CN107365782A - A kind of genetic engineering bacterium and its application - Google Patents

A kind of genetic engineering bacterium and its application Download PDF

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CN107365782A
CN107365782A CN201710757239.7A CN201710757239A CN107365782A CN 107365782 A CN107365782 A CN 107365782A CN 201710757239 A CN201710757239 A CN 201710757239A CN 107365782 A CN107365782 A CN 107365782A
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李必金
胡丹
吴涛
刁刘洋
李岩
王成
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Langfang Meihua Bio Technology Development Co Ltd
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12Y302/01141—4-Alpha-D-{(1->4)-alpha-D-glucano} trehalose trehalohydrolase (3.2.1.141)
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    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99015—(1->4)-Alpha-D-glucan 1-alpha-D-glucosylmutase (5.4.99.15)

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Abstract

The invention belongs to gene engineering technology field, discloses a kind of genetic engineering bacterium and its application.Genetic engineering bacterium of the present invention can express MTSase and MTHase simultaneously in same strain bacterium, and regulate and control the expression of two kinds of enzymes in translation skill, realize the high efficient expression of the varying level of two kinds of enzymes.Genetic engineering bacterium of the present invention can ferment simultaneously prepares two kinds of enzymes of MTSase and MTHase, it is only necessary to which a set of equipment is with regard to that can meet production requirement.And two kinds of expression of enzymes amount proportionings of MTSase and MTHase are fixed, and production is more stable.The present invention adjusts the expression of the MTSase and MTHase enzymes of the genetic engineering bacterium by molecular biology method so that two kinds of enzymes obtain optimum proportioning, and synergy is strong, and highest conversion ratio is obtained in the most short time.Experiment shows the product production trehalose obtained using genetic engineering bacterium of the present invention expression, and the conversion ratio of trehalose is up to 87%.

Description

A kind of genetic engineering bacterium and its application
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of genetic engineering bacterium and its application, more particularly, to The genetic engineering bacterium of a kind of malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase coexpression and its in sea Application in algae sugar production.
Background technology
Trehalose is the irreducibility disaccharide formed by two pyranoid ring glucose molecules by α -1, the condensation of 1 glycosidic bond, With characteristic of biological activity is protected under extreme conditions, the good reputation of " sugar of life " is known as in scientific circles, and be widely used in In food, medicine and cosmetic industry.The production method of trehalose mainly includes microorganism extraction process, fermentation method, enzyme turn at present Change method and method of gene recombination.Enzyme transforming process is to be adapted to industrialization due to the advantages that technique is simple, high conversion rate, cheap cost The desirable route of production.
The biosynthesis pathway of enzymatic clarification trehalose has a variety of, wherein trehalose-6-phosphate synthase and trehalose -6- It is raw material that phosphate conversion method, trehalose phosphorylase conversion method, which are required to energy-rich compound, and cost is higher and phosphorylase It is unstable, be not suitable for industrialized production.Trehalose synthase conversion method is to pass through intramolecular transglucosidation by substrate of maltose Trehalose is directly produced, the enzyme can act on maltose generation trehalose and its back reaction trehalose generation maltose, from fat Bacillus (Pimelobacter species.R48), Pseudomonas putidas (Pseudomonas putida H626) and aquatic The trehalose synthase (Trehalose synthase, EC5.4.99.16) of extraction in Thermophilic Bacteria (The rmus aquaticus), It can be catalyzed in maltose molecule and turn glycosides effect, the maltose of α-Isosorbide-5-Nitrae glucosides key connection is converted into α -1,1 glycosidic bond, maltose It is transformed into trehalose.For trehalose synthase conversion method using maltose as direct substrate, technique is simple, but reaction temperature is low (general For 20-30 DEG C), the reaction time is longer, and trehalose synthase catalysis is a reversible reaction, when reaction proceeds to certain journey The conversion of maltose and trehalose can reach relative equilibrium when spending, therefore conversion ratio only up to reach 60% or so.And malt Oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose hydrolase (MTHase) conversion method, can be formed sediment with straight chain Powder is substrate, after conversion end product be trehalose and a small amount of glucose, maltose and maltotriose, this pair of enzyme system Do not need phosphate to coexist, and more than 80% can reach to Starch Conversion rate, be preferable industrialized production approach.
The researcher of Japanese woods primaryization research institute and Japanese kylin company is from arthrobacterium (Arthrabacter Sp.Q36), it is purified into sulfolobus acidocaldarius (Sulfolbus acidocaldarius) and sulphur ore deposit sulfolobus solfataricus and this turn Change related enzyme, and illustrate the enzyme system and trehalose synthesis mechanism of this trehalose route of synthesis.The enzyme system includes wheat Bud oligosaccharide based mycose synthetase (Maltooligosyl trehalose synthase, MTSase, EC5.4.99.15) and wheat Bud oligosaccharide based mycose hydrolase (Maltooligosyl trehalose trehalohyrolase, MTHase, EC3.2.1.141).MTSase and MTHase cooperates with Fructus Hordei Germinatus oligose or the amylose synthesis of different DP values (DP >=3) Go out trehalose, the process is to phosphate and no dependence.Wherein MTSase acts on the C1-OH of substrate reducing end under neutral, produces α-Isosorbide-5-Nitrae glycosidic bond to α -1, the intramolecular transglycosylation of 1 glycosidic bond, form intermediate product malt oligosaccharide based mycose； MTHase α-Isosorbide-5-Nitrae glycosidic bonds that then exclusively Fructus Hordei Germinatus oligose base is connected with trehalose in the inscribe intermediate product, it is allowed to decompose production Raw trehalose and the new Fructus Hordei Germinatus oligose for reducing by 2 glucose units, reducing the new Fructus Hordei Germinatus oligose of 2 glucose units can be used as newly Substrate carry out next round reaction, so alternately and repeatedly carry out 2 kinds of enzyme reaction cans and convert Fructus Hordei Germinatus oligose or amylose Into predominantly trehalose, and a small amount of glucose, maltose, the product of maltotriose.Such as coordinate other amylolytic enzymes, such as Alpha-amylase, isoamylase, glucoamylase etc., the conversion ratio that Starch Conversion is trehalose can be made up to more than 80%.Double enzymes Method is the main method of current trehalose industrialized production.
Because MTHase substrate specificity is MTSase reaction product, therefore two kinds of being converted with comparison trehalose for enzyme have Significantly affect, when the timings of MTSase mono-, MTHase deficiencies, transglycosylation is fast, and MTHase hydrolysis is slow, causes intermediate product wheat Bud oligosaccharide based mycose accumulates；When MTHase excess, transglycosylation speed is slow, and intermediate product malt oligosaccharide based mycose is not Foot, trehalose conversion is slow, and reaction time is grown.Therefore need to find two kinds of enzymes and suitably match, so as to obtain highest marine alga Sugared conversion ratio.
Japanese woods original discloses a kind of new non-reducing saccharide generation enzyme and its system in Chinese patent CN100352920C Standby and application, i.e. MTSase, this enzyme can be from Rhizobium sp.M-11 (FERMBP4130) and Arthrobacter Obtained in the cultures of microorganism such as sp.Q36 (FERMBP4316), can when it acts on reproducibility Starch Fraction hydrolysate Non-reducing saccharide of the generation with trehalose structure.When glucoamylase and alpha-Glucosidase act on this irreducibility During carbohydrate, it is easy to produce trehalose.Japanese woods is former to disclose a kind of trehalose release in Chinese patent CN1204249C simultaneously Enzyme, i.e. MTHase, this enzyme are also from Rhizobium sp.M-11 (FERMBP4130) and Arthrobacter sp.Q36 Etc. (FERMBP4316) obtained in the culture of microorganism, it can specific for hydrolysis there is trehalose structure as an end list Key in the non-reduced polysaccharide that position and glucose polymerization degree are 3 or higher between trehalose moiety and remaining glycosyl part, then By column chromatographic isolation and purification trehalose, last condensing crystallizing, which is dried, obtains finished product trehalose.
Southern Yangtze University researcher discloses a kind of high yield Fructus Hordei Germinatus oligose base marine alga in Chinese patent CN106190880A The genetic engineering bacterium of sugared synzyme and its application, using pPIC3.5k as carrier, using Pichia pastoris KM71 as expression place It is main, the malt oligosaccharide based mycose synthetase in expression Sulfolobus acidocaldarius sources, obtain recombinant bacterium pPIC3.5k-treY/P.pastoris.Fermentation production malt oligosaccharide based mycose synthetase is lived up to 3128.7U/mL.Exist simultaneously A kind of genetic engineering bacterium of high yield malt oligosaccharide based mycose hydrolase is disclosed in Chinese patent CN105969713A and its is answered With with pET-32a (+) for carrier, with E.coli Origami (DE3) for expressive host, constructing high yield Fructus Hordei Germinatus oligose Ji Hai The genetic engineering bacterium of algae glycosylhydrolase, enzymatic production is carried out in fermentation tank using the bacterial strain as production bacterial strain, enzyme activity can reach 204U/ mL。
A kind of malt oligosaccharide based mycose is individually disclosed in Chinese patent CN103194432A and CN103194434A to close Into enzyme and malt oligosaccharide based mycose hydrolase, the gene of enzyme, the recombinant expression carrier containing the gene and recombination engineering and The preparation method of enzyme, use pET-24a (+) as carrier, using E.coli BL21 as expressive host, the MTSase of fermentation expression and MTHase enzymes have higher optimal reactive temperature and heat endurance, have relatively low optimal pH, reduce microbiological contamination risk, improve The stability of production, significantly improve the effect of itself and Pullulanase synergy reproducibility glucidtemns production trehalose Rate, significantly improve trehalose production efficiency.
But be mostly at present two plants of bacterium of structure using two enzymes method conversion production trehalose, Fructus Hordei Germinatus oligose Ji Hai is expressed respectively Algae sugar synzyme (MTSase) and malt oligosaccharide based mycose hydrolase (MTHase), are fermented by two fermentation tanks respectively Producing enzyme, then crush, centrifuge two kinds of enzyme liquids of preparation, added according to a certain percentage according to enzyme activity after measure enzyme activity and enzyme is carried out in substrate Solution conversion, enzyme processed is fermented respectively, it is necessary to which two complete equipments, equipment investment are big, it is necessary to every batch of measure enzyme activity, two kinds of enzymes using two plants of bacterium Adding proportion every batch is required for adjusting, and enzyme adds proportioning is improper can cause to digest conversion ratio reduction, and final influence production is stable.I.e. Make to have two kinds of enzymes of expression in same strain bacterium, but the expression of enzyme is not regulated and controled, cause the expression ratio of two kinds of enzymes not Properly, influence finally to digest conversion ratio.
The content of the invention
In view of this, it is an object of the invention to for prior art the defects of, build a kind of malt oligosaccharide based mycose The genetic engineering bacterium of synzyme (MTSase enzymes) and malt oligosaccharide based mycose hydrolase (MTHase enzymes) coexpression, realizes two The high efficient expression of kind enzyme varying level, and produced applied to trehalose.
To realize the purpose of the present invention, the present invention adopts the following technical scheme that：
A kind of gene, by the DNA molecular of coding malt oligosaccharide based mycose synthetase, ribosome bind site sequence (RBS Sequence), coding malt oligosaccharide based mycose hydrolase DNA molecular be connected in sequence.The gene can co-express malt Oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose hydrolase (MTHase).
In some embodiments, it is described coding malt oligosaccharide based mycose hydrolase DNA molecular be from Arthrobacter sp.Q36 treY genes.The DNA molecular of the coding malt oligosaccharide based mycose hydrolase derives from Arthrobacter sp.Q36 treZ genes.
Further, its nucleotide sequence of DNA molecular such as SEQ of the coding malt oligosaccharide based mycose synthetase IDNO:Shown in 1.Its nucleotide sequence of DNA molecular such as SEQ IDNO of the coding malt oligosaccharide based mycose hydrolase:3 institutes Show；Its nucleotide sequence of ribosome bind site sequence such as SEQ IDNO:5-9 is shown in any one.
It is the carrier comprising said gene present invention also offers a kind of recombinant vector.Wherein, institute in the recombinant vector State DNA molecular of the DNA molecular positioned at coding malt oligosaccharide based mycose hydrolase of coding malt oligosaccharide based mycose synthetase Upstream.
In one embodiment of the invention, the recombinant vector, it is that the gene is connected to pET-28a (+) expression Obtained on carrier.
Present invention also offers a kind of genetic engineering bacterium, includes above-mentioned recombinant vector.The recombinant vector is transformed into place Malt oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose hydrolase (MTHase) table altogether are obtained in main bacterium The genetic engineering bacterium reached.
Further, in some embodiments, the ribosomes in the recombinant vector of the genetic engineering bacterium combines Site sequence such as SEQ IDNO:Shown in 7.
Further, in some embodiments, the ribosome binding site in recombinant vector described in the genetic engineering bacterium Point sequence such as SEQ IDNO:Shown in 7, carrier is pET-28a (+), and the Host Strains are E.coli BL21 (DE3) bacterial strain, life Entitled Escherichia coli SH003.
Present invention also offers the preparation method of the genetic engineering bacterium, including：
Step A, the gene sequence of coding malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase is obtained Row, and connection ribosome bind site sequence；
Step B, recombinant vector conversion Host Strains are built, wherein the recombinant vector is included by coding Fructus Hordei Germinatus oligose base marine alga The DNA molecular of sugared synzyme, ribosome bind site sequence, encode the DNA molecular of malt oligosaccharide based mycose hydrolase successively Be formed by connecting gene, and the DNA molecular of the coding malt oligosaccharide based mycose synthetase is positioned at coding malt oligosaccharide based mycose The upstream of the DNA molecular of hydrolase；
The present invention by molecular biology method from the double expression of enzymes of transcription and translation Level tune come build MTSase and MTHase coexpression bacterial strain, realizes the requirement of two kinds of enzyme variable expressions, so as to control two kinds of enzymes suitably matching, with up to The efficient production of trehalose under to the transformation system.Wherein, the double expression of enzymes of the transcriptional level control are double by single promoter Enzyme duplex is expressed to build co-expression plasmid.The double expression of enzymes of translation skill regulation and control are by changing ribosome bind site sequence The interval of (RBS sequences) between initiation codon, and then translation initiation speed is influenceed, protein table is adjusted in translation skill Up to amount.
The double enzyme duplex expression ways of single promoter of the present invention, it is few using the T7 promoters carried in pET systems, malt The RBS sequences of glycosyl trehalose synthetase gene (treY) use the RBS sequences carried in pET systems, by optimizing malt widow Interval between the RBS sequences and initiation codon of glycosyl hydrolysis of trehalose enzyme gene (treZ) front end regulates and controls the different table of the enzyme Up to amount, interval is set in 4bp~12bp.Pass through RBS Calculatorv1.1 (https://www.denovodna.com/ Software/ different RBS intensity and translation initiation speed TIR) are predicted, so as to design different RBS sequences, builds double enzyme series connection Expression plasmid, obtain satisfactory enzyme proportioning.
Carrier described in preparation method step B of the present invention includes but is not limited to pGEX series, pET series, pQ E series With pMAL series.In some embodiments, the carrier is pET serial carriers.In a specific embodiment, the carrier For pET-28a (+).
Host Strains described in preparation method step B of the present invention are E. coli expression strains, can be Rosetta series Or BL21 series bacterial strains.In some embodiments, the Host Strains are E.coli BL21 (DE3) bacterial strain.
Further, present invention also offers the product that described genetic engineering bacterium obtains through induced expression.It is of the present invention Genetic engineering bacterium co-expresses MTSase and MTHase after induction, obtains the MTSase and MTHase of specific proportioning double enzymes mixing Product.
Present invention also offers the preparation method of the product, is inoculated in after the genetic engineering bacterium is activated in culture medium Induced expression.
Wherein, in some embodiments, the activation adds 37 DEG C of vibrations in TB culture mediums for the genetic engineering bacterium Overnight incubation.
In some embodiments, the inoculation is is inoculated in culture medium by 10% inoculum concentration, cultivates to OD600 and is 6~10.
In some embodiments, the induction is addition derivant induced expression.
Preferably, the derivant is 0.3~0.5mmol/L IPTG or 3~7g/L lactose.
Preferably, the inducing temperature is 25-30 DEG C, induction time 12-16h.
Thalline is collected by centrifugation after the preparation method induced expression of product of the present invention, after ultrasonication, centrifugation again is gone Fall bacterial chip, produce the MTSase and MTHase of specific proportioning double enzyme mix products.
In above-mentioned preparation method, those skilled in the art can select to be adapted to production according to existing Escherichia coli culture medium The culture medium of enzyme either further optimizes to culture medium.
Genetic engineering bacterium of the present invention co-expresses MTSase and MTHase after induction, the specific proportioning of acquisition MTSase and MTHase double enzyme mix products, enzymolysis conversion can be carried out according to a certain percentage by, which adding in substrate, is used for trehalose life Production.Therefore the product obtained through induced expression present invention also offers the genetic engineering bacterium or described genetic engineering bacterium is in life Produce the application in trehalose.
Present invention also offers a kind of production method of trehalose, using linear dextrin or amylose as substrate, adds institute The product conversion that the genetic engineering bacterium stated obtains through induced expression.
Using linear dextrin or amylose as substrate, concentration of substrate 10%~30%, in 40-60 DEG C of pH5.0-7.0, temperature Under, the product that the genetic engineering bacterium obtains through induced expression is added, converts 24-72h.
In one embodiment of the invention, the concentration of substrate is 10%~30%.
In one embodiment of the invention, the conversion pH5.0-7.0, conversion temperature are 40-60 DEG C, transformation time For 24-72h.
In one embodiment of the invention, the product addition is 10~20mL/Kg dries or 10~20g/Kg Dry.I.e. per Kg linear dextrins or amylose adds 10~20mL genetic engineering bacteriums through induced expression enzyme liquid or per Kg straight chains paste Essence or amylose add 10~20g genetic engineering bacteriums through induced expression enzyme dry.
In certain embodiments, it is described to be converted into 10% amylose, 2~2.5mL genetic engineering bacteriums are added through luring Expression enzyme liquid or 2~2.5g genetic engineering bacteriums are led through induced expression enzyme dry, in pH6.0 phosphoric acid buffer liquid system, 40~ 60 DEG C, 24h is converted under the conditions of 200rpm.
Compared with prior art, the present invention at least has with next advantage：
1st, genetic engineering bacterium of the present invention can express MTSase and MTHase simultaneously in same strain bacterium, and turn over The expression of two kinds of enzymes of level modulation is translated, realizes the high efficient expression of the varying level of two kinds of enzymes；
2nd, genetic engineering bacterium of the present invention can ferment simultaneously prepares two kinds of enzymes of MTSase and MTHase, and technique is simple, Equipment investment is low, it is only necessary to which a set of equipment is with regard to that can meet production requirement.And two kinds of expression of enzymes amount proportionings of MTSase and MTHase Fixed, production is more stable.
3rd, the present invention adjusts the expression of the MTSase and MTHase enzymes of the genetic engineering bacterium by molecular biology method It is horizontal so that two kinds of enzymes obtain optimum proportioning, and synergy is most strong, and highest conversion ratio is obtained in the most short time.Test table The bright product obtained of being expressed using genetic engineering bacterium of the present invention produces trehalose, and the conversion ratio of trehalose is up to 87%.
Embodiment
The invention discloses a kind of genetic engineering bacterium and its application.Those skilled in the art can use for reference present disclosure, fit When modified technique parameter is realized.In particular, all similar replacements and change for a person skilled in the art It is it will be apparent that they are considered as being included in the present invention.The method and product of the present invention is entered by preferred embodiment Description is gone, related personnel can substantially not depart from present invention, method described herein changed in spirit and scope It is dynamic or suitably change with combining, to realize and using the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention Case is clearly and completely described, it is clear that and described embodiment is only part of the embodiment of the present invention, rather than all Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art institute under the premise of creative work is not made The every other embodiment obtained, belongs to the scope of protection of the invention.
The formula of involved TB culture mediums (W/V) is in the embodiment of the present invention：1.2% peptone, 2.4% dusty yeast, 0.213%KH2PO4, 1.643%K2HPO4·3H2O, 0.5% glycerine, pH7.0.
Technology used in the present invention, such as PCR amplification techniques, design of primers technology, vector construction technology, engineering bacteria structure The technology that technology, detection technique, electrophoretic techniques etc. are comparative maturity in genetic engineering is built, those skilled in the art can be according to existing Technology is realized.Equipment used or reagent, carrier, enzyme etc. in operation, unless otherwise instructed, it can obtain in the market Arrive.The performance evaluation of all genetic engineering bacteriums induces measure producing enzyme effect under identical conditions in the present invention, followed by Enzyme liquid converts linear dextrin or amylose production trehalose.
Embodiment 1：Obtain target gene
Arthrobacterium Arthrobacter sp.Q36 are searched from NCBI, obtain coding MTSase and MTHase gene orders TreY and treZ, MTSase nucleotide sequence such as SEQ IDNO:Shown in 1, amino acid sequence such as SEQ ID NO:Shown in 2. MTHase nucleotide sequence such as SEQ ID NO:Shown in 3, amino acid sequence such as SEQ ID NO:Shown in 4.
Embodiment 2：Connect RBS sequences Designs
Separately design 5 groups of RBS sequences, being distinguished as 5 kinds of RBS sequences is different from ATG spacer region, is respectively designated as rbs- 1 (sequence such as SEQ ID NO:5), rbs-2 (sequence such as SEQ ID NO:6), rbs-3 (sequence such as SEQ ID NO:7)、rbs-4 (sequence such as SEQ ID NO:And rbs-5 (sequence such as SEQ ID NO 8):9), its sequence and corresponding T.I.R values are shown in Table 1.Separately Outside, rbs-1 is identical with RBS sequences used by expression malt oligosaccharide based mycose synthetase gene, its sequence such as SEQ ID NO:5, corresponding T.I.R values are shown in Table 1.The RBS sequences intensity be by RBS Calculator v1.1 (https:// www.denovodna.com/software/) calculated and obtained.
Table 1：Different RBS and its translation initiation speed
RBS sequences Sequence number MRNA sequence T.I.R[au]
rbs-1 SEQ ID NO:5 aataattttgtttaactttaagaaggagatatacc 1055.70
rbs-2 SEQ ID NO:6 aataattttgtttaactttaagaaggagatat 429.70
rbs-3 SEQ ID NO:7 aataattttgtttaactttaagaaggagatataccat 912.40
rbs-4 SEQ ID NO:8 aataattttgtttaactttaagaaggagatataccata 704.10
rbs-5 SEQ ID NO:9 aataattttgtttaactttaagaaggagatataccatacc 366.70
Embodiment 3：The structure of recombinant strains
Malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose water are expressed using pET-28a (+) carrier simultaneously Enzyme is solved, expression way is the double enzyme expressing in series of single promoter, and malt oligosaccharide based mycose synthetase is located at Fructus Hordei Germinatus oligose base marine alga The upstream of glycosylhydrolase, the spacer region of two enzyme coding genes is RBS sequences, and promoter is T7 promoters.Fructus Hordei Germinatus oligose Ji Hai The RBS sequences of algae sugar synthase gene are fixed as rbs-1, and rbs-1 is the RBS sequences of pET carriers itself.Fructus Hordei Germinatus oligose base marine alga The RBS sequences of glycosylhydrolase gene take rbs-1, rbs-2, rbs-3, rbs-4 and rbs-5 respectively, and 5 kinds of different RBS sequences are led to Design primer is crossed by pcr amplification primer to be entered.
It is suitable that malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase gene are introduced by primer Restriction enzyme site, it is attached with expression vector pET-28a (+), so as to obtain purpose expression plasmid.Primer and restriction enzyme site are shown in Table 2.By this operation, obtain 5 kinds combination double expression of enzymes systems, and by corresponding expression vector be named as pET-treYZ1, PET-treYZ2, pET-treYZ3, pET-treYZ4 and pET-treYZ5,5 kinds of expression vectors are then converted into Host Strains respectively In E.coli BL21 (DE3), kalamycin resistance flat board is coated with, screening obtains the gene for expressing MTSase and MTHase simultaneously Engineering bacteria, corresponding strain name are that (i.e. MTSase and MTHase pass through SEQ ID NO to SH001:5 catenation sequence is connected Connect), (i.e. MTSase and MTHase pass through SEQ ID NO to SH002:6 catenation sequence is attached), SH003 (i.e. MTSase and MTHase passes through SEQ ID NO:7 catenation sequence is attached), (i.e. MTSase and MTHase pass through SEQ ID NO to SH004: 8 catenation sequence is attached) and SH005 (i.e. MTSase and MTHase pass through SEQ ID NO:9 catenation sequence is connected Connect).
The primer sequence of table 2
Primer Primer sequence Restriction enzyme site
PtreY-f ggaattccatatgccggcatccacctaccgcc NdeI
PtreY-r cgggatccaaacaaaattattttaggtttcgaccagcagtgcg BamHI
PtreZ-rbs1 cgggatccaagaaggagatataccatgaatcgccgttttccggt BamHI
PtreZ-rbs2 cgggatccaagaaggagatatatgaatcgccgttttccggt BamHI
PtreZ-rbs3 cgggatccaagaaggagatataccatatgaatcgccgttttccggt BamHI
PtreZ-rbs4 cgggatccaagaaggagatataccataatgaatcgccgttttccggt BamHI
PtreZ-rbs5 cgggatcc aagaaggagatataccataccatgaatcgccgttttccggt BamHI
PtreZ-A cccaagcttttattccagacgcacaatcgcc HindIII
Embodiment 4：It is prepared by different ratio crude enzyme liquid
Recombination engineering bacteria SH001~005 built is cultivated in TB culture mediums respectively, vibrated in 37 DEG C of water-baths Overnight incubation, transferred by 10% inoculum concentration in the 500mL triangular flasks equipped with 50mLTB culture mediums, continue culture to OD600 For 8 when, add IPTG final concentration of 0.3mmolL-1, 30 DEG C of induced expression 12-16h.By recombinant bacterium respectively through being collected by centrifugation, And ultrasonication, centrifuging and taking supernatant is the crude enzyme liquid for MTSase and the MTHase coexpression for obtaining different ratio again.According to egg White appliances swimming band intensity calculates protein content, obtains different strains enzymatic production ratio.
The different strains enzymatic production ratio of table 3
Strain name MTSase and MTHase expression quantity ratios
SH001 1：1
SH002 1：0.41
SH003 1：0.86
SH004 1：0.67
SH005 1：0.35
Embodiment 5：Trehalose converts
Using linear dextrin or amylose as substrate, concentration of substrate 30%, under pH5.0-6.0, temperature 45 C, add respectively Enter MTSase the and MTHase crude enzyme liquids prepared by bacterial strain SH001~SH005 fermentations, convert 60-72h, add carbohydrase afterwards The remaining unreacted substrate of conversion is glucose, then using high performance liquid chromatography detection content of trehalose, calculates different ratio The trehalose conversion ratio of enzyme liquid.As a result such as table 5 below.
The trehalose conversion ratio of the different ratio enzyme liquid of table 5
Strain name Conversion ratio
SH001 63%
SH002 55%
SH003 87%
SH004 75%
SH005 50%
As a result show, be separately added into MTSase and MTHase crude enzyme liquids prepared by bacterial strain SH001~SH005 fermentations, conversion 60-72h, it is glucose to add saccharification enzymatic conversion residue unreacted substrate afterwards, trehalose conversion ratio 50~87%.
Sequence table
<110>Langfang plum blossom biotechnology development corporation, Ltd.
<120>A kind of genetic engineering bacterium and its application
<130> MP1709817
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2274
<212> DNA
<213> Arthrobacter sp. Q36
<400> 1
atgccggcat ccacctaccg cctgcaaatc tcagccgaat tcaccctgtt tgacgccgcc 60
cgtatcgttc cgtatctgca ccgcctgggc gcggattggc tgtatctgag cccgctgctg 120
gaaagtgaat ccggcagctc tcatggttac gatgtggttg accactcacg tgttgacgca 180
gcacgcggcg gtccggaagg tctggcggaa ctgtcgcgtg cagctcatga acgcggcatg 240
ggtgtcgtgg ttgatattgt tccgaaccac gtgggcgttg cgaccccgaa agccaatcgt 300
tggtggtggg acgtgctggc acgtggtcag cgtagtgaat atgccgatta ctttgatatc 360
gactgggaat tcggcggtgg ccgtctgcgc ctgccggttc tgggtgatgg tccggacgaa 420
ctggatgcgc tgcgtgttga tggcgacgaa ctggtctatt acgaacatcg ctttccgatt 480
gcagaaggta ccggtggcgg tacgccgcgt gaagtgcatg atcgccagca ctatgaactg 540
atgtcttggc gtcgcgcaga tcacgacctg aactaccgtc gctttttcgc ggttaatacc 600
ctggcagcag tccgtgtgga agatccgcgc gtcttcgatg acacgcatcg tgaaattggc 660
cgctggatcg cagaaggcct ggtcgacggt ctgcgtgtgg accacccgga tggtctgcgt 720
gcaccgggtg attatctgcg tcgcctggca gaactggcac agggtcgtcc gatttgggtg 780
gaaaaaatta tcgaaggtga cgaacgcatg ccgccgcaat ggccgattgc aggcaccacg 840
ggttacgatg cactggctgg catcgaccgt gttctggtcg atccggcggg tgaacatccg 900
ctgacccaga tcgttgatga agcagctggc tcaccgcgtc gctgggcaga actggtcccg 960
gaacgtaaac gtgcagtggc acgtggtatt ctgaactcgg aaatccgtcg cgtggcacgt 1020
gaactgggcg aagtggcagg tgacgttgaa gatgctctgg ttgaaattgc ggccgcactg 1080
tcagtctatc gttcgtacct gccgtttggt cgcgaacacc tggatgaagc agtggctgca 1140
gcacaggcag ctgcaccgca actggaagca gacctggcag cagttggtgc tgcactggcc 1200
gatccgggta acccggccgc actgcgcttt cagcaaacca gcggcatgat tatggcaaaa 1260
ggtgtcgaag ataatgcttt ctatcgttac ccgcgcctga ccagcctgac ggaagtgggc 1320
ggtgacccgt ctctgtttgc catcgatgct gcggccttcc atgcagctca gcgtgatcgt 1380
gcagcacgtc tgccggaaag tatgaccacg ctgaccacgc acgacaccaa acgctccgaa 1440
gatacccgtg cgcgcattac ggcactggct gaagcgccgg aacgttggcg tcgctttctg 1500
accgaagttg gcggtctgat cggcacgggt gatcgcgtcc tggaaaatct gatttggcaa 1560
gccatcgtgg gcgcatggcc ggctagtcgt gaacgcctgg aagcctatgc actgaaagca 1620
gctcgtgaag ccggcgaatc caccgattgg atcgatggtg acccggcatt cgaagaacgt 1680
ctgacccgcc tggtgacggt tgcggtcgaa gaaccgctgg tgcatgaact gctggaacgc 1740
ctggttgatg aactgaccgc ggccggctat tccaacggtc tggcagctaa actgctgcag 1800
ctgctggcac cgggtacccc ggacgtgtac cagggtacgg aacgttggga tcgcagcctg 1860
gtcgatccgg acaatcgtcg cccggtggat tttgcggccg catctgaact gctggaccgt 1920
ctggatggcg gttggcgtcc gccggtggac gaaaccggtg cagttaaaac gctggtcgtg 1980
agccgtgctc tgcgtctgcg tcgcgatcgt ccggaactgt ttaccgcata tcatccggtt 2040
acggcacgtg gtgctcaggc agaacacctg attggtttcg atcgtggcgg tgcaatcgca 2100
ctggctaccc gtctgccgct gggtctggct gcagccggcg gttggggcga caccgttgtc 2160
gatgtgggtg aacgttctct gcgcgatgaa ctgacgggcc gtgaagcgcg cggtgccgct 2220
cgtgtcgctg aactgtttgc tgactatccg gtcgcactgc tggtcgaaac ctaa 2274
<210> 2
<211> 775
<212> PRT
<213> Arthrobacter sp. Q36
<400> 2
Met Arg Thr Pro Val Ser Thr Tyr Arg Leu Gln Ile Arg Lys Gly Phe
1 5 10 15
Thr Leu Phe Asp Ala Ala Lys Thr Val Pro Tyr Leu His Ser Leu Gly
20 25 30
Val Asp Trp Val Tyr Leu Ser Pro Val Leu Thr Ala Glu Gln Gly Ser
35 40 45
Asp His Gly Tyr Asp Val Thr Asp Pro Ser Ala Val Asp Pro Glu Arg
50 55 60
Gly Gly Pro Glu Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Ala Ala
65 70 75 80
Gly Met Gly Val Leu Ile Asp Ile Val Pro Asn His Val Gly Val Ala
85 90 95
Thr Pro Ala Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg
100 105 110
Gln Ser Arg Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu Ala Gly
115 120 125
Gly Arg Ile Arg Leu Pro Val Leu Gly Ser Asp Asp Asp Leu Asp Gln
130 135 140
Leu Glu Ile Arg Asp Gly Glu Leu Arg Tyr Tyr Asp His Arg Phe Pro
145 150 155 160
Leu Ala Glu Gly Thr Tyr Ala Glu Gly Asp Ala Pro Arg Asp Val His
165 170 175
Ala Arg Gln His Tyr Glu Leu Ile Gly Trp Arg Arg Ala Asp Asn Glu
180 185 190
Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu Ala Gly Val Arg
195 200 205
Val Glu Ile Pro Ala Val Phe Asp Glu Ala His Gln Glu Val Val Arg
210 215 220
Trp Phe Arg Glu Asp Leu Ala Asp Gly Leu Arg Ile Asp His Pro Asp
225 230 235 240
Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val Thr
245 250 255
Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln
260 265 270
Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu
275 280 285
Ala Asp Val Asp Arg Val Leu Val Asp Pro Arg Gly Gln Glu Pro Leu
290 295 300
Asp Arg Leu Asp Ala Ser Leu Arg Gly Gly Glu Pro Ala Asp Tyr Gln
305 310 315 320
Asp Met Ile Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His
325 330 335
Ser Glu Ile Leu Arg Leu Ala Arg Leu Val Pro Gly Asp Ala Asn Val
340 345 350
Ser Ile Asp Ala Gly Ala Asp Ala Leu Ala Glu Ile Ile Ala Ala Phe
355 360 365
Pro Val Tyr Arg Thr Tyr Leu Pro Glu Gly Ala Glu Val Leu Lys Glu
370 375 380
Ala Cys Glu Leu Ala Ala Arg Arg Arg Pro Glu Leu Asp Gln Ala Ile
385 390 395 400
Gln Ala Leu Gln Pro Leu Leu Leu Asp Thr Asp Leu Glu Leu Ala Arg
405 410 415
Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu Asp
420 425 430
Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly Thr Leu Thr Glu Val Gly
435 440 445
Ala Asp Pro Thr Glu Phe Ala Val Glu Pro Asp Glu Phe His Ala Arg
450 455 460
Leu Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met Thr Thr Leu Ser
465 470 475 480
Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val
485 490 495
Ile Ser Glu Val Ala Gly Asp Trp Glu Lys Ala Leu Asn Arg Leu Arg
500 505 510
Asp Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Ala Leu Leu Trp Gln
515 520 525
Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Tyr Tyr
530 535 540
Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Asn Trp Thr Asp
545 550 555 560
Pro Ala Pro Ala Phe Glu Glu Lys Leu Lys Ala Ala Val Asp Ala Val
565 570 575
Phe Asp Asn Pro Ala Val Gln Ala Glu Val Glu Ala Leu Val Glu Leu
580 585 590
Leu Glu Pro Tyr Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln
595 600 605
Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp
610 615 620
Asp Arg Ser Leu Thr Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Asp
625 630 635 640
Asp Arg Arg Ala Ala Leu Glu Gln Leu Asp Ala Gly Asp Leu Pro Ala
645 650 655
Ser Phe Thr Asp Glu Arg Thr Lys Leu Leu Val Thr Ser Arg Ala Leu
660 665 670
Arg Leu Arg Arg Asp Arg Pro Glu Leu Phe Thr Gly Tyr Arg Pro Val
675 680 685
Leu Ala Ser Gly Pro Ala Ala Gly His Leu Leu Ala Phe Asp Arg Gly
690 695 700
Thr Ala Ala Ala Pro Gly Ala Leu Thr Leu Ala Thr Arg Leu Pro Tyr
705 710 715 720
Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu Leu Asn
725 730 735
Thr Ala Met Lys Asp Glu Leu Thr Gly Ala Gly Phe Gly Pro Gly Ala
740 745 750
Val Lys Ile Ala Asp Ile Phe Arg Ser Phe Pro Val Ala Leu Leu Val
755 760 765
Pro Gln Thr Gly Gly Glu Ser
770 775
<210> 3
<211> 1728
<212> DNA
<213> Arthrobacter sp. Q36
<400> 3
atgaatcgcc gttttccggt ctgggctccg caagccgctc aagtgacgct ggtcgtgggc 60
caaggtcgtg ccgaactgcc gctgacgcgc gatgaaaacg gttggtgggc actgcagcaa 120
ccgtgggacg gcggtccgga cctggtggat tatggctacc tggttgatgg caaaggtccg 180
tttgcagacc cgcgttcact gcgtcagccg cgtggtgttc atgaactggg ccgcgaattt 240
gatccggctc gttatgcatg gggtgatgac ggttggcgtg gtcgtgacct gaccggtgcc 300
gtcatttacg aactgcatgt gggtaccttt acgccggaag gcacgctgga ttcggcaatc 360
cgtcgcctgg accacctggt tcgtctgggc gtggatgctg ttgaactgct gccggtcaac 420
gcgtttaatg gtacccatgg ctggggttat gatggcgtcc tgtggtacgc tgtgcacgaa 480
ccgtatggcg gtccggaagc gtaccagcgc ttcgtggatg cctgccatgc acgtggtctg 540
gcagtggttc aagacgtcgt gtataaccat ctgggtccga gtggcaatca cctgccggat 600
tttggtccgt acctgggttc cggtgcagca aacacgtggg gtgacgcgct gaatctggat 660
ggcccgctgt cagacgaagt ccgtcgctat attatcgata acgccgtgta ctggctgcgc 720
gacatgcacg cagatggtct gcgtctggat gctgttcatg cactgcgtga cgctcgtgca 780
ctgcacctgc tggaagaact ggcagctcgc gtggatgaac tggcaggtga actgggtcgt 840
ccgctgaccc tgattgcaga aagcgacctg aacgatccga aactgatccg ttctcgcgcg 900
gcccatggct atggtctgga tgcgcagtgg gatgacgatg tccatcacgc cgtgcacgca 960
aatgttaccg gtgaaacggt tggctattac gctgattttg gcggtctggg tgcgctggtg 1020
aaagtttttc aacgcggttg gttccatgat ggcacctgga gctctttccg tgaacgtcat 1080
cacggtcgtc cgctggaccc ggatattccg tttcgtcgcc tggtggcctt cgcacaggac 1140
cacgatcaag ttggtaatcg cgccgtcggc gatcgtatgt cagcacaggt tggtgaaggt 1200
tcgctggcag ctgcagcagc actggtgctg ctgggtccgt ttaccccgat gctgttcatg 1260
ggtgaagaat ggggcgcacg taccccgtgg caatttttca cgagccatcc ggaaccggaa 1320
ctgggtgaag ctacggcgcg tggccgcatt gctgaatttg cgcgtatggg ttgggatccg 1380
gcagttgtcc cggacccgca ggatccggca accttcgcac gtagtcatct ggattggtcc 1440
gaaccggaac gtgaaccgca cgcaggtctg ctggcatttt atacggatct gatcgccctg 1500
cgtcgcgaac tgccggtgga cgcaccggca cgcgaagttg acgctgatga agcgcgtggt 1560
gtgtttgctt tctctcgcgg cccgctgcgt gtcaccgtgg cactgcgtcc gggtccggtt 1620
ggcgtcccgg aacatggcgg tctggttctg gcgtatggcg aagtccgtgc tggtgcggct 1680
ggtctgcatc tggatggtcc gggtgcggcg attgtgcgtc tggaataa 1728
<210> 4
<211> 598
<212> PRT
<213> Arthrobacter sp. Q36
<400> 4
Met Thr His Thr Tyr Pro Arg Glu Ala Ala Lys Pro Val Leu Gly Pro
1 5 10 15
Ala Arg Tyr Asp Val Trp Ala Pro Asn Ala Glu Ser Val Thr Leu Leu
20 25 30
Ala Gly Gly Glu Arg Tyr Ala Met Gln Arg Arg Ala Glu Thr Gly Pro
35 40 45
Glu Asp Ala Gly Trp Trp Thr Ala Ala Gly Ala Pro Thr Asp Gly Asn
50 55 60
Val Asp Tyr Gly Tyr Leu Leu Asp Gly Asp Glu Thr Pro Leu Pro Asp
65 70 75 80
Pro Arg Thr Arg Arg Gln Pro Asp Gly Val His Ala Leu Ser Arg Thr
85 90 95
Phe Asp Pro Ser Ala Tyr Ser Trp Gln Asp Asp Ala Trp Gln Gly Arg
100 105 110
Glu Leu Gln Gly Ala Val Ile Tyr Glu Leu His Leu Gly Thr Phe Thr
115 120 125
Pro Glu Gly Thr Leu Glu Ala Ala Ala Gly Lys Leu Asp Tyr Leu Ala
130 135 140
Gly Leu Gly Val Asp Phe Ile Glu Leu Leu Pro Val Asn Ala Phe Asn
145 150 155 160
Gly Thr His Asn Trp Gly Tyr Asp Gly Val Gln Trp Phe Ala Val His
165 170 175
Glu Ala Tyr Gly Gly Pro Glu Ala Tyr Gln Arg Phe Val Asp Ala Ala
180 185 190
His Ala Ala Gly Leu Gly Val Ile Gln Asp Val Val Tyr Asn His Leu
195 200 205
Gly Pro Ser Gly Asn Tyr Leu Pro Arg Phe Gly Pro Tyr Leu Lys Gln
210 215 220
Gly Glu Gly Asn Thr Trp Gly Asp Ser Val Asn Leu Asp Gly Pro Gly
225 230 235 240
Ser Asp His Val Arg Arg Tyr Ile Leu Asp Asn Leu Ala Met Trp Leu
245 250 255
Arg Asp Tyr Arg Val Asp Gly Leu Arg Leu Asp Ala Val His Ala Leu
260 265 270
Lys Asp Glu Arg Ala Val His Ile Leu Glu Asp Phe Gly Ala Leu Ala
275 280 285
Asp Gln Ile Ser Ala Glu Val Gly Arg Pro Leu Thr Leu Ile Ala Glu
290 295 300
Ser Asp Leu Asn Asn Pro Arg Leu Leu Tyr Pro Arg Asp Val Asn Gly
305 310 315 320
Tyr Gly Leu Glu Gly Gln Trp Ser Asp Asp Phe His His Ala Val His
325 330 335
Val Asn Val Thr Gly Glu Thr Thr Gly Tyr Tyr Ser Asp Phe Asp Ser
340 345 350
Leu Ala Ala Leu Ala Lys Val Leu Arg Asp Gly Phe Phe His Asp Gly
355 360 365
Ser Tyr Ser Ser Phe Arg Glu Arg His His Gly Arg Pro Ile Asn Phe
370 375 380
Ser Ala Val His Pro Ala Ala Leu Val Val Cys Ser Gln Asn His Asp
385 390 395 400
Gln Ile Gly Asn Arg Ala Thr Gly Asp Arg Leu Ser Gln Thr Leu Pro
405 410 415
Tyr Gly Ser Leu Ala Leu Ala Ala Val Leu Thr Leu Thr Gly Pro Phe
420 425 430
Thr Pro Met Leu Leu Met Gly Glu Glu Tyr Gly Ala Ser Thr Pro Trp
435 440 445
Gln Phe Phe Thr Ser His Pro Glu Pro Glu Leu Gly Lys Ala Thr Ala
450 455 460
Glu Gly Arg Ile Lys Glu Phe Glu Arg Met Gly Trp Asp Pro Ala Val
465 470 475 480
Val Pro Asp Pro Gln Asp Pro Glu Thr Phe Arg Arg Ser Lys Leu Asp
485 490 495
Trp Ala Glu Ala Ala Glu Gly Asp His Ala Arg Leu Leu Glu Leu Tyr
500 505 510
Arg Ser Leu Thr Ala Leu Arg Arg Ser Thr Pro Asp Leu Thr Lys Leu
515 520 525
Gly Phe Glu Asp Thr Gln Val Ala Phe Asp Glu Asp Ala Arg Trp Leu
530 535 540
Arg Phe Arg Arg Gly Gly Val Gln Val Leu Leu Asn Phe Ser Glu Gln
545 550 555 560
Pro Val Ser Leu Asp Gly Ala Gly Thr Ala Leu Leu Leu Ala Thr Asp
565 570 575
Asp Ala Val Arg Leu Glu Gly Glu Arg Ala Glu Leu Gly Pro Leu Ser
580 585 590
Ala Ala Val Val Ser Asp
595
<210> 5
<211> 35
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
aataattttg tttaacttta agaaggagat atacc 35
<210> 6
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
aataattttg tttaacttta agaaggagat at 32
<210> 7
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
aataattttg tttaacttta agaaggagat ataccat 37
<210> 8
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
aataattttg tttaacttta agaaggagat ataccata 38
<210> 9
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
aataattttg tttaacttta agaaggagat ataccatacc 40

Claims (10)

1. a kind of gene, it is characterised in that by the DNA molecular of coding malt oligosaccharide based mycose synthetase, ribosome binding site Point sequence, the DNA molecular of coding malt oligosaccharide based mycose hydrolase are connected in sequence.
2. gene according to claim 1, it is characterised in that the DNA of the coding malt oligosaccharide based mycose synthetase Its nucleotide sequence of molecule such as SEQ IDNO:Shown in 1；Its core of the DNA molecular of the coding malt oligosaccharide based mycose hydrolase Nucleotide sequence such as SEQ IDNO:Shown in 3；Its nucleotide sequence of ribosome bind site sequence such as SEQ IDNO:5-9 is any Shown in one.
3. a kind of recombinant vector, includes the carrier of the gene of claim 1 or 2, the coding malt oligosaccharide based mycose closes DNA molecular into enzyme is located at the upstream for the DNA molecular for encoding malt oligosaccharide based mycose hydrolase.
4. a kind of genetic engineering bacterium, it is characterised in that include recombinant vector described in claim 3.
5. the preparation method of genetic engineering bacterium described in claim 4, it is characterised in that including：
Step A, the gene order of coding malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase is obtained, with And connection ribosome bind site sequence；
Step B, recombinant vector conversion Host Strains are built；Wherein described recombinant vector is included and closed by coding malt oligosaccharide based mycose DNA molecular, ribosome bind site sequence, the DNA molecular of coding malt oligosaccharide based mycose hydrolase into enzyme are sequentially connected Into gene, the DNA molecular of the coding malt oligosaccharide based mycose synthetase is positioned at coding malt oligosaccharide based mycose hydrolysis The upstream of the DNA molecular of enzyme.
6. the product that the genetic engineering bacterium described in claim 4 obtains through induced expression.
7. the preparation method of product described in claim 6, it is characterised in that be inoculated in culture after the genetic engineering bacterium is activated Induced expression in base.
8. application of the product described in genetic engineering bacterium described in claim 4 or claim 6 in trehalose is produced.
9. a kind of production method of trehalose, it is characterised in that using linear dextrin or amylose as substrate, add claim The 6 product conversions.
10. production method according to claim 9, it is characterised in that the concentration of substrate is 10%~30%；Described turn Change pH5.0-7.0；Conversion temperature is 40-60 DEG C；Transformation time is 24-72h；Product addition is described in the claim 6 10~20mL/Kg dries or 10~20g/Kg dries.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116904381A (en) * 2023-07-31 2023-10-20 江南大学 Construction and application of recombinant escherichia coli producing adipic acid

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