3
Synthesize the laccase gene sequence containing SacI and XhoI restriction enzyme sites with downstream primer of the sequence for SEQ ID NO.4；
(2) by the SacI/XhoI of the PCR primer of previous step purification insertion vector pET-23a Jing after SacI, XhoI double digestion
Between restriction enzyme site, the expression plasmid pET-23a-fmb-L103 containing laccase gene is obtained；
(3) escherichia coli expression host strain BL21 will be converted containing fmb-L103 expression plasmids pET-23a-fmb-L103
(DE3) pLysS, picking petite after cultivating 10-11 hours at 37 DEG C access the training of the 50ml LB liquid containing ampicillin
Foster base, 30 DEG C of overnight incubations of 70-90rpm, according to 1:40 volume ratio takes seed liquor and is added to containing ampicillin
100mlLB fluid mediums, 35 DEG C of 180rpm add IPTG (100 μ g/ml of final concentration) when vibrating 2-3 hours to OD600 about 0.6
Induction, was collected by centrifugation thalline after 5 hours；
(4) thalline that abduction delivering is collected, with the resuspended thalline of phosphate buffer, ultrasonic disruption thalline, 4 DEG C,
10000 × g is centrifuged 10min, collects supernatant as crude enzyme liquid, will process the crude enzyme liquid for obtaining according to Ni-NTA His Tag
Kit description carries out purification and obtains described restructuring laccase.
The described preferred 8%-12% of the weight/mass percentage composition into lactalbumin in film liquid, further preferred 10%；Glycerol
With lactalbumin quality ratio preferably 1:4-1:5, further preferred 1:4；Ferulic acid weight/mass percentage composition is preferably into film liquid quality
1%-1.2%, further preferred 1%, the restructuring preferred 10U/g-20U/g of laccase amount into film liquid, further preferred 15U/g；Into
The preferred 5.5-6 of film liquid pH, further preferred 5；Preferably 85 DEG C of reaction temperature, response time preferred 20min.
A kind of method for preparing lactalbumin membrane, adds lactalbumin, glycerol, restructuring laccase and ferulic acid in pure water
Into film liquid, into film liquid in 80-90 DEG C of heating in water bath 15-25min, pour into and be covered with the glass culture dish of plastic packaging film, 50-55 DEG C is done
Dry 36-48h takes off film；The aminoacid sequence of wherein described restructuring laccase is as shown in SEQ ID NO.2.
Described restructuring paint is preferably prepared via a method which：
(1) with dead paddy bacillus cereuss fmb-103 genomic DNAs template, forward primer of the sequence for SEQ ID NO.3
Synthesize the laccase gene sequence containing SacI and XhoI restriction enzyme sites with downstream primer of the sequence for SEQ ID NO.4；
(2) by the SacI/XhoI of the PCR primer of previous step purification insertion vector pET-23a Jing after SacI, XhoI double digestion
Between restriction enzyme site, the expression plasmid pET-23a-fmb-L103 containing laccase gene is obtained；
(3) escherichia coli expression host strain BL21 will be converted containing fmb-L103 expression plasmids pET-23a-fmb-L103
(DE3) pLysS, picking petite after cultivating 10-11 hours at 37 DEG C access the training of the 50ml LB liquid containing ampicillin
Foster base, 30 DEG C of overnight incubations of 70-90rpm, according to 1:40 volume ratio takes seed liquor and is added to containing ampicillin
100mlLB fluid mediums, 35 DEG C of 180rpm add IPTG (100 μ g/ml of final concentration) when vibrating 2-3 hours to OD600 about 0.6
Induction, was collected by centrifugation thalline after 5 hours；
(4) thalline that abduction delivering is collected, with the resuspended thalline of phosphate buffer, ultrasonic disruption thalline, 4 DEG C,
10000 × g is centrifuged 10min, collects supernatant as crude enzyme liquid, will process the crude enzyme liquid for obtaining according to Ni-NTA His Tag
Kit description carries out purification and obtains described restructuring laccase.
The described preferred 8%-12% of the weight/mass percentage composition into lactalbumin in film liquid, further preferred 10%；Glycerol
With lactalbumin quality ratio preferably 1:4-1:5, further preferred 1:4；Ferulic acid weight/mass percentage composition is preferably into film liquid quality
1%-1.2%, further preferred 1%, the restructuring preferred 10U/g-20U/g of laccase amount into film liquid, further preferred 15U/g；Into
The preferred 5.5-6 of film liquid pH, further preferred 5；Preferably 85 DEG C of reaction temperature, response time preferred 20min.
The described method for preparing lactalbumin membrane, further preferably weighs during lactalbumin powder is dissolved in ultra-pure water and is configured to
Weight/mass percentage composition is 10% solution, and stirring adds glycerol, ferulic acid and described restructuring laccase, magnetic agitation to make which abundant
PH is adjusted with NaOH or HCl solution after dissolving film liquid is formed into 5, will be permanent as 85 DEG C into after the process of film liquid ultrasonic degas
30, min are heated in warm water domain, film forming in coating film forming or importing plane container.
Beneficial effect：
The present invention is used as cross-linking agent by adding heat stability restructuring laccase oxygenase, while using high-temperature heating treatment breast
The processing method of albumin substrate, makes the catalytic effect of enzyme preparation reach coordinative role with the effect of protein heat denatured, makes
Standby lactalbumin membrane.Prepared lactalbumin membrane, green edible, the equal table of tensile strength, light transmission, absorption of water, plasticity
Reveal good property, performance is better than the matched group using fungal laccase process and unused ferment treatment, with important application valency
Value.
Description of the drawings
Fig. 1 restructuring fmb-103 laccase gene expression vector establishment processes
Fig. 2 is without the ferulic acid lactalbumin film-formation result sour with addition ferulic acid
A is without ferulic acid, without the lactalbumin film-formation result of enzyme
B is addition ferulic acid, without the lactalbumin film-formation result of enzyme
Fig. 3 is without heat stability restructuring laccase and the lactalbumin film-formation result for adding enzyme
A is the lactalbumin film-formation result without heat stability restructuring laccase
B is the lactalbumin film-formation result for adding heat stability restructuring laccase
The lactalbumin film-formation result of Fig. 4 addition heat stability restructuring laccases and ferulic acid
Biomaterial preservation information
Dead paddy bacillus cereuss fmb-103, Classification And Nomenclature are Bacillus vallismortis, are preserved in Chinese micro- life
Thing culture presevation administration committee common micro-organisms center, preservation date are on 06 08th, 2012, court of preservation address Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of positive area's North Star West Road 1, preserving number are CGMCC No.6198.
Specific embodiment
The invention will be further elaborated by the following examples.
Embodiment 1：The clone of dead paddy bacillus cereuss (Bacillus vallismortis) fmb-103 laccase genes
Dead paddy bacillus cereuss (Bacillus vallismortis) fmb-103 thalline (CGMCC are collected by centrifugation
No.6198), dead paddy bacillus cereuss (Bacillus is extracted with Shanghai life work genome DNA extracting reagent kit
Vallismortis) the genomic DNA of fmb-103.
Two are designed according to listed bacillus cereuss laccase gene (No.GU972592.1) in Genebank data bases to draw
Thing：
Forward primer F-1：5’-ATGACACTTGAAAAATTTGTGGATGC-3’(SEQ ID NO.3)；
Downstream primer R-1：5’-TTATTTATGGGGATCAGTTATATC-3’(SEQ ID NO.4)；
In 50 μ l systems, primer final concentration is respectively 1 μM, the final concentration of 0.2mM of dNTPs, fmb-103 strain gene groups
DNA10ng, 2U Pfu archaeal dna polymerases.Amplification program is 94 DEG C of 3min；30 × (94 DEG C of 40s, 53 DEG C of 50s, 72 DEG C of 90s)；72
℃10min.Agarose gel electrophoresiies, cut glue, are reclaimed using Shanghai life work test kit, by the PCR primer for reclaiming with
TaKaRapMD19-simple-T vector connect, and Transformed E .coli DH5 α are applied to containing IPTG, X-gal, ampicillin
LB flat boards, 37 DEG C of culture 13-14 hours choose white colony, and shaken cultivation is extracted plasmid, is sent to after determining successful connection
Hai Shenggong is sequenced, and analyzes sequencing result with computer software DNAMAN, obtains length of the sequence as shown in SEQ ID NO.1
For the ORF of 1542bp, i.e. fmb-103 laccase genes (fmb-L103), the protein being made up of 514 aminoacid is encoded,
Sequence is as shown in SEQ ID NO.2.
Embodiment 2：Dead paddy bacillus cereuss (Bacillus vallismortis) fmb-103 laccase gene prokaryotic expressions
The structure (accompanying drawing 1) of carrier
According to the laccase gene sequence for obtaining, two primers are designed, forward primer adds SacI recognition sequences, downstream primer
Plus XhoI recognition sequences (underscore part is restriction enzyme recognition sequence):
Forward primer F-2：5′-CGCGAGCTCATGACACTTGAAAAATTTGTGGATGC-3′(SEQ ID NO.5),
Downstream primer R-2:5′-CCGCTCGAGTTATTTATGGGGATCAGTTATATC-3′(SEQ ID NO.6),
Each composition is added according to following PCR system, laccase gene is expanded:
PCR programs are：94℃3min；30×(94℃40s；53℃50s；72℃90s)；72℃10min.
Work PCR primer Purification Kit PCR primer, plus SacI, XhoI double digestion are given birth to Shanghai, inactivation, ethanol are sunk
Form sediment, ddH2O weights are molten, the carrier pET-23a connections for limiting enzymic digestion identical with appropriate use, convert bacillus coli DH 5 alpha.From turn
Change the several bacterium colonies of random picking on flat board, access LB fluid mediums, shaken cultivation extracts plasmid in a small amount, and electrophoresis is stagnant with electrophoresis
Plasmid afterwards enters performing PCR checking for template, and life work sequencing in Shanghai is sent to after determining successful connection.
Embodiment 3：Dead paddy bacillus cereuss (Bacillus vallismortis) fmb-103 laccase genes are in large intestine bar
Expression in bacterium
Escherichia coli expression host strain BL21 will be converted containing fmb-L103 expression plasmids pET-23a-fmb-L103
(DE3) pLysS, picking petite after cultivating 10-11 hours at 37 DEG C access the training of the 50ml LB liquid containing ampicillin
Foster base, 30 DEG C of overnight incubations of 70-90rpm, according to 1:40 volume ratio takes seed liquor and is added to containing ampicillin
100ml LB fluid mediums, 35 DEG C of 180rpm add IPTG (100 μ g/ of final concentration when vibrating 2-3 hours to OD600 about 0.6
Ml) induce.Thalline is collected by centrifugation after 1.5 hours.Broken thalline, is collected by centrifugation supernatant, obtains the dead paddy bacillus cereuss of restructuring
The crude enzyme liquid of (Bacillus vallismortis) laccase (fmb-rL103).
Laccase activity measure adopts 2,2 '-azine-two (3- ethyl-benzothiazole -6- sulfonic acid), and (ABTS) method is slightly modified：
Reaction system cumulative volume 3mL, including 5.0 citrate-phosphate salt buffers of 2.45mL 0.2mol/L pH, 0.5mL 6mmol/L
The enzyme extract of ABTS and 50 μ l 5.0 citrate-phosphate salt buffers of pH suitably dilution, 45 DEG C of front 3min for determining reaction
The incrementss of interior reactant liquor light absorption value OD at the 420nm, do blank to inactivate enzyme liquid.Will be 1 μm of ol of interior generation per minute anti-
The enzyme amount needed for thing is answered to be defined as an enzyme activity unit.Laccase activity computing formula：Laccase activity (U)=VAlways×ΔOD/(VEnzyme
×ε×Δt×10-6) × total enzyme enzyme liquid extension rate；Wherein, ε=3.6 × 104mol/cm；Δt：3min；ΔOD：In 3min
The changing value of absorbance OD；VAlways：In enzyme reaction, the cumulative volume of reactant liquor；VEnzyme：In enzyme reaction, the volume of enzyme liquid.Experiment repeats 3
It is secondary, average.
Engineering bacteria after pET-23a-fmb-L103 conversion BL21 (DE3) pLysS, laccase of recombinating are measured using said method
Yield be 12000U/ml fermentation liquids.
Embodiment 4：Dead paddy bacillus cereuss (Bacillus vallismortis) fmb-103 laccase (fmb- of restructuring
Isolating and purifying rL103)
The fmb-rL103 crude enzyme liquids obtained in example 3 are carried out using NTA (nickel post, GE Products) affinity chromatography
Isolate and purify.
Add 5mM imidazoles (final concentration) in sample, strengthen adsorption column.
Before loading, chromatographic column is balanced with 20mM imidazoles.Sample crosses three column materials, reaches fmb-rL103 and affinity column material
Expect well-bound purpose.
After end of the sample, with 100mM imidazoles eluting (respectively with 10 bed volumes), collect eluent and survey enzyme activity.It is above-mentioned
Restructuring laccase fmb-rL103 enzyme activity prepared by purification is 3.6U/mg albumen.
Embodiment 5：The preparation flow of lactalbumin packaging film
Weigh during lactalbumin powder is dissolved in ultra-pure water and be configured to weight/mass percentage composition 10%, glycerol and whey protein proportions 1:
4 are slowly added to obtain in whipping process film liquid, magnetic agitation which is fully dissolved after with NaOH or HCl solution adjust pH to
5, ferulic acid weight/mass percentage composition is 1%, adds restructuring fmb-rLac, and enzyme amount 15U/g is into film liquid.Will be permanent as 85 DEG C into film liquid
20min is heated in warm water domain, after liquid cooling to be filmed, ultrasonic degas process is poured into and is covered with the glass culture dish of plastic packaging film,
20 DEG C of reaction 2h of room temperature, 55 DEG C are dried 36h and take off film.
Embodiment 6：The measure of lactalbumin film properties
The measure of film organoleptic indicator：The stickiness of the color of subjective appreciation film, surface flatness and film, if easily take off film.
The measure of film thickness (mm)：On tested film, random (the most edge of membrane removal) takes five points, uses digimatic calipers
Its thickness is measured, its meansigma methods is taken and is recorded.
The measure of film folding line (FM)：By film doubling, the folding line of film is judged according to folding line degree, with 0-4 as index, 0
Indicate without folding line, 1 represents that folding line is slight, 2 represent film folding line substantially, 3 represent portion fractures after film doubling, and 4 represent film doubling
Part complete rupture.
The measure of light transmittance：Sample is cut into into the rectangle of certain specification (0.8mm*4.5cm), the interior light of cuvette is pasted into
Sliding surface, determines its transmittance i.e. light transmittance under the wavelength of 500nm, using empty cuvette as control.Each sample does 2 and puts down
OK.
Film water dissolubility is determined：Film sample (2cm × 2cm) is dried to constant weight under the conditions of 105 DEG C, according to dry, wet after weighing
The difference of weight calculates moisture. and the film for having determined moisture is put in the beaker for filling 30mL water, is dissolved at room temperature
24h, then film is dried to constant weight under the conditions of 105 DEG C, weigh, water solublity calculated according to the difference of dry weight twice.
Moisture-vapor transmission is determined:Diameter 3cm, the circular open plastic cup of depth 4cm are taken, the CaC1 for adding 3g to be dried2, will
The membrane sample of diameter 7cm is cut into, rim of a cup is covered, the interface between film and cup is sealed with paraffin.Each cup is placed in into bottom then
In the exsiccator of the distilled water of addition 1000mL, (25 DEG C, relative humidity is 100%).Weigh 1 time per 12h, continue 1 week.By cup
The incrementss of weight determine the transit dose of vapor.Vapor transfer rate (WVTR) is calculated by ASTM method (E96-93,1993)
With moisture-vapor transmission (WVP).
Measuring Mechanical Properties：Thin film is cut into into the strip of 4cm*2cm specifications, and is fixed on measure on physical property instrument.Arrange
The initial distance of two fixture of physical property instrument is 20mm, and speed is 1mm/sec.After measure terminates, recording film break moment power (g) and
Film rupture when two fixtures apart from L.Each sample takes three parallel samples and determines.
Tensile strength TS (g)：When being broken with film, the power of moment represents tensile strength.
Elongation at break E (%) is calculated as follows：
E=(L1-L0)/L0
In formula：The elongation at break (%) of E-film；
The distance (mm) of two folder fixtures when L1-film ruptures；
Distance (mm) when L0-two fixture is initial.
According to the method described above, to laccase treatment of recombinating, the lactalbumin of the two methods preparation for not increasing group laccase treatment
Film properties index is to such as table 1：
Lactalbumin film properties prepared by 1 different disposal of table
Claims (8)
1. a kind of lactalbumin membrane, it is characterised in that the lactalbumin membrane is by lactalbumin, glycerol, weight are added in pure water
Group laccase and ferulic acid obtain into film liquid, into film liquid in 80-90 DEG C of heating in water bath 15-30min, pour the glass training for being covered with plastic packaging film into
In foster ware, 50-55 DEG C is dried 36-48h and takes off obtained by film；The aminoacid sequence such as SEQ ID NO.2 of wherein described restructuring laccase
It is shown；Wherein, the described weight/mass percentage composition into lactalbumin in film liquid is 8%-12%, glycerol and lactalbumin quality ratio
For 1:4-1:5, ferulic acid weight/mass percentage composition is into the 1%-1.2% of film liquid quality, restructuring laccase amount be 10U/g-20 U/g into
Film liquid.
2. lactalbumin membrane according to claim 1, it is characterised in that the encoding gene such as SEQ of described restructuring laccase
Shown in ID NO.1.
3. lactalbumin membrane according to claim 2, it is characterised in that described restructuring laccase is prepared via a method which：
（1）With dead paddy bacillus cereuss fmb-103 genomic DNAs template, forward primer and sequence of the sequence for SEQ ID NO.3
It is classified as the downstream primer laccase gene sequence of the synthesis containing SacI and XhoI restriction enzyme sites of SEQ ID NO.4；
（2）By the SacI/XhoI enzyme action of the PCR primer of previous step purification insertion vector pET-23a Jing after SacI, XhoI double digestion
Between site, the expression plasmid pET-23a-fmb-L103 containing laccase gene is obtained；
（3）Escherichia coli expression host strain BL21 will be converted containing fmb-L103 expression plasmids pET-23a-fmb-L103
(DE3) pLysS, picking petite after cultivating 10-11 hours at 37 DEG C access the training of the 50ml LB liquid containing ampicillin
Foster base, 30 DEG C of overnight incubations of 70-90rpm, according to 1:40 volume ratio takes seed liquor and is added to containing ampicillin
100ml LB fluid mediums, 35 DEG C of 180rpm vibrate when 2-3 hours are 0.6 to OD600 and add IPTG to induce, and are centrifuged after 5 hours
Collects thalline；The final concentration of 100 μ g/ml of IPTG；
（4）The thalline that abduction delivering is collected, with the resuspended thalline of phosphate buffer, ultrasonic disruption thalline, 4 DEG C, 10 000
× g is centrifuged 10 min, collects supernatant as crude enzyme liquid, the crude enzyme liquid that process is obtained is said according to Ni-NTA His Tag Kit
Bright book carries out purification and obtains described restructuring laccase.
4. lactalbumin membrane according to claim 1, it is characterised in that the described quality hundred into lactalbumin in film liquid
Content is divided to be 10%, glycerol is 1 with whey protein proportions:4, ferulic acid weight/mass percentage composition is 1%, and restructuring laccase enzyme amount is 15U/
G, is 5 into film liquid pH.
5. a kind of method for preparing lactalbumin membrane, it is characterised in that add in pure water lactalbumin, glycerol, restructuring laccase and
Ferulic acid into film liquid, into film liquid in 80-90 DEG C of heating in water bath 15-25min, pour into and be covered with the glass culture dish of plastic packaging film,
50-55 DEG C is dried 36-48h and takes off film；The aminoacid sequence of wherein described restructuring laccase is as shown in SEQ ID NO.2；Wherein institute
The weight/mass percentage composition into lactalbumin in film liquid stated is 8%-12%, and glycerol and lactalbumin quality ratio are 1:4-1:5, Ah
Wei's acid weight/mass percentage composition is 1%-1.2%, and restructuring heat stability enzyme amount is 10U/g-20 U/g.
6. the method for lactalbumin membrane being prepared according to claim 5, it is characterised in that described restructuring laccase is by the following method
Prepare：
（1）With dead paddy bacillus cereuss fmb-103 genomic DNAs template, forward primer and sequence of the sequence for SEQ ID NO.3
It is classified as the downstream primer laccase gene sequence of the synthesis containing SacI and XhoI restriction enzyme sites of SEQ ID NO.4；
（2）By the SacI/XhoI enzyme action of the PCR primer of previous step purification insertion vector pET-23a Jing after SacI, XhoI double digestion
Between site, the expression plasmid pET-23a-fmb-L103 containing laccase gene is obtained；
（3）Escherichia coli expression host strain BL21 will be converted containing fmb-L103 expression plasmids pET-23a-fmb-L103
(DE3) pLysS, picking petite after cultivating 10-11 hours at 37 DEG C access the training of the 50ml LB liquid containing ampicillin
Foster base, 30 DEG C of overnight incubations of 70-90rpm, according to 1:40 volume ratio takes seed liquor and is added to containing ampicillin
100ml LB fluid mediums, 35 DEG C of 180rpm vibrate when 2-3 hours are 0.6 to OD600 and add IPTG to induce, after 5 hours from
Heart collects thalline；The final concentration of 100 μ g/ml of IPTG；
（4）The thalline that abduction delivering is collected, with the resuspended thalline of phosphate buffer, ultrasonic disruption thalline, 4 DEG C, 10 000
× g is centrifuged 10 min, collects supernatant as crude enzyme liquid, the crude enzyme liquid that process is obtained is said according to Ni-NTA His Tag Kit
Bright book carries out purification and obtains described restructuring laccase.
7. the method for preparing lactalbumin membrane according to claim 5, it is characterised in that described into milk surum egg in film liquid
White weight/mass percentage composition is 10%, and glycerol is 1 with whey protein proportions:4, ferulic acid weight/mass percentage composition is 1%, laccase of recombinating
Enzyme amount is 15U/g, is 5 into film liquid pH.
8. the method for preparing lactalbumin membrane according to claim 7, it is characterised in that weigh lactalbumin powder be dissolved in it is super
The solution that weight/mass percentage composition is 10% is configured in pure water, stirring adds glycerol, ferulic acid and described restructuring laccase, magnetic force
Stirring adjusts pH with NaOH or HCl solution after which is fully dissolved and forms into film liquid to 5, will process into film liquid ultrasonic degas
Afterwards, as heating 20min in 85 DEG C of constant temperature waters, coating film forming or import film forming in plane container.
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| CN201510066436.5A CN104672919B (en) | 2015-02-09 | 2015-02-09 | Method for preparing whey protein film from thermally stable recombinant laccase |
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| CN107325316B (en) * | 2017-07-27 | 2021-02-12 | 泰兴市东圣生物科技有限公司 | High-performance microbial-source polyglucosyl-based degradable membrane and preparation method thereof |
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| CN108570459B (en) * | 2018-04-10 | 2022-10-04 | 南京农业大学 | Method for producing recombinant bacterial laccase by high-efficiency fermentation |
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| CN115444141A (en) * | 2021-06-08 | 2022-12-09 | 天津大学 | High oxidation resistance soybean protein isolate-gallic acid compound and preparation method thereof |
| CN114703247B (en) * | 2022-06-07 | 2022-08-23 | 北京幸福能量健康科技有限公司 | High-absorptivity composite protein composition and preparation method and application thereof |
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