CN106086131B - A kind of processing and utilization method of the exceeded rice of cadmium - Google Patents
A kind of processing and utilization method of the exceeded rice of cadmium Download PDFInfo
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Abstract
The invention discloses a kind of processing and utilization methods of the exceeded rice of cadmium, the high gene of the polypeptide (BMP) and malt oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose hydrolase (MTHase) activity for selecting metal compatibility good by half design and rational and confirmatory experiment carries out artificial synthesized, building sequence is the affine polypeptide BMP of SEQ NO.1 metal, the MTHase that the MTSase and sequence that sequence is SEQ NO2 are SEQ NO3 shows the genetic engineering bacterium on Pichia pastoris surface respectively, fermented culture respectively, centrifugal dehydration and freeze-drying obtain 3 primary yeast genetic engineering bacterium thallus, then it adds it to respectively in the hydrolyzate and liquefying starch cream of the separating obtained cadmium albumen of cadmium rice after carrying out de- cadmium or catalysis reaction and carries out respective handling again, Achieve the purpose that cadmium rice takes off cadmium while producing and meets food polypeptide and seaweed sugar product.
Description
Technical field
The invention belongs to food processings and field of biotechnology, and in particular to a kind of to utilize biotechnology by the exceeded rice of cadmium
Drop cadmium and the method for being processed into rice protein peptide and trehalose.
Background technique
Cadmium rice refers generally to the rice of cadmium content exceeded (Chinese national standard 0.2mg/kg).In the past few decades, it is chased after due to unilateral
It asks the growth of GDP and environmental protection is thinked little of, Chinese soil pollution is increasingly severe, and the heavy metals exceeding standards such as rice cadmium are also increasingly
Seriously；Pan Genxing team, Nanjing agricultural university in 2008 purchases sample in the multiple markets above county level in the whole nation at random, the results showed that 10%
The commercially available rice cadmium of left and right is exceeded；Report that the ground such as Hunan, Jiangxi flow to the cadmium rice highest in Guangdong in Guangdong cadmium rice disturbance in 2013
Unexpectedly more than 6 times of national standard；Since difficulty is administered in land pollution, significant period of time China will face the processing of a large amount of cadmium rices from now on
Handle problem；Yang Jurong's et al. studies have shown that cadmium in rice mainly in conjunction with protein, it is less in conjunction with starch；And show
Go cadmium method to focus mostly on either physically or chemically, rare biological method removes cadmium, more lack by cadmium rice take off after cadmium simultaneously plus
Work at the products such as high value added product such as protein peptides, trehalose research.
Trehalose (Trehalose) is irreducibility made of being connected by two molecule glucoses with the glycosidic bond of α, α-1,1-
Disaccharides is known as " sugar of life " and " 20 since it has the excellent performances such as the degeneration-resistant protective effect excellent to organism
Centurial novel carbohydrate ", method for large scale production receives significant attention；Trehalose is primarily now with two enzymes method or single enzyme process
Production, two methods cut both ways, and two enzymes method is made from starch, and cost of material is low, but enzyme is at high cost, and most double enzyme reaction temperature
It spends lower；Single enzyme process is using maltose as raw material, and cost is relatively low for enzyme, but the purity of maltose is very big on conversion ratio height influence, and
High-purity malt sugar price is high, and therefore, cost of material is high.
The yeast cell of display protein or enzyme has the advantages that immobilization is reusable, and has preparation simply, can
The characteristics of High Density Cultivation obtains a large amount of thallus, and cost is relatively low, thus since last century end comes out, development is swift and violent, has perhaps
Multiple protein or enzyme are by the report of successful expression；(the Cd based on display technique such as Feng Xia2+It is inhaled in conjunction with peptide screening and yeast heavy metal
Attached research [J] Henan Agricultural Sciences, 2013,42 (9): 142-145) report shows dodecapeptide in saccharomyces cerevisiae EBY100
On, induction obtains that Cd can be adsorbed for 24 hours afterwards2+Saccharomycete, but it is to Cd2+Adsorption rate it is not high, only 30.4%；It is previous single, double
Production by Enzymes trehalose substantially utilizes Escherichia coli to produce single, double enzyme, and the catalysis reaction time is long, and enzyme cannot repeat
It utilizes, inventor, which once announces in CN104046665A, is applied to single Production by Enzymes seaweed using yeast display trehalose synthetase
Sugar, but it is not directed to two enzymes method, CN201510431597.X is reported while being shown malt oligosaccharide based mycose synthetase
(MTSase) and malt oligosaccharide based mycose hydrolase (MTHase) is in saccharomycete surface conversion Starch Production trehalose, but the two
Ratio and the inconvenient change with the variation of the different conditions such as raw material, still have any problem in actual production.
Summary of the invention
The present invention is directed to overcome the deficiencies of the prior art and provide a kind of processing and utilization method of exceeded rice of cadmium.
In order to achieve the above object, technical solution provided by the invention are as follows:
The processing and utilization method of the exceeded rice of cadmium includes the following steps:
(1) starch liquefacation enzyme will be added to decompose 30-60 minutes in 100-110 DEG C of conditions after the exceeded rice in steep of cadmium, defibrination
Afterwards, the starch milk that DE value is 3-12 is obtained, obtaining solid quality degree after removing cadmium albumen with flame filter press is
20-30% starch milk, the cadmium albumen being removed are retained spare；
(2) by after resulting cadmium albumen proteases for decomposing, with the Pichia pastoris gene work for showing metal affinity peptide (BMP)
Journey bacterium adhesion protein enzyme decomposes the heavy metals such as the cadmium of separate out, is then centrifuged the yeast removed after inhaling cadmium, collects
To drop cadmium rice protein peptide, drop cadmium rice protein peptide is used as food or feedstuff；
Resulting starch milk is pressed into 2-8 parts of displaying Fructus Hordei Germinatus oligoses of addition in every 200-300 parts of starch milks in parts by weight
The pichia yeast genetic engineering bacteria of base trehalose synthetase (MTSase) and 1-3 parts of displaying malt oligosaccharide based mycose hydrolases
(MTHase) pichia yeast genetic engineering bacteria carries out mistake after 15-20h of catalysis reaction under the conditions of 40-60 DEG C, pH4.5-7
Filter separation, will be separated by filtration rear resulting reaction solution again through centrifugal filtration, ion-exchange, be concentrated to get trehalose syrup；
Wherein, the DNA encoding sequence of the metal affinity peptide is as shown in SEQ NO.1；The malt oligosaccharide based mycose closes
At the DNA encoding sequence of enzyme as shown in SEQ ID NO.2；The DNA encoding sequence of the malt oligosaccharide based mycose hydrolase is such as
Shown in SEQ ID NO.3.The pichia yeast genetic engineering bacteria for showing metal affinity peptide is the preparation method is as follows: synthesis metal parent
With Peptide D NA sequence, then the DNA clone that synthesis is obtained further is subcloned into pPIC9K carrier on pUC57 carrier,
It is transformed into Pichia pastoris (Pichia Pastoris) GS115 (Pichia pastoris is commercial goods) again, obtains showing metal parent
With the pichia yeast genetic engineering bacteria of peptide.By the pichia yeast genetic engineering bacteria for showing metal affinity peptide successively in YPD and
In BMGY culture medium distinguish 16-32h of amplification cultivation and 96-120h, make metal affinity peptide yield with saccharomycete proliferation substantially
Increase.
It is described show malt oligosaccharide based mycose synthetase pichia yeast genetic engineering bacteria the preparation method is as follows: synthesis
Malt oligosaccharide based mycose synthetase DNA sequence dna, the DNA clone that synthesis is obtained is on pUC57 carrier, and then further sub- gram
It is grand then to be converted in Pichia pastoris (Pichia Pastoris) GS115 into pPIC9K carrier, it obtains showing Fructus Hordei Germinatus oligose Ji Hai
The pichia yeast genetic engineering bacteria of algae sugar synzyme.By the Pichia pastoris gene for showing malt oligosaccharide based mycose synthetase
Engineering bacteria successively distinguishes 16-32h of amplification cultivation and 96-120h in YPD and BMGY culture medium, makes malt oligosaccharide based mycose
Synthesis production of enzyme is significantly increased with the proliferation of saccharomycete.
It is described show malt oligosaccharide based mycose hydrolase pichia yeast genetic engineering bacteria the preparation method is as follows: synthesis
Malt oligosaccharide based mycose synthetase DNA sequence dna, the DNA clone that synthesis is obtained is on pUC57 carrier, and then further sub- gram
It is grand then to be converted in Pichia pastoris (Pichia Pastoris) GS115 into pPIC9K carrier, it obtains showing Fructus Hordei Germinatus oligose Ji Hai
The pichia yeast genetic engineering bacteria of algae glycosylhydrolase.By the Pichia pastoris gene for showing malt oligosaccharide based mycose hydrolase
Engineering bacteria successively distinguishes 16-32h of amplification cultivation and 96-120h in YPD and BMGY culture medium, makes malt oligosaccharide based mycose
Hydrolysis production of enzyme is significantly increased with the proliferation of saccharomycete.
Preferably, the pichia yeast genetic engineering bacteria of the displaying malt oligosaccharide based mycose synthetase after being separated by filtration
After showing that the pichia yeast genetic engineering bacteria of malt oligosaccharide based mycose hydrolase is rinsed, recycled again, it is recycled and reused for being catalyzed
Starch milk produces trehalose.
Preferably, the trehalose syrup that trehalose mass percent concentration is 20-70% is made in trehalose syrup, or
Person is further concentrated by seaweed syrup, crystallization, is centrifuged, obtains crystalline trehalose of the purity 98% or more after drying.
The invention will be further described below:
The technical problems to be solved by the invention are first is that the building better yeast gene engineering bacteria of adsorption effect comes more effectively
The cadmium of cadmium rice proteolysis separate out is sloughed on ground, second is that constructing the Fructus Hordei Germinatus oligose base seaweed by Starch Conversion at trehalose respectively
Sugared synzyme MTSase and malt oligosaccharide based mycose hydrolase MTHase is showed in the genetic engineering bacterium on saccharomycete surface, from skill
Production and application is further facilitated in art, is reduced the processing and utilization cost of rice production trehalose, is promoted the processing added value of cadmium rice,
Cadmium rice is reduced because the economic loss caused by can only selling cheap cannot be eaten.
The present invention is improved on the basis of forefathers' gene order by the methods of half design and rational, one system of design synthesis
New gene order is arranged, sequent synthesis and the building of multiple related gene engineering bacterias are carried out, cadmium parent is then selected by confirmatory experiment
It is shown for the BMP of SEQ NO.1 on Pichia pastoris surface with the good DNA sequence dna of property, and the higher DNA sequence dna of activity is SEQ
The MTSase and DNA sequence dna of NO.2 is that the MTHase of SEQ NO3 is shown respectively on Pichia pastoris surface, passes through these genetic engineerings
The fermented and cultured of bacterium obtains a large amount of yeast gene engineering bacterias, is added separately to cadmium rice protein hydrolyzate and rice starch liquefaction
De- cadmium and catalysis reaction are carried out in cream respectively, meets food industry polypeptide and lower to reach cadmium rice and take off cadmium while producing
The purpose of cost seaweed sugar product.
Wherein, show that the Pichia pastoris of metal affinity peptide BMP makes in this way: by metal affinity peptide DNA sequence dna SEQ
NO:1 is artificial synthesized, and the gene cloning for then obtaining synthesis is further subcloned into pPIC9K carrier on pUC57 carrier
In, then it is transformed into Pichia pastoris (Pichia Pastoris) GS115, the Pichia pastoris for showing BMP is obtained, then respectively successively
Distinguish 16-32h of amplification cultivation and 96-120h again in YPD and BMGY culture medium, centrifugal dehydration or freeze-drying obtain big
Amount shows the pichia yeast genetic engineering bacteria of BMP-GS115.
Wherein, malt oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose hydrolase are respectively shown
(MTHase) Pichia pastoris makes in this way: by the MTSase's of DNA sequence dna SEQ NO.2 and DNA sequence dna SEQ NO.3
MTHase is artificial synthesized, and then the gene that synthesis obtains is cloned in respectively on pUC57 carrier, is further subcloned into pPIC9K
In carrier, then it is transformed into Pichia pastoris (Pichia Pastoris) GS115, is shown finishing for MTSase and MTHase respectively
Red yeast, then successively distinguishes 16-32h of amplification cultivation and 96-120h again in YPD and BMGY culture medium respectively, and centrifugation is de-
Water or freeze-drying obtain the pichia yeast genetic engineering bacteria for largely showing MTSase or MTHase.
Cadmium rice albumen water is added to by 1-6 parts of saccharomycete of the displaying affine polypeptide of SEQ NO.1 metal by weight
Solution liquid be stirred de- cadmium 16-for 24 hours, be then centrifuged for de- cadmium yeast and peptide separation, take off cadmium rate up to 81% or more, energy
Enough achieve the purpose that cadmium rice protein sloughing most of cadmium.
The starch milk of gained solid content 20-30% is pressed every 200-300 parts by weight, 2-8 parts of exhibitions are respectively added
Show the pichia yeast genetic engineering bacteria and 1-3 parts of displaying sequences of sequence SEQ NO:2 malt oligosaccharide based mycose synthetase
The pichia yeast genetic engineering bacteria of SEQNO:3 malt oligosaccharide based mycose hydrolase under the conditions of 40-60 DEG C, pH4.5-7 into
Row catalysis reaction 15-20h after, be separated by filtration, gained reaction solution through centrifugal filtration, ion-exchange, be concentrated to get trehalose sugar
Slurry.
Compared with prior art, of the invention to be advantageous in that: by the conversion ratio of Starch Production trehalose reach 81% with
On, reaction temperature is 40-60 DEG C, and the reaction time shortens, as long as 15-20h, and separating obtained genetic engineering bacterium is logical after reaction
It repeats after crossing washing and filtering for catalytic starch cream production trehalose, after reusing 5 times, enzyme activity still keeps former
63.8% come, thus trehalose production cost substantially reduces.
Specific embodiment
The present invention is more specifically described in detail below by way of specific embodiment, but embodiments of the present invention are unlimited
Routine techniques progress can refer to for not specifically specified technological parameter in this.
1 Pichia pastoris of embodiment shows metal rabphilin Rab MBP
Metal affinity peptide DNA sequence dna SEQ NO.1 is artificial synthesized, and the gene cloning for then obtaining synthesis is carried in pUC57
It on body, is further subcloned into pPIC9K carrier, then is transformed into Pichia pastoris (Pichia Pastoris) GS115, opened up
Show the Pichia pastoris of BMP, then successively distinguishes 16-32h of amplification cultivation and 96-again in YPD and BMGY culture medium respectively
120h, centrifugal dehydration or freeze-drying obtain the pichia yeast genetic engineering bacteria for largely showing BMP-GS115.
2 Pichia pastoris of embodiment shows that metal rabphilin Rab takes off Cadmium treated
The exceeded rice of cadmium is soaking, defibrination, liquefaction, filter residue is cadmium albumen obtained by filters pressing, which the weight such as is added
Water after, 1-2% protease hydrolytic 16-is added by weight for 24 hours, then with NaOH tune pH to 8-10, by weight will
The saccharomycete MBP of the metal affinity peptide of 1-3% displaying SEQ NO:1 is added to cadmium rice protein hydrolyzate and is stirred 12-for 24 hours,
It is separated with centrifuge, gained precipitating is rich cadmium yeast, collects supernatant acid adding tune pH to 6-8, is after gained precipitating is dry
Rice protein peptide can be used as food or feedstuff.
3 Yeast engineering bacteria of embodiment takes off the measurement of cadmium degree
Cd is measured by National Standard Method atomic absorption spectrophotometer2+Then mass of ion concentration calculates absorptivity, Jin Erji
The adsorption rate to cadmium is calculated, measurement result is as shown in table 1:
1 MBP Yeast engineering bacteria of table takes off cadmium effect
4 Pichia pastoris of embodiment shows malt oligosaccharide based mycose synthetase (MTSase)
As shown in SEQ NO2, gene cloning carries malt oligosaccharide based mycose synthetase (MTSase) DNA sequence dna in pUC57
On body, successively MTSase is subcloned into pPIC9K carrier, using plastic recovery kit recycling target gene fragment and
PPIC9K carrier segments, are eluted with water respectively, are attached reaction with T4DNA Ligase later, then are transformed into DH5a competence
In cell, it is coated on the LB (Miller broth (1%NaCl): 1%peptone, 0.5% containing amp and kana resistance
Yeast extract, and 1%NaCl) 37 DEG C are carried out on solid medium is incubated overnight；Picked clones carry out shaking bacterium, plasmid
Extracting, and carry out the identification of Eco RI and Not I double digestion；After constructing pPIC9K-MTSase carrier, it is transformed into Bi Shi ferment
In female GS115.
5 Pichia pastoris of embodiment shows bud oligosaccharide based mycose synthetase (MTHase)
As shown in SEQ NO3, gene cloning carries malt oligosaccharide based mycose hydrolase (MTHase) DNA sequence dna in pUC57
On body, successively MTHase is subcloned into pPIC9K carrier, using plastic recovery kit recycling target gene fragment and
PPIC9K carrier segments, are eluted with water respectively, are attached reaction with T4DNALigase later, then are transformed into DH5a competence
In cell, it is coated on the LB (Miller broth (1%NaCl): 1%peptone, 0.5% containing amp and kana resistance
Yeast extract, and 1%NaCl) 37 DEG C are carried out on solid medium is incubated overnight；Picked clones carry out shaking bacterium, plasmid
Extracting, and carry out the identification of Eco RI and Not I double digestion；After constructing pPIC9K-MTSase carrier, it is transformed into Bi Shi ferment
In female GS115.
Genetic engineering bacterium amplification cultivation
1), the picking single colonie from plate is inoculated in YPD culture medium, and 30 DEG C, 250rpm cultivates 16-32h；
2) it, is transferred to by 1% inoculum concentration in fresh BMGY culture medium, 30 DEG C, 250rpm cultivates 5d, adds every for 24 hours
Methanol induction is collected fermentation thalli, is washed 3 times with pH8.0 buffer, is resuspended with a small amount of buffer and is closed to get recombination trehalose
It is at enzyme gene engineering bacteria bacterium solution, its centrifugal dehydration or freeze-drying is spare.
Embodiment 6 produces trehalose using rice starch cream
Gained is consolidated into the starch milk containing 20-30% by weight by 2-6 parts of displaying sequences of every 200-300 parts each additions
The pichia yeast genetic engineering bacteria of SEQNO:2 malt oligosaccharide based mycose synthetase (MTSase) and 1-3 parts of displaying sequences
The pichia yeast genetic engineering bacteria of SEQNO:3 malt oligosaccharide based mycose hydrolase (MTHase) is in 40-60 DEG C, pH4.5-7
Under the conditions of carry out catalysis reaction 15-20h after, be separated by filtration, gained reaction solution through centrifugal filtration, ion-exchange, be concentrated to get sea
Algae sugar syrup.
Then the analysis of various sugared contents is carried out to reaction product with HPLC, the results are shown in Table 2:
Conversion ratio and opposite enzyme activity of the double enzymes of yeast display to starch after table 2 is reused
The production of 7 crystalline trehalose of embodiment
Trehalose syrup obtained by above-mentioned production can need that trehalose of the trehalose concentration 20-70% is made according to market
Syrup can also obtain crystalline trehalose of the purity 98% or more after concentrated, crystallization, centrifugation, drying.
Claims (9)
1. a kind of processing and utilization method of the exceeded rice of cadmium, which is characterized in that described method includes following steps:
(1) starch liquefacation enzyme will be added to decompose 30-after sixty minutes in 100-110 DEG C of conditions after the exceeded rice in steep of cadmium, defibrination, obtained
The starch milk for being 3-12 to DE value obtains the shallow lake that solid quality degree is 20-30% after being filtered to remove cadmium albumen
Powder cream, the cadmium albumen being removed are retained spare；
(2) it by after resulting cadmium albumen proteases for decomposing, is adsorbed with the pichia yeast genetic engineering bacteria for showing metal affinity peptide
The heavy metal of proteases for decomposing separate out, is then centrifuged the yeast removed after inhaling cadmium, and collection obtains drop cadmium rice egg
Drop cadmium rice protein peptide is used as food or feedstuff by white peptide；
Resulting starch milk is pressed into 2-8 parts of displaying Fructus Hordei Germinatus oligose Ji Hai of addition in every 200-300 parts of starch milks in parts by weight
The Pichia pastoris base of the pichia yeast genetic engineering bacteria of algae sugar synzyme and 1-3 parts of displaying malt oligosaccharide based mycose hydrolases
Because of engineering bacteria, it is separated by filtration after 15-20h of catalysis reaction are carried out under the conditions of 40-60 DEG C, pH4.5-7, after being separated by filtration
Resulting reaction solution again through centrifugal filtration, ion-exchange, be concentrated to get trehalose syrup；
The DNA encoding sequence of the metal affinity peptide is as shown in SEQ NO.1；The DNA of the malt oligosaccharide based mycose synthetase
Coded sequence is as shown in SEQ ID NO.2；The DNA encoding sequence of the malt oligosaccharide based mycose hydrolase such as SEQID NO.3
It is shown.
2. the method as described in claim 1, which is characterized in that the pichia yeast genetic engineering bacteria for showing metal affinity peptide
The preparation method is as follows: synthesis metal affinity peptide DNA sequence dna, the DNA clone that synthesis is obtained is on pUC57 carrier, then into one
Step is subcloned into pPIC9K carrier, then is transformed into Pichia pastoris GS115, and the Pichia pastoris base for showing metal affinity peptide is obtained
Because of engineering bacteria.
3. method according to claim 2, which is characterized in that by the pichia pastoris gene engineering for showing metal affinity peptide
Bacterium successively distinguishes 16-32h of amplification cultivation and 96-120h in YPD and BMGY culture medium, makes metal affinity peptide yield with yeast
The proliferation of bacterium is significantly increased.
4. the method as described in claim 1, which is characterized in that the displaying malt oligosaccharide based mycose synthetase finishes red ferment
Female genetic engineering bacterium the preparation method is as follows: synthesis malt oligosaccharide based mycose synthetase DNA sequence dna, will the obtained DNA of synthesis
It is cloned on pUC57 carrier, is then further subcloned into pPIC9K carrier, then convert in Pichia pastoris GS115, is opened up
Show the pichia yeast genetic engineering bacteria of malt oligosaccharide based mycose synthetase.
5. method as claimed in claim 4, which is characterized in that show that finishing for malt oligosaccharide based mycose synthetase is red for described
Yeast gene engineering bacteria successively distinguishes 16-32h of amplification cultivation and 96-120h in YPD and BMGY culture medium, makes Fructus Hordei Germinatus oligose
Base trehalose synthetase yield is significantly increased with the proliferation of saccharomycete.
6. the method as described in claim 1, which is characterized in that the displaying malt oligosaccharide based mycose hydrolase finishes red ferment
Female genetic engineering bacterium the preparation method is as follows: synthesis malt oligosaccharide based mycose synthetase DNA sequence dna, will the obtained DNA of synthesis
It is cloned on pUC57 carrier, is then further subcloned into pPIC9K carrier, then convert in Pichia pastoris GS115, is opened up
Show the pichia yeast genetic engineering bacteria of malt oligosaccharide based mycose hydrolase.
7. method as claimed in claim 6, which is characterized in that show that finishing for malt oligosaccharide based mycose hydrolase is red for described
Yeast gene engineering bacteria successively distinguishes 16-32h of amplification cultivation and 96-120h in YPD and BMGY culture medium, makes Fructus Hordei Germinatus oligose
Base hydrolysis of trehalose production of enzyme is significantly increased with the proliferation of saccharomycete.
8. method as described in any one of claim 1 to 7, which is characterized in that the displaying Fructus Hordei Germinatus oligose after being separated by filtration
The pichia yeast genetic engineering bacteria of base trehalose synthetase and the Pichia pastoris gene for showing malt oligosaccharide based mycose hydrolase
After engineering bacteria is rinsed again, recycled, it is recycled and reused for catalytic starch cream production trehalose.
9. method as described in any one of claim 1 to 7, which is characterized in that trehalose quality hundred is made in trehalose syrup
Dividing specific concentration is 20-70% trehalose syrup, or after seaweed syrup is further concentrated, crystallization, centrifugation, drying
Crystalline trehalose to purity 98% or more.
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