CN107475271A - 6 phosphotrehalose UDP-transglucosylase synzyme caused by the microbacterium of deep-sea and 6 phosphotrehalose UDP-transglucosylase phosphates - Google Patents

6 phosphotrehalose UDP-transglucosylase synzyme caused by the microbacterium of deep-sea and 6 phosphotrehalose UDP-transglucosylase phosphates Download PDF

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CN107475271A
CN107475271A CN201710849502.5A CN201710849502A CN107475271A CN 107475271 A CN107475271 A CN 107475271A CN 201710849502 A CN201710849502 A CN 201710849502A CN 107475271 A CN107475271 A CN 107475271A
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mstps
mstpp
phosphotrehalose udp
transglucosylase
udp
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汤熙翔
许薷方
黄平
于丽波
林文珍
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Third Institute of Oceanography SOA
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Abstract

6 phosphotrehalose UDP-transglucosylase synzyme caused by the microbacterium of deep-sea and 6 phosphotrehalose UDP-transglucosylase phosphates, two kinds of enzymes are two enzymes in the actinomyces Microbacterium sediminis sp.nov.YLB 01 in deep-sea source TPS/TPP trehaloses synthesis path, 6 phosphotrehalose UDP-transglucosylase synzyme and 6 phosphotrehalose UDP-transglucosylase phosphates respectively.Clone described two enzymes, and by cloned sequence insertion vector pET 28a (+).After plasmid vector is transformed into competence E.coli BL21 cells, induced expression is carried out.The restructuring enzyme liquid His purification column Ni Sepharose obtainedTM6Fast Flow are separated and purified, and obtain the recombinase of purifying.The two kinds of recombinases provided still have higher activity under cryogenic, such as under the conditions of 4 DEG C, reach 72.78% and 50.20% activity.

Description

6- phosphotrehalose UDP-transglucosylases synzyme caused by the microbacterium of deep-sea and 6- phosphotrehalose UDP-transglucosylase phosphoric acid Esterase
Technical field
The present invention relates to genetic engineering, more particularly, to the microbacterium (Microbacterium from deep-sea Sediminis sp.nov.) YLB-01 trehaloses synthesis TPS/TPP approach in two novel enzymes, be 6- phosphoric acid marine algas respectively 6- obtained by the encoding gene and recombination expression of sugared synzyme (msTPS) and 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) Phosphotrehalose UDP-transglucosylase synzyme (msTPS) and 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) and preparation method thereof.
Background technology
Trehalose is a kind of irreducibility disaccharide, is linked by two glucose monomers with 1,1 glycosidic bond[1], molecular formula For C12H22O11·2H20, relative molecular mass Mr=378.33.Trehalose is in many organisms, with free state or various The form of glycolipid is present, and is the important composition composition of cell membrane, and it acts not only as the carbon source of some organisms, and can be with Regulate and control the growth of organism.Trehalose is not only a kind of energetic supersession deposit material in organism, and by maintaining albumen The stability of the large biological molecules such as matter, nucleic acid, trehalose are sent out in some species adapt to adverse circumstances (such as deep-sea extreme environment) Important function is waved[2].In addition, trehalose has unique physics and chemical property, it is extremely steady to become a kind of processing characteristics Fixed raw-food material, it is adapted to the process of different based foods[3], there is wide application in field of food[1]。
At present, the production method of trehalose mainly includes chemical synthesis, microorganism extraction method, microbe fermentation method, base Because of engineering method and Enzyme optrode etc..Enzyme optrode production trehalose is to utilize transglycosylation possessed by trehalose synthetase, Corresponding substrate catalysis generation trehalose is acted in vitro.Hitherto it is found that the route of synthesis of trehalose is extremely in organism Rare 5, including TPS/TPP approach, TreS approach, TreY/TreZ approach, TreT approach and TreP approach.Wherein with TPS/ The distribution of TPP approach is most extensive, most common in bacterium, archeobacteria, fungi, insect and plant[4-6], also cause its be found compared with It is early, also it is most widely used[7].This approach is with G6P (glucose-6-phosphate, G6P) and uridine 5'-diphosphate Glucose (UDP-glucose, UDPG) is substrate, first through 6- phosphotrehalose UDP-transglucosylase synzyme (Trehalose-6-phosphate Synthase, TPS) catalysis glucosyl group transfer reaction generation 6- phosphotrehalose UDP-transglucosylases (Trehalose-6-phosphate, T6P), After T6P is catalyzed through 6- phosphotrehalose UDP-transglucosylases phosphate (Trehalose-6-phosphate phosphatase, TPP) Dephosphorylation and generate trehalose[8-11].Be initially be cloned into from Escherichia coli be separately encoded 6- phosphotrehalose UDP-transglucosylases synzyme and The gene OtsA and OtsB of 6- phosphotrehalose UDP-transglucosylase phosphates[12], the osmolarity resistance of this 2 genes and E.coli cells is close It is related and gain the name[9], this approach is also referred to as OtsA/OtsB approach.
Bibliography：
[1]Elbein A D,Pan Y T,Pastuszak I,et al.New insights on trehalose:a multifunctional molecule[J].glycobiology.2003,13(4):17R-27R.
[2] Zhang Yuhua, Ling Peixue, nationality are kept tie the present Research and its application prospect [J] food and medicine of trehaloses .2005(03).
[3] processing characteristics of Liu Hong plums trehaloses and application [J] light science and technologies .2013 (08) in food service industry
[4]Pan Y T,Carroll J D,Elbein A D.Trehalose-phosphate synthase of Mycobacterium tuberculosis[J].european journal of biochemistry.2002,269(24): 6091-6100.
[5]Valenzuela-Soto E M,Marquez-Escalante J A,Iturriaga G,et al.Trehalose 6-phosphate synthase from Selaginella lepidophylla:purification and properties[J].Biochemical and biophysical research communications.2004, 313(2):314-319.
[6]Silva Z,Alarico S,Da Costa M.Trehalose biosynthesis in Thermus thermophilus RQ-1:biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase[J].extremophiles.2005,9(1):29- 36.
[7] Yang Yan, Lee increase ripple trehalose TPS/TPP biosynthesis pathways Study on Evolution [J] scientific and technological information exploitation with Economic .2008 (16)
[8]Cabib E,Leloir L F.The biosynthesis of trehalose phosphate.[J].The Journal of biological chemistry.1958,231(1):259-275.
[9]Kaasen I,Falkenberg P,Styrvold O B,et al.Molecular cloning and physical mapping of the otsBA genes,which encode the osmoregulatory trehalose pathway of Escherichia coli:evidence that transcription is activated by katF (AppR)[J].Journal of bacteriology.1992,174(3):889-898.
[10]Lunn J E,Delorge I,Figueroa C M,et al.Trehalose metabolism in plants[J].plant journal.2014,79(4SI):544-567.
[11]Paul M J,Primavesi L F,Jhurreea D,et al.Trehalose metabolism and signaling[M].2008:59,417-441.
[12] Li Lei, Ding Hongbiao, Yu Xiaobin, the recombination expression research of trehalose enzyme process route of synthesis and its enzyme gene is waited [J] biotechnologys circulate a notice of .2007 (03)
[13]De Smet K A,Weston A,Brown I N,et al.Three pathways for trehalose biosynthesis in mycobacteria.[J].Microbiology(Reading,England).2000,146(Pt1): 199-208.
The content of the invention
The first object of the present invention is to provide 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) gene.
The second object of the present invention is to provide 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) gene.
The third object of the present invention is the recombinase for providing 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS).
The fourth object of the present invention is the recombinase for providing 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP).
The fifth object of the present invention is to provide 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) and 6- phosphotrehalose UDP-transglucosylase phosphates The preparation method of enzyme (msTPP).
6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) gene is Microbacterium sediminis Sp.nov.YLB-01 6- phosphotrehalose UDP-transglucosylase synthase genes, its nucleotide sequence is as shown in SEQ ID NO.1.
6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) gene is Microbacterium sediminis Sp.nov.YLB-01 6- phosphotrehalose UDP-transglucosylase phosphatase genes, its nucleotide sequence is as shown in SEQ ID NO.3.
The recombinase of the 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) is Microbacterium sediminis The recombinase of sp.nov.YLB-01 6- phosphotrehalose UDP-transglucosylase synzyme, the recombinase still have higher work under cryogenic Property.
The recombinase of the 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) is Microbacterium sediminis The recombinase of sp.nov.YLB-01 6- phosphotrehalose UDP-transglucosylase phosphates, the recombinase still have higher under cryogenic Activity.
The 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) and the production bacterial strain of 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) For Microbacterium sediminis sp.nov.YLB-01, be State Oceanic Administration Bureau The Third Oceanography Institute's living resources with Hereditary key lab Tang Xi Xiangs seminar is from the Chinese flight number (Nov.2008) of ocean No.1 research ship 20 in PSW The one plant of new spherical bacterium of Gram-positive isolated in the halmeic deposit that 2327m is gathered deeply, is one plant of type strain, in It is preserved in China typical culture collection center within 2010, deposit number is：CCTCC NO：AB2010363, address are Wuhan, Wuhan University, postcode 430072.
The 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) and the preparation method of 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) Comprise the following steps：
1) msTPS or msTPP genes are cloned, SEQ in upstream and downstream primer used in msTPS gene clonings such as sequence table Shown in ID NO.5 and SEQ ID NO.6；SEQ ID NO.7 in upstream and downstream primer used in msTPP gene clonings such as sequence table Shown in SEQ ID NO.8；
2) PCR primer is used in homologous recombination strategy insertion vector pET-28a (+), builds the gene containing msTPS or msTPP Recombinant plasmid；
3) recombinant plasmid transformed is entered in competent cell E.coli DH5 α, to expand plasmid；
4) recombinant plasmid successfully constructed is chosen, is converted into competent cell E.coli BL21；
5) choose positive clone molecule and carry out protein induced expression；
6) restructuring msTPS or msTPP albumen presses His purification column Ni SepharoseTM6 Fast Flow operational manuals Isolated and purified.
The present invention has cloned Microbacterium sediminis sp.nov.YLB-01 6- phosphotrehalose UDP-transglucosylases synthesis The encoding gene of enzyme (msTPS) and 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP), and build table in E.coli BL21 cells Both enzymes are reached.It is respectively 40 DEG C and 7.5 to recombinate msTPS optimal reactive temperatures and pH, but is still had under cryogenic Higher activity, such as at 4 DEG C, enzyme activity has reached 72.78%.In addition, Mg2+、Co2+Or Ba2+Its activity can be greatly facilitated, Mn2+、Zn2+、Ca2+And Hg2+Activity is not influenceed.To G6P and uridine 5'-diphosphate grape under the conditions of the present invention The K of sugarmValue is respectively 1.38mM and 1.53mM, maximum reaction velocity VmaxFor 62.5U/minmg.It is most suitable anti-to recombinate msTPP enzymes It is respectively 30 DEG C and 7.5 to answer temperature and pH, and similarly, the enzyme also has higher activity at low temperature, can be protected under the conditions of 4 DEG C Hold 50.20% activity.It is in Mg2+Activity reaches highest, Co under the conditions of existing2+Or Ba2+There is larger promotion to make activity With Mn2+、Zn2+、Ca2+Restructuring enzymatic activity is not influenceed, to the K of 6- phosphotrehalose UDP-transglucosylases under the conditions of the present inventionmFor 2.45mM, Maximum reaction velocity VmaxFor 82.64U/minmg.
Brief description of the drawings
Fig. 1 is msTPS encoding genes amplification of the present invention.Wherein swimming lane M is Trans2K Plus DNA Marker, Swimming lane 1 is mstps gene amplification products.
Fig. 2 is recombinant plasmid TPS@pET-28a of the present invention plasmid PCR amplification.Wherein swimming lane M is Trans2K Plus DNA Marker, swimming lane 1 are plasmid TPS@pET-28a amplified productions.
Fig. 3 is the plasmid map for the recombinant plasmid TPS@pET-28a that the present invention is built.
Fig. 4 is SDS-PAGE detection restructuring msTPS induced expression situation.Wherein swimming lane M is TaKaRa protein Marker, swimming lane 1 are the total proteins of the recombinant bacterial strain before induction, and swimming lane 2 is the total protein of the recombinant bacterial strain after induction.
Fig. 5 is the restructuring situation that SDS-PAGE detects recombinant plasmid TPS@pET-28a.Wherein swimming lane M is TaKaRa Protein Marker, swimming lane 1 are recombinant plasmid TPS@pET-28a total proteins, and swimming lane 2 is the total protein of empty plasmid.
Fig. 6 is restructuring msTPS purifying situations.Wherein swimming lane M is TaKaRa protein Marker, and swimming lane 1 is thick enzyme Liquid, swimming lane 2 are eluents, and swimming lane 3 is refined solution.
Fig. 7 is that temperature influences on restructuring msTPS enzymatic activitys.
Fig. 8 is that pH influences on restructuring msTPS enzymatic activitys.
Fig. 9 is that metal ion influences on restructuring msTPS enzymatic activitys.
Figure 10 is to recombinate msTPS to scheme G6P Lineweaver-Burk.
Figure 11 is to recombinate msTPS to scheme UDPG Lineweaver-Burk.
Figure 12 is msTPP encoding genes amplification of the present invention.Wherein swimming lane M is Trans2K Plus DNA Marker, Swimming lane 1 is mstpp gene amplification products.
Figure 13 is recombinant plasmid TPP@pET-28a of the present invention plasmid PCR amplification.Wherein swimming lane M is Trans2K Plus DNA Marker, swimming lane 1 are plasmid TPS@pET-28a amplified productions.
Figure 14 is the plasmid map for the recombinant plasmid TPP@pET-28a that the present invention is built.
Figure 15 is SDS-PAGE detection restructuring msTPP induced expression situation.Wherein swimming lane M is TaKaRa protein Marker, swimming lane 1 are the total proteins of the recombinant bacterial strain before induction, and swimming lane 2 is the total protein of the recombinant bacterial strain after induction.
Figure 16 is the restructuring situation that SDS-PAGE detects recombinant plasmid TPP@pET-28a.Wherein swimming lane M is TaKaRa Protein Marker, swimming lane 1 are recombinant plasmid TPP@pET-28a total proteins, and swimming lane 2 is the total protein of empty plasmid.
Figure 17 is restructuring msTPP purifying situations.Wherein swimming lane M is TaKaRa protein Marker, and swimming lane 1 is thick enzyme Liquid, swimming lane 2 are eluents, and swimming lane 3 is refined solution.
Figure 18 is that temperature influences on restructuring msTPP enzymatic activitys.
Figure 19 is that pH influences on restructuring msTPP enzymatic activitys.
Figure 20 is that metal ion influences on restructuring msTPP enzymatic activitys.
Figure 21 is to recombinate msTPP to scheme T6P Lineweaver-Burk.
Embodiment
With reference to specific embodiments and the drawings, the present invention is expanded on further.But present disclosure is not limited thereto, Method operating according to a conventional method unless otherwise specified in the present embodiment, agents useful for same unless otherwise specified using conventional examination Agent or the reagent configured according to a conventional method.The reagent used in the present embodiment is mainly molecular biology experiment reagent, various limits Property restriction endonuclease processed, archaeal dna polymerase, dNTP etc. are Dalian treasured bioengineering Co., Ltd product, bacillus coli DH 5 alpha, Escherichia coli BL21 and plasmid pET-28a preserves for this laboratory.Remaining reagent is that domestic analysis is pure, and instrument is molecular biology and base Because of engineering experiment room apparatus ＆ equipment in common use.In Shanghai, Mei Ji biological medicine science and technology has with gene sequencing for all primer sequence synthesis Limit company.
Embodiment 1Microbacterium sediminis YLB-01 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) and 6- The clone of the encoding gene of phosphotrehalose UDP-transglucosylase phosphate (msTPP)
(1) culture of bacterium
Microbacterium sediminis YLB-01 bacterium solutions are inoculated in TSB culture mediums according to 1 ︰ 100 ratio, 18h is cultivated in 28 DEG C, 180rpm shaking table to logarithmic phase.
(2) bacterial genomes DNA extraction
The extraction of the gDNA is carried out using OMEGA bacterial genomes DNA extraction kit, all centrifugation steps are to use Desk centrifuge centrifuges at room temperature.
(3) amplification of target gene
1) amplimer
MsTPS encoding gene amplimers are：
TPS-F：5′-GTGCCGCGCGGCAGCCATATGGCTAGCGACCGTGCCGATCTTGTCGTCG-3′
TPS-R：5′-CATTTGCTGTCCACCAGTCATGCTAGCTCATTCGCGCGGCTTCTTCCTC-3′
Dashed part is homology arm, is matched completely with design insertion point position upstream and downstream 27bp sequences respectively, italicized item For Nhe I restriction enzyme sites.
MsTPP encoding gene amplimers are：
TPP-F：5′-TGGTGCCGCGCGGCAGCCATATGGCTAGCACCGAGCCCACCACCGACTG-3
TPP-R：5′-CCATTTGCTGTCCACCAGTCATGCTAGCTCAGGATGCCCGCAGATCGGC-3′
Dashed part is homology arm, and respectively with designing insertion point position upstream 29bp, downstream 28bp sequences are matched completely, tiltedly Body portion is Nhe I restriction enzyme sites.
2) PCR reaction systems：Referring to table 1.
Table 1
3) PCR cycle condition：94 DEG C, 2min；98 DEG C, 10s, 68 DEG C, 120s, repeat 35 circulations；68 DEG C, 7min.
4) 1% detected through gel electrophoresis of PCR primer, testing result is as shown in figs. 1 and 12.
(4) PCR primer sequencing identification
Whether the extension increasing sequence to identify, which undergos mutation, is sequenced to the PCR primer of msTPS encoding genes amplification, is sequenced Primer is as follows：
TPS-1470F：5′-GACCGTGCCGATCTTGTCGTCG-3′
TPS-1470R：5′-TCATTCGCGCGGCTTCTTCCTC-3′
Whether the extension increasing sequence to identify, which undergos mutation, is sequenced to the PCR primer of msTPP encoding genes amplification, is sequenced Primer is as follows：
TPP-789F：5′-ACCGAGCCCACCACCGACTG-3′
TPP-789R：5′-TCAGGATGCCCGCAGATCGGC-3′
(5) purifying of PCR primer
It will identify that correct product carries out PCR primer purifying with reference to JaRa PCR primer purification kit specification.Purify DNA can be immediately available for subsequent experimental or be frozen in -20 DEG C.
The construction recombination plasmid TPS@pET-28a of embodiment 2 and recombinant plasmid TPP@pET-28a
(1) carrier pET-28a (+) linearisation
Plasmid vector pET-28a (+) is carried out single restriction enzyme site (Nhe I) digestion be in order to prepare linearized vector, Linearized vector is most important to subsequent homo reconstruction experiment, and the carrier not linearized can produce very high background.Linearly Carrier after change passes through gel purification.Reaction system is fully mixed after overnight in 37 DEG C of constant-temperature metal baths according to table 2 Digestion.It is whether linear with 1% agarose gel electrophoresis checking carrier after digestion.
Table 2
(2) recovery of digestion products
Glue reclaim purifying is carried out to the linear carrier obtained after digestion, entered according to Omega gel reclaims kit specifications Row operation.
(3) PCR primer is connected with carrier pET-28a (+)
PCR primer after purification is inserted in the carrier pET-28a (+) linearized using homologous recombination strategy, homologous heavy Group reaction system is referring to table 3.
Table 3
By 25 DEG C of incubation 30min of reaction system, convert immediately or -20 DEG C freeze, subsequent transformation.
(4)CaCl2The preparation of method E.coli DH5 α and E.coli BL21 competent cells
1) picking E.coli DH5 α single bacteriums are fallen within 5ml LB fluid nutrient mediums, 37 DEG C, be incubated overnight under the conditions of 200rpm (12h or so).The bacteria suspension is inoculated in 100ml LB fluid nutrient mediums with the ratios of 1 ︰ 100,37 DEG C, train under the conditions of 200rpm Support to OD600 to 0.5 or so (2~3h).
2) bacterium solution is transferred in 50ml centrifuge tubes, ice bath 10min, 5min is centrifuged under the conditions of 4 DEG C, 5000g.
3) precooling deionized water washing thalline is used, 4 DEG C, centrifuge 5min under the conditions of 5000g.
4) supernatant is abandoned, with the 0.05mol/L CaCl of 10ml precoolings2Solution gently suspension cell, ice bath 15min, 4 DEG C, 5min is centrifuged under the conditions of 5000g.
5) supernatant is abandoned, with the 0.05mol/L CaCl containing 15% glycerine of 4ml precoolings2Solution gently suspension cell, ice bath 5min, competent cell suspension.
6) 100 μ l aliquots are distributed into, are frozen immediately in -70 DEG C.
(5) conversion of recombinant plasmid and the screening of positive clone molecule
The conversion reference of recombinant plasmid《Molecular Cloning:A Laboratory guide》The specific steps of (third edition), by recombinant plasmid transformed In E. coli DH5 α, operating procedure is as follows：
1) -70 DEG C of 100 μ l DH5 α competent cells frozen are taken, the gently suspension cell after thawing completely on ice.
2) 10 μ l connection liquid are added, are gently mixed, ice bath 30min.
3) 42 DEG C of heat shock 90sec., ice bath 2min.
4) plus 600 μ l are free of the LB fluid nutrient mediums of any antibiotic, 37 DEG C, cultivate 1h under the conditions of 250rpm, make cell Recovery.
5) 5min is centrifuged under the conditions of room temperature, 4000rpm, 500 μ l supernatant nutrient solutions is absorbed, cell is suspended.
6) bacterium solution is coated in screening flat board (the LB plates for containing 50 μ g/ml kanamycins).
7) after flat board front being placed into 1h (the excessive liquid of absorption) at 37 DEG C, inversion is incubated overnight.
(6) screening of positive clone molecule
Next day picking single bacterium colony, it is inoculated in the LB fluid nutrient mediums containing 50 μ g/ml kanamycins, 220rpm, 37 DEG C of bars After being incubated overnight under part, plasmid is extracted.The extraction of plasmid is entered according to the raw a small amount of extraction agent box specifications of work (China) DNA OK.
To its correctness of institute's upgrading grain progress plasmid PCR preliminary identification.And will verify that correct plasmid send sequencing, wherein surveying Sequence primer sequence is as follows：
T7：5’-TAATACGACTCACTATAGGG-3’
T7ter：5’-GCTAGTTATTGCTCAGCGG-3’
Recombinant plasmid TPS@pET-28a and recombinant plasmid TPP@pET-28a specification are as shown in Fig. 3 and Figure 14.Restructuring Plasmid TPS@pET-28a and recombinant plasmid TPP@pET-28a plasmid PCR amplification are as shown in Fig. 2 and Figure 12.
The recombinant bacterial strain of embodiment 3 is built and protein induced expression
(1) msTPS recombinant bacterial strains structure and protein induced expression
The 1 correct plasmid of μ l sequence verifications is taken, converts in E.coli BL21, positive clone molecule is protected after culture Kind.Recombinant bacterial strain is inoculated in LB fluid nutrient mediums of the 5ml containing 50 μ g/ml kanamycins, 2ml bacterium solutions are taken after being incubated overnight by 1 ︰ 100 are inoculated in LB fluid nutrient mediums of the 200ml containing kanamycins；It is 0.6 to be cultivated under the conditions of 37 DEG C, 220rpm to OD600 When, IPTG solution is added to final concentration of 1mM；5min is centrifuged under the conditions of 4 DEG C, 6000g after 30 DEG C, 180rpm cultures 4h, Collect thalline.Thalline is resuspended in 10ml Binding Buffer (20mM Tris-HCl pH7.4,0.5M NaCl, 20mM miaows Azoles) in, addition lysozyme to final concentration of 1mg/ml, after cracking 1h on ice, ultrasonication 15min, thalline is cracked completely, 4 DEG C, centrifuge 30min under the conditions of 8000g, supernatant is crude enzyme liquid.1 μ l empty plasmid pET-28a (+) separately are taken, are entered by the above process Row processing, the control strain as recombinant bacterial strain induced expression.Recombinant bacterial strain induced expression result is as shown in Figure 4.
(2) msTPP recombinant bacterial strains structure and protein induced expression
The 1 correct plasmid of μ l sequence verifications is taken, is converted in E.coli BL21, to positive clone molecule conservation.By recombinant bacterium Strain is inoculated in LB fluid nutrient mediums of the 5ml containing 50 μ g/ml kanamycins and is incubated overnight, and takes 2ml overnight cultures to be connect by 1 ︰ 100 Kind in LB fluid nutrient mediums of the 200ml containing kanamycins, 37 DEG C, 220rpm is cultivated to OD600 is 0.6~0.8, IPTG is added To final concentration of 1mM, the Fiber differentiation 4h under the conditions of 30 DEG C, 180rpm, 6000g centrifugation 5min collect thalline.Thalline is resuspended In 10ml Binding Buffer (20mM Tris-HCl pH7.4,0.5M NaCl, 20mM imidazoles), lysozyme is added extremely Final concentration of 1mg/ml, after cracking 1h on ice, ultrasonication 15min makes thalline crack completely, 8000g centrifugation 30min, in collection It is clear to obtain recombinating msTPP crude enzyme liquids.Separately take 1 μ l empty plasmid pET-28a (+), convert in E.coli BL21, press more than Method is handled, the control as recombinant bacterial strain induced expression.Recombinant bacterial strain induced expression result is as shown in figure 15.
(3) recombinant protein isolates and purifies
Leave and take that recombinant bacterial strain induction is front and rear respectively and control group induction after after each 1ml of nutrient solution, and recombinant bacterial strain induction Supernatant carries out SDS-PAGE electrophoresis, detects expression of recombinant proteins situation.
1) post separation is crossed
His purification column Ni Sepharose are pressed in the purifying of restructuring TPS enzymes and restructuring TPP enzymesTM6 Fast Flow operations are said Bright book is carried out.
2) enzyme liquid removes imidazoles and high concentration NaCl
Imidazoles and NaCl containing high concentration in restructuring msTPS after separation, its activity may be influenceed.First dialysed using 3k Bag removes imidazoles and salt, reuses 10kDa super filter tubes and it is concentrated.
SDS-PAGE electrophoresis is carried out to concentrate, detects separation and purification of protein effect.Testing result such as Fig. 6 and Figure 17 institutes Show.
Embodiment 4 recombinates msTPS zymology Quality Research
(1) msTPS enzyme activity determination methods are recombinated
MsTPS enzyme activity determinations method is with reference to De Smet, K.A. etc.[13]Correlative study method adjusted it, in G6P Under conditions of enough, the UDP contents discharged after UDPG reacts in system are determined.
Assay method is divided into two steps：(1) UDPG and G6P is reacted under msTPS catalytic action, discharges UDP；(2) phosphorus The catalytic liquid that sour enol pyruvic acid (PEP), NADH (NADH) and the first step obtain swashs in pyruvic acid Reacted under the catalysis of enzyme (PyK) and lactic dehydrogenase (L-LDH), light absorption value is determined at 340nm, it is bent according to UDP standards Line computation goes out the UDP contents that reaction system is discharged.
1) first step reaction (100 μ l systems)
1.5ml EP pipes are placed in mixture of ice and water, add 50mM Tris-HCl (pH 7.4) buffer solution, 10mM G6P, 5mM UDPG, 2mM MgCl2,100ng msTPS.Gentle centrifugation mixes, 40 DEG C of incubation 30min.Boiling water boiling 5min, makes MsTPS is inactivated, as cooled on ice, 13,000rpm/min, 10min centrifugations.One enzyme activity unit (U) is defined as：It is anti-at this Under the conditions of answering, conversion enzyme amount needed for 1 μm of ol UDPG per minute.
2) second step reaction (200 μ l systems)
1.5ml EP pipes are placed in mixture of ice and water, add 50mM Tris-HCl (pH 7.4) buffer solution, 30 μ l first Step reaction supernatant, 2.5mM PEP, 0.5mM NADH, 2mM MgCl2, 3U/ml PyK, 3U/ml LDH.Gentle centrifugation mixes, 37 DEG C of incubation 30min.Boiling water boiling 5min, as cooled on ice, 13,000r/min, 1min centrifugations.Reaction solution is transferred to 96 holes In ELISA Plate, light absorption value has been determined in 20min.
(2) msTPS optimum temperatures determine
Set reaction temperature be respectively：4 DEG C, 12 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C.Determined by upper methods described Enzyme activity, to measure enzyme activity under optimum temperature as 100%, the enzyme activity under different temperatures is calculated, each thermograde is set Put three repetitions.
Temperature influences as shown in Figure 7 on restructuring msTPS enzymatic activitys.As a result show to recombinate msTPS activity with reaction temperature Increase and increase, activity reaches maximum at 40 DEG C, and enzymatic activity drastically declines with the rise of reaction temperature afterwards.Restructuring MsTPS optimal reactive temperature is 40 DEG C.In 4~40 DEG C of scope inner enzyme vigors more than the 60% of highest enzyme activity, this says Understand that the enzyme can still keep of a relatively high enzyme activity at low temperature, it may be possible to a kind of cold adapted.
(3) msTPS optimal pHs determine
Set pH gradient be respectively：5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0, when pH scopes 5.0~ Between 7.5, using Mes-NaOH buffer solutions, pH scopes are between 7.0~9.0, using Tris-HC1 buffer solutions.By the upper side Method determines enzyme activity, to measure enzyme activity under optimum pH as 100%, calculates the enzyme activity under different pH value, every pH ladders Degree sets 3 repetitions.
PH influences as shown in Figure 8 on restructuring msTPS enzymatic activitys.It is 7.5 to recombinate msTPS optimal pHs, and has wider pH It is worth scope, between pH 6.5~8, activity still reaches more than the 70% of maximum activity.When pH value is higher than 8, restructuring enzymatic activity shows Writing reduces.
(4) influence of the metal ion to msTPS activity
It is separately added into final concentration of 2mM Mg2+, Co2+, Mn2+, Ba2+, Zn2+, K+, Ca2+, Hg2+, Mg2+/Ca2+.To be not added with The reaction solution of any metal ion determines enzyme activity, each cation sets 3 weights as negative control by upper methods described It is multiple.
Metal ion influences as shown in Figure 9 on restructuring msTPS enzymatic activitys.MsTPS is recombinated without any metal ion In reaction solution, only faint activity, in Mg2+、Co2+Or Ba2+Under the conditions of existing, restructuring enzymatic activity reaches highest.K+ pairs Restructuring enzymatic activity has certain facilitation, but effect is not so good as Mg2+、Co2+Or Ba2+。Mn2+、Zn2+、Ca2+And Hg2+To recombinase Activity does not influence, and Ca2+With Mg2+It will not suppress Mg in the presence of simultaneously2+Effect.
(5) measure of msTPS Enzyme kinetic parameters
1) setting G6P concentration is respectively 1mM, 1.5mM, 2mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, UDPG constant concentrations are 5mM；
2) setting UDPG concentration be respectively 1mM, 1.5mM, 2mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, G6P constant concentration are 5mM.
3) by upper methods described measure enzyme activity, the reaction speed in two groups of reactions under different concentration of substrate [S] is determined respectively Spend V.Both inverses are obtained, to mapping, to draw straight line, msTPS is calculated respectively to G6P's and UDPG according to curvilinear equation KmValue and Vmax。
Figure 10 is to recombinate msTPS to scheme G6P Lineweaver-Burk.The regression equation that curve can be tried to achieve by figure is y= 0.022x+0.016, modified R2=0.99148.Michaelis constant K of the restructuring msTPS enzymes to G6P can be drawn by the equationm= 1.38mM and maximum reaction velocity Vmax=62.5U/minmg.
Figure 11 is that restructuring msTPS schemes to UDPG Lineweaver-Burk.The regression equation that curve can be tried to achieve by figure is y =0.0252x+0.0156, modified R2=0.99338.Michaelis constant K of the restructuring msTPS enzymes to UDPG can be drawn by the equationm= 1.62mM and maximum reaction velocity Vmax=64.1U/minmg.
Embodiment 5 recombinates msTPP zymologic property research
(1) msTPP enzyme activity determination methods are recombinated
The 1 correct plasmid of μ l sequence verifications is taken, is converted in E.coli BL21, to positive clone molecule conservation.By recombinant bacterium Strain is inoculated in LB fluid nutrient mediums of the 5ml containing 50 μ g/ml kanamycins and is incubated overnight, and takes 2ml overnight cultures to be connect by 1 ︰ 100 Kind in LB fluid nutrient mediums of the 200ml containing kanamycins, 37 DEG C, 220rpm is cultivated to OD600 is 0.6~0.8, IPTG is added To final concentration of 1mM, the Fiber differentiation 4h under the conditions of 30 DEG C, 180rpm, 6000g centrifugation 5min collect thalline.Thalline is resuspended In 10ml Binding Buffer (20mM Tris-HCl pH7.4,0.5M NaCl, 20mM imidazoles), lysozyme is added extremely Final concentration of 1mg/ml, after cracking 1h on ice, ultrasonication 15min makes thalline crack completely, 8000g centrifugation 30min, in collection It is clear to obtain recombinating msTPP crude enzyme liquids.Separately take 1 μ l empty plasmid pET-28a (+), convert in E.coli BL21, press more than Method is handled, the control as recombinant bacterial strain induced expression.It is as shown in figure 17 to recombinate msTPP purifying situation.
(2) msTPP optimum temperatures determine
Temperature is respectively set to 4 DEG C, 12 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C.According to method described above point Inorganic phosphorus concentration is not determined, to measure inorganic phosphorus concentration under optimum temperature as 100%, calculates relative inorganic phosphorus concentration, Mei Yiwen Spend gradient and 3 repetitions are set.
MsTPP optimum temperature measurement results are shown in Figure 18.As a result show recombinate msTPP activity with increasing for reaction temperature and Increase, at 30 DEG C, enzymatic activity to maximum, afterwards activity decline with the rise of reaction temperature；Recombinate msTPP optimal reaction temperature Spend for 30 DEG C；In 4~50 DEG C of scope inner enzyme vigors more than the 50% of highest enzyme activity, this illustrates the enzyme compared with low temperature Remain to keep of a relatively high enzyme activity.
(3) measure of msTPP optimal pH is recombinated
PH is respectively set to 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0, is separately added into 50mM differences pH It is worth buffer solution.Inorganic phosphorus concentration is determined respectively according to above method, to measure inorganic phosphorus concentration under optimum pH as 100%, meter The relative inorganic phosphorus concentration under different pH value is calculated, each pH gradient sets three repetitions.
Inorganic phosphorus concentration is determined under different pH condition, to measure inorganic phosphorus concentration under optimum pH as 100%, is calculated Relative inorganic phosphorus concentration under different pH value.It is vertical with respect to inorganic phosphorus concentration using the pH value of differential responses buffer solution as abscissa Coordinate, make pH- with respect to inorganic phosphorus concentration curve map (referring to Figure 19).As a result it is 7.5 to show the optimal pH for recombinating msTPP, and With wider pH value range, between pH 6~8.5, activity still reaches more than the 70% of maximum activity；It is higher than 8.5 in pH value When, restructuring enzymatic activity significantly reduces.
(4) influence of the metal ion to restructuring msTPP activity
It is separately added into final concentration of 2mM Mg2+, Co2+, Mn2+, Ba2+, Zn2+, K+, Ca2+, Hg2+, Mg2+/Ca2+.To be not added with Any metal ion system is negative control, and inorganic phosphorus concentration is determined respectively according to above method, and each cation sets 3 Repeat.
MsTPP is recombinated in the reaction solution without any metal ion, only very faint activity, in Mg2+In the presence of Under conditions of, restructuring enzymatic activity reaches highest；Co2+Or Ba2+There is larger facilitation to restructuring enzymatic activity, but effect is not so good as Mg2+；K+Or Hg2+To maintaining restructuring enzymatic activity to have certain effect；Mn2+、Zn2+、Ca2+Restructuring enzymatic activity is not influenceed, and Ca2+With Mg2+It will not suppress Mg in the presence of simultaneously2+Effect (referring to Figure 20).
(5) measure of msTPP Enzyme kinetic parameters is recombinated
It is respectively 1mM, 1.5mM, 2mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM to set T6P concentration.Press The reaction speed V under different T6P concentration [S] is determined according to method described above.Both inverses are obtained, to mapping, to draw Straight line, Ks of the restructuring msTPP to T6P is calculated according to curvilinear equationmValue and Vmax。
Figure 21 is that restructuring msTPP schemes to T6P Lineweaver-Burk.The regression equation that curve can be tried to achieve by figure is y= 0.0297x+0.0121, modified R2=0.98163.Michaelis constant K of the restructuring msTPS enzymes to UDPG can be drawn by the equationm= 2.45mM and maximum reaction velocity Vmax=82.64U/minmg.
The invention discloses two kinds of trehalose synthesis related gene order of enzyme and the expression and purification method of both enzymes. Two kinds of described enzymes are the actinomyces Microbacterium sediminis sp.nov.YLB-01 in deep-sea source respectively Two enzymes in TPS/TPP trehaloses synthesis path, 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) and 6- phosphotrehalose UDP-transglucosylase phosphates Enzyme (msTPP).Two kinds of described enzymes of clone, and by cloned sequence insertion vector pET-28a (+).Plasmid vector is transformed into impression After state E.coli BL21 cells, induced expression is carried out.The restructuring enzyme liquid His purification column Ni Sepharose obtainedTM 6 Fast Flow are separated and purified, the recombinase that can be purified.It is demonstrated experimentally that two kinds of restructuring provided by the present invention Enzyme still has higher activity under cryogenic, such as under the conditions of 4 DEG C, respectively reached 72.78% (msTPS) and 50.20% (msTPP) activity.
Sequence table
<110>State Oceanic Administration Bureau The Third Oceanography Institute
<120>6- phosphotrehalose UDP-transglucosylases synzyme caused by the microbacterium of deep-sea and 6- phosphotrehalose UDP-transglucosylase phosphates
<141> 2017-09-14
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1473
<212> DNA
<213> Microbacterium sediminis sp. nov.YLB-01
<400> 1
atggaccgtg ccgatcttgt cgtcgtagcc aaccgcctgc cggtcgaccg tgtggtcgac 60
accgacggca ccgagacctg gcggcgttcg ccggggggtc tcgtgaccgc gctcgagcag 120
gtgatggcga agaccgacgg cgcgtgggtc ggatggggcg gcaaggccga tctcgacatc 180
gagccgttcg acttcgaggg cacgctgctc gtgccggtgc cgctgagcgc cgaggacctg 240
aagtactact acgagggctt ctcgaacggc acgatctggc cgctgtacca cgacgtcatc 300
gcggcgccgg tgtatcgccg gtcgtggtgg gacgcctacg tgcgcgtgaa ccggaggttc 360
gccgaggcgg cggccgaggt ggccgatcaa gagggcttcg tgtgggtgca ggactaccaa 420
ctgcagctcg tcccgcagat gctgcgcgag ctgcggccgg atctgacgat cggctacttc 480
caccacattc cgttcccggc atacggcctg tactcgcagc tgccgtggcg gcgccaggtg 540
ctcgaggggc tgctcggtgc cgacgtgctc ggcttccagc gggtggccga cgcgggcaac 600
ttccagcggt cggtgcggcg catcttcaag taccccaccc gcgcgaacga gatcgaggtg 660
cccgcgggcg acggcggatc gcgcagcgtg atcgcggcgc ccttcccgat ctcgatcgac 720
gcgcaggcgt tcatcgagct gtcgcagcgc cccgacatcc gggcgcgcgc cgtcgagatc 780
cgcgagcagt tgggcaaccc gaagaagatc ctgctgggcg tcgaccgcct cgactacacc 840
aagggcatcc ggcaccggct caaggcgttc ggggagctcc tcgatgacgg cgcggtgacc 900
gtcggcgagg tggcgctcgt gcagatcgcc agcccgagcc gccccggcgt cgaggcgtat 960
caggacctcc gcgacgagat cgagctcacc gtcggccgca tcaacggcga ctacgacacg 1020
atgcaccaca cggcgatccg ctacctgcat cagggcttcc cgcgcgagga gatggtcgcg 1080
ctgtacctgg ccgccgacgt gatgctgatc acggcgctgc gcgacggcat gaacctcgtc 1140
gccaaggagt acgtggcggt gcgcaccgac aatcgcggcg tgctcgtgct gagcgagttc 1200
gccggcgccg ccgacgagct gactacggcg ctgctcgtga acccccacga catcgacggc 1260
atgaaggacc tgatcctccg cgcgatcaac atgcccgagg ccgagcagtc gcgtcgcatg 1320
cgcgcgatgc gccgcaaggt gctcgagaac gacgtcgagg cgtggggccg cagcttcatg 1380
aaggccgtcg acgccgtgcg gaacaagcgc gccacgggcg ccccgattcc gcgagaggtc 1440
aagcccaccc cgaggaagaa gccgcgcgaa tga 1473
<210> 2
<211> 490
<212> PRT
<213> Microbacterium sediminis sp. nov.YLB-01
<400> 2
Met Asp Arg Ala Asp Leu Val Val Val Ala Asn Arg Leu Pro Val Asp
1 5 10 15
Arg Val Val Asp Thr Asp Gly Thr Glu Thr Trp Arg Arg Ser Pro Gly
20 25 30
Gly Leu Val Thr Ala Leu Glu Gln Val Met Ala Lys Thr Asp Gly Ala
35 40 45
Trp Val Gly Trp Gly Gly Lys Ala Asp Leu Asp Ile Glu Pro Phe Asp
50 55 60
Phe Glu Gly Thr Leu Leu Val Pro Val Pro Leu Ser Ala Glu Asp Leu
65 70 75 80
Lys Tyr Tyr Tyr Glu Gly Phe Ser Asn Gly Thr Ile Trp Pro Leu Tyr
85 90 95
His Asp Val Ile Ala Ala Pro Val Tyr Arg Arg Ser Trp Trp Asp Ala
100 105 110
Tyr Val Arg Val Asn Arg Arg Phe Ala Glu Ala Ala Ala Glu Val Ala
115 120 125
Asp Gln Glu Gly Phe Val Trp Val Gln Asp Tyr Gln Leu Gln Leu Val
130 135 140
Pro Gln Met Leu Arg Glu Leu Arg Pro Asp Leu Thr Ile Gly Tyr Phe
145 150 155 160
His His Ile Pro Phe Pro Ala Tyr Gly Leu Tyr Ser Gln Leu Pro Trp
165 170 175
Arg Arg Gln Val Leu Glu Gly Leu Leu Gly Ala Asp Val Leu Gly Phe
180 185 190
Gln Arg Val Ala Asp Ala Gly Asn Phe Gln Arg Ser Val Arg Arg Ile
195 200 205
Phe Lys Tyr Pro Thr Arg Ala Asn Glu Ile Glu Val Pro Ala Gly Asp
210 215 220
Gly Gly Ser Arg Ser Val Ile Ala Ala Pro Phe Pro Ile Ser Ile Asp
225 230 235 240
Ala Gln Ala Phe Ile Glu Leu Ser Gln Arg Pro Asp Ile Arg Ala Arg
245 250 255
Ala Val Glu Ile Arg Glu Gln Leu Gly Asn Pro Lys Lys Ile Leu Leu
260 265 270
Gly Val Asp Arg Leu Asp Tyr Thr Lys Gly Ile Arg His Arg Leu Lys
275 280 285
Ala Phe Gly Glu Leu Leu Asp Asp Gly Ala Val Thr Val Gly Glu Val
290 295 300
Ala Leu Val Gln Ile Ala Ser Pro Ser Arg Pro Gly Val Glu Ala Tyr
305 310 315 320
Gln Asp Leu Arg Asp Glu Ile Glu Leu Thr Val Gly Arg Ile Asn Gly
325 330 335
Asp Tyr Asp Thr Met His His Thr Ala Ile Arg Tyr Leu His Gln Gly
340 345 350
Phe Pro Arg Glu Glu Met Val Ala Leu Tyr Leu Ala Ala Asp Val Met
355 360 365
Leu Ile Thr Ala Leu Arg Asp Gly Met Asn Leu Val Ala Lys Glu Tyr
370 375 380
Val Ala Val Arg Thr Asp Asn Arg Gly Val Leu Val Leu Ser Glu Phe
385 390 395 400
Ala Gly Ala Ala Asp Glu Leu Thr Thr Ala Leu Leu Val Asn Pro His
405 410 415
Asp Ile Asp Gly Met Lys Asp Leu Ile Leu Arg Ala Ile Asn Met Pro
420 425 430
Glu Ala Glu Gln Ser Arg Arg Met Arg Ala Met Arg Arg Lys Val Leu
435 440 445
Glu Asn Asp Val Glu Ala Trp Gly Arg Ser Phe Met Lys Ala Val Asp
450 455 460
Ala Val Arg Asn Lys Arg Ala Thr Gly Ala Pro Ile Pro Arg Glu Val
465 470 475 480
Lys Pro Thr Pro Arg Lys Lys Pro Arg Glu
485 490
<210> 3
<211> 792
<212> DNA
<213> Microbacterium sediminis sp. nov.YLB-01
<400> 3
atgaccgagc ccaccaccga ctggcgcacc gccctggagc agatcgcccg caccgacacg 60
ctgctcgtcg ccctcgactt cgacggcacg ctcgcgccgc tgcaggacga tccgatgcag 120
tcccgtgccc tgccggagtc ggcggcggcg atcgcgcggc tggcggacct cccggcgacg 180
atcgtggcgt acgtgtcggg gcggagcctg ccggatctgc ggatcatcgg cgagcacgac 240
gacgcgtcga aggtgtggct ggcgggctcg cacggggtgg agtactggcg gcccgccgac 300
gccgtggtca ccgagctcga cgagcccgac gagacgcacg agcgcgagct gctggagcgg 360
ctcaacgccg aggctaagca gctcgtcgac ggcttcgagg gcgcgtggat cgaggacaag 420
agcgtggggt tcgcgctgca cacgcggctc accccgcccg acatcgcccc ggggatccag 480
ggcgtcatcg acgcgctcgt cgcccgcgag gcgcccggct ggcggcgccg cccgggccac 540
aacctcatcg agtactcgtg gcgccacgag ggcaaggacg cggcggtggc gcggctgcgg 600
gagcagaccg gcgcgagcgc ggtgctgttc gcgggcgacg acgtcaccga cgaggacgcc 660
ctcggcagcc tgcaggagca cgacctcggc gtgcgcgtgg ggccggggga gacctcggcg 720
cgcgtgcgcg tggccgacgc ccacgagttc gcggagctgc tcagcgcgct ggccgatctg 780
cgggcatcct ga 792
<210> 4
<211> 263
<212> PRT
<213> Microbacterium sediminis sp. nov.YLB-01
<400> 4
Met Thr Glu Pro Thr Thr Asp Trp Arg Thr Ala Leu Glu Gln Ile Ala
1 5 10 15
Arg Thr Asp Thr Leu Leu Val Ala Leu Asp Phe Asp Gly Thr Leu Ala
20 25 30
Pro Leu Gln Asp Asp Pro Met Gln Ser Arg Ala Leu Pro Glu Ser Ala
35 40 45
Ala Ala Ile Ala Arg Leu Ala Asp Leu Pro Ala Thr Ile Val Ala Tyr
50 55 60
Val Ser Gly Arg Ser Leu Pro Asp Leu Arg Ile Ile Gly Glu His Asp
65 70 75 80
Asp Ala Ser Lys Val Trp Leu Ala Gly Ser His Gly Val Glu Tyr Trp
85 90 95
Arg Pro Ala Asp Ala Val Val Thr Glu Leu Asp Glu Pro Asp Glu Thr
100 105 110
His Glu Arg Glu Leu Leu Glu Arg Leu Asn Ala Glu Ala Lys Gln Leu
115 120 125
Val Asp Gly Phe Glu Gly Ala Trp Ile Glu Asp Lys Ser Val Gly Phe
130 135 140
Ala Leu His Thr Arg Leu Thr Pro Pro Asp Ile Ala Pro Gly Ile Gln
145 150 155 160
Gly Val Ile Asp Ala Leu Val Ala Arg Glu Ala Pro Gly Trp Arg Arg
165 170 175
Arg Pro Gly His Asn Leu Ile Glu Tyr Ser Trp Arg His Glu Gly Lys
180 185 190
Asp Ala Ala Val Ala Arg Leu Arg Glu Gln Thr Gly Ala Ser Ala Val
195 200 205
Leu Phe Ala Gly Asp Asp Val Thr Asp Glu Asp Ala Leu Gly Ser Leu
210 215 220
Gln Glu His Asp Leu Gly Val Arg Val Gly Pro Gly Glu Thr Ser Ala
225 230 235 240
Arg Val Arg Val Ala Asp Ala His Glu Phe Ala Glu Leu Leu Ser Ala
245 250 255
Leu Ala Asp Leu Arg Ala Ser
260
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 5
tcattcgcgc ggcttcttcc tc 22
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 6
gaccgtgccg atcttgtcgt cg 22
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 7
tcaggatgcc cgcagatcgg c 21
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 8
accgagccca ccaccgactg 20

Claims (6)

  1. 6- phosphotrehalose UDP-transglucosylases synzyme 1. (msTPS) gene is Microbacterium sediminis sp.nov.YLB-01 6- phosphotrehalose UDP-transglucosylase synthase genes, its nucleotide sequence is as shown in SEQ ID NO.1.
  2. 6- phosphotrehalose UDP-transglucosylases phosphate 2. (msTPP) gene is Microbacterium sediminis sp.nov.YLB- 01 6- phosphotrehalose UDP-transglucosylase phosphatase genes, its nucleotide sequence is as shown in SEQ ID NO.3.
  3. 3. the recombinase of 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) is Microbacterium sediminis The recombinase of sp.nov.YLB-01 6- phosphotrehalose UDP-transglucosylase synzyme.
  4. 4. the recombinase of 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) is Microbacterium sediminis The recombinase of sp.nov.YLB-01 6- phosphotrehalose UDP-transglucosylase phosphates.
  5. 5. the production bacterial strain of 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) and 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP), it is special Sign is for Microbacterium sediminis sp.nov.YLB-01, to be one plant of new spherical bacterium of Gram-positive, be One plant of type strain, was preserved in China typical culture collection center in 2010, and deposit number is：CCTCC NO： AB2010363。
  6. 6. 6- phosphotrehalose UDP-transglucosylases synzyme (msTPS) as claimed in claim 5 and 6- phosphotrehalose UDP-transglucosylases phosphate (msTPP) Preparation method, it is characterised in that comprise the following steps：
    1) msTPS or msTPP genes are cloned, SEQ ID in upstream and downstream primer used in msTPS gene clonings such as sequence table Shown in NO.5 and SEQ ID NO.6；In upstream and downstream primer used in msTPP gene clonings such as sequence table SEQ ID NO.7 and Shown in SEQ ID NO.8；
    2) PCR primer is used in homologous recombination strategy insertion vector pET-28a (+), builds the weight of the gene containing msTPS or msTPP Group plasmid；
    3) recombinant plasmid transformed is entered in competent cell E.coli DH5 α, to expand plasmid；
    4) recombinant plasmid successfully constructed is chosen, is converted into competent cell E.coli BL21；
    5) choose positive clone molecule and carry out protein induced expression；
    6) restructuring msTPS or msTPP albumen presses His purification column Ni SepharoseTM6Fast Flow operational manuals are divided From purifying.
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CN108220266A (en) * 2018-03-14 2018-06-29 国家海洋局第三海洋研究所 Prawn liver sausage born of the same parents' worm trehalose-6-phosphate synthase and preparation method thereof
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Application publication date: 20171215