CN101323858A - Xylose isomerase, and encoding gene and use thereof - Google Patents
Xylose isomerase, and encoding gene and use thereof Download PDFInfo
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Abstract
The invention relates to a xylose isomerase and the coded protein and the application thereof, pertaining to the technical field of enzyme genetic engineering. A coded xylose isomerase DNA sequence is obtained from mutation at partial positions of a xylose isomerase gene xylA(Genebank EU643621) in Sorangium cellulosum 157-2. The invention also relates to enzyme coded by the DNA sequence and the application of the coded enzyme in the fields of chemical industry, food and medicine. In addition, compared with the traditional xylose isomerase, the activity of xylose isomerase of the invention in saccharomyces cerevisiae is increased by 2.8 times.
Description
Technical field
The present invention relates to a kind of xylose isomerase and encoding gene thereof and application, belong to the enzyme gene engineering technology field.
Background technology
Xylose isomerase (D-Xylose Isomerase, XI) catalysis five-carbon sugar D-wood sugar is converted into the D-xylulose in vivo, in external catalysis hexose D-conversion of glucose is the isomerization reaction of D-fructose, claim glucose isomerase (GlucoseIsomerase again, GI), extensively be present in bacterium, the small part fungi.
Lignocellulose (lignocelluiosic) is a renewable resources the abundantest on the earth, mainly is present in the wooden waste of agricultural byproducts and forest products industry.Its main component is: Mierocrystalline cellulose (cellulose), hemicellulose (hemicellulose) and xylogen (lignin).Mierocrystalline cellulose is through not branch crystalloid polymer that β-1,4 glycosidic link connects into by glucosyl residue; Hemicellulose is to comprise that multiple five-carbon sugars such as wood sugar, pectinose, glucose, semi-lactosi, seminose are main noncrystalline heterogeneous polymer, and wherein wood sugar content accounts for 6%-28% in different material, is only second to glucose content.The reaction of reversible xylose isomerase changes into xylulose to xylose isomerase, and xylulose enters tricarboxylic acid cycle, fermentative production of ethanol.In addition, glucose isomerization reaction is industrially to prepare high fructose syrup (high fructose corn syrup, committed step HFCS) with starch on a large scale.
The xylose isomerase gene in multiple source is by clonal expression, as Escherichia coli, Bacillus subtilis, Thermotoga species, Streptomyces species, Actinoplanes missouriensis, Thermus species and Arthrobacter species or the like.But the xylose isomerase gene that has only a few up to now and mostly be thermophilic bacterium is at alcohol production tradition bacterial strain---obtain activity expression in the yeast saccharomyces cerevisiae (Saccharomyces cevisiae), and, generally owing to the active too low rate-limiting step that becomes the xylose metabolism approach, therefore the exploitation and the enzyme molecular modification of novel xylose isomerase are very necessary under the ethanol fermentation temperature about 30 ℃.
Middle warm type bacterial fibers heap capsule bacterium (Sorangium cellulosum) is the bacterial classification of unique energy degraded cellulose in the slime bacteria, extensively distributes in soil.S.cellulosum can be implied with the growth and the macromolecule substrate of degrading effectively on the simple inorganic salt flat board of crystalline cellulose (as filter paper) or xylan.In the previous work, we have cloned the xylose isomerase gene xylA of S.celluiosum, and it can show activity in yeast saccharomyces cerevisiae, but it is not high enough to live than enzyme, thereby are necessary further to transform.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of xylose isomerase and encoding gene thereof and application are provided.
A kind of dna sequence dna of the xylose isomerase of encoding, it is that 53 of xylose isomerase gene xylA (Genebank EU643621) in sorangium cellulosum (Sorangium cellulosum) 157-2 sport T by C; 219 sport A by C; 243 sport A by G; 265 sport G by A; 437 sport T by G; 503 sport A by T; 596 sport A by G; 635 sport T by A; 755 sport G by A; 914 sport T by G; 930 sport T by C; 1006 sport A by G; 1175 sport A by G; 1255 sport G by A.
A kind of dna sequence dna of the xylose isomerase of encoding, gene order is as follows:
atgaccgtcg?tgattggaaa?caaagggtac?ttccccggtg?tcggcaagat?cctctacgag 60
gggcccgagt?ccgacaaccc?cgtcgccttc?aagtggtacg?acgagagccg?cgtcgtcgcc 120
ggcaagccca?tgaaggacca?cttcaagttc?gccgtgtgtt?actggcacac?cttctgtggt 180
cgtgggcacg?accccttcgg?tccggggacg?atcaacttac?cctgggacga?ggggaaggac 240
ccactgaccc?gcgctcgcat?gaaggcggac?gcgaacttcg?agttcatcac?caagctgggc 300
gcgacgcact?actgtttcca?cgacatcgac?ctggtggagg?agggcgacag?ccgcgccgag 360
acgtcgcgcc?gccttcaggg?catcgtcggg?tacgccaagc?agaagcaagc?gcagtcgggc 420
gtcaagctgc?tgtgggtcac?ggcgaacttg?ttctccaacc?cgcgctacat?gaacggcgcc 480
tccacgaacc?cggatttctc?cgaggtggcc?tacgccggcg?cccagctgaa?ggacgccctc 540
gacgccacca?tcgcgctgaa?cggtgagaac?tacgtgttct?ggggcggccg?cgaggattac 600
atgagcctgc?tgaacacgga?catgaagcgc?gagatggagc?acttcgcgcg?cttcttgacc 660
atggcgcgcg?actacgcgcg?cgcccggggc?ttcaagggga?ccttcttcat?cgagcccaag 720
ccgatggagc?cgtcgaagca?ccagtacgac?ttcggcgccg?agaccgtgat?cgggttcctg 780
cgccagcacg?gcctggacaa?ggacttcaag?ctcaacctgg?agacgaacca?cgcgacgctc 840
gctggccaca?ccatggagca?cgagatgcag?gtggccgcgg?acgccggcat?gctcggcagc 900
atcgacgcga?acctcggcga?ctaccagaat?gcgtgggaca?ccgatcagtt?cccctacaac 960
atcaacgaga?cggtggagat?gatgctcatc?ctcctccgca?gcggcaggtt?ccagggtggc 1020
ggcgtcaact?tcgacgcgaa?gcgccgccgc?aactcgacgg?acctgatcga?caccttccac 1080
ggccacatcg?gcggcatgga?cgtgttcgcc?cgcgcgctca?tcatcgccgg?cgacctgatc 1140
cgcaagtcgc?cgctcgagtc?gatgcgcaag?gctcattacg?cgtcgttcga?cggcggcaag 1200
ggcgcggagt?tcgagcaggg?gaagctcacg?ctggagcagc?tggccgagat?cggcgacgcc 1260
ggcggcgagg?tcaagctcac?gagcggtcag?caagagctgt?acgagaacat?cgtcaatcgg 1320
tggatccgg 1329。
A kind of xylose isomerase, it is the mutant enzyme of the xylose isomerase gene xylA encoded polypeptides (Genebank ACC99633) in sorangium cellulosum (Sorangium cellulosum) 157-2, relatively preceding with sudden change, have the amino-acid residue in 12 sites that variation has taken place, it comprises: 18 sport L by P, 73 sport L by F, 89 sport A by T, 146 sport V by G, 168 sport E by V, 199 sport D by G, 212 sport M by K, 252 sport G by D, 305 sport L by R, 336 sport R by G, 392 sport H and 419 by R and sport D by N.
A kind of xylose isomerase, aminoacid sequence is as follows:
MTVVIGNKGY?FPGVGKILYE?GPESDNPVAF?KWYDESRVVA?GKPMKDHFKF?AVCYWHTFCG 60
RGHDPFGPGT?INLPWDEGKD?PLTRARMKAD?ANFEFITKLG?ATHYCFHDID?LVEEGDSRAE 120
TSRRLQGIVG?YAKQKQAQSG?VKLLWVTANL?FSNPRYMNGA?STNPDFSEVA?YAGAQLKDAL 180
DATIALNGEN?YVFWGGREDY?MSLLNTDMKR?EMEHFARFLT?MARDYARARG?FKGTFFIEPK 240
PMEPSKHQYD?FGAETVIGFL?RQHGLDKDFK?LNLETNHATL?AGHTMEHEMQ?VAADAGMLGS 300
IDANLGDYQN?AWDTDQFPYN?INETVEMMLI?LLRSGRFQGG?GVNFDAKRRR?NSTDLIDTFH 360
GHIGGMDVFA?RALIIAGDLI?RKSPLESMRK?AHYASFDGGK?GAEFEQGKLT?LEQLAEIGDA 420
GGEVKLTSGQ?QELYENIVNR?WIR 443。
The present invention also protects the genophore that contains above-mentioned dna fragmentation.
The present invention also protects the transformant that contains the said gene carrier.
Above-mentioned xylose isomerase, dna sequence dna, genophore and transformant are in the application of chemical industry, food, field of medicaments.
Xylose isomerase gene mxylA provided by the invention is that the clone obtains xylose isomerase gene xylA (Genebank EU643621) from sorangium cellulosum (Sorangium cellulosum) 157-2, introduce catastrophe point by fallibility PCR with the external molecular orientation evolvement technology that DNA reorganization combines then, screening obtains, and concrete steps are as follows:
The acquisition of i, goal gene:
(1) from sorangium cellulosum (Sorangium cellulosum) 157-2, extract total DNA:
Collect the 0.1g thalline, adding 0.75mL GTE damping fluid (25% sucrose, 50mmol/L Tris-HCl, the 100mmol/L ethylenediamine tetraacetic acid (EDTA), pH 8.0) and an amount of granulated glass sphere, vortex oscillation, fully mixing thalline.Shift thalline to the centrifuge tube of 5mL, add 0.5mL GTE, add 10%SDS (sodium laurylsulfonate) 150 μ L behind the vortex oscillation device mixing, the Proteinase K 15 μ L of 20mg/mL, abundant mixing, 37 ℃ of insulation 1h.Put upside down mixing during this time at regular intervals.Add 0.25mL 5mol/LNaCl, leave standstill 10min behind the mixing, add 0.25mL CTAB (cetyl trimethylammonium bromide)/NaCl (10%CTAB/0.7mol/L NaCl) then, mixing, 65 ℃ of insulation 20min (CTAB puts into 65 ℃ of water-baths in advance and is incubated).Isopyknic phenol/chloroform/primary isoamyl alcohol extracting twice is gently mixed the centrifugal 10min of 30min left and right sides 5000g, and supernatant is transferred to new pipe.Add the dehydrated alcohol of 2 times of volumes, put upside down mixing, room temperature leaves standstill 10min, and the centrifugal 10min of 10000g adds 70% washing with alcohol, and drying at room temperature 20min adds 200 μ L TE ,-20 ℃ of preservations.
(2) according to the open reading frame of sorangium cellulosum 157-2 xylose isomerase gene (ORF) design of amplification primers:
Primer 1:5 '-ATCG
AGATCTATGACCGTCGTGATTGGA-3 '
Primer 2: 5 '-TTCG
GAGCTCTTACCGGATCCACCGATT-3 ';
Total DNA with acquisition in the step (1) is that template is carried out pcr amplification, and the PCR reaction conditions is: 94 ℃ of sex change 2min; 94 ℃ of sex change 1min; 62 ℃ of annealing 1min; 72 ℃ are extended 90sec; Reaction is carried out 30 and is followed badly, and last circulation extension time is 5min.In the PCR reaction system, dAP concentration is 0.2mmol/L, and dGTP concentration is 0.2mmol/L, and dTTP concentration is 1mmol/L, and dCTP concentration is 1mmol/L, MgCl
2Concentration is 7mmol/L, MnCl
2Be concentration 0.3mmol/L.After reclaiming purifying, be the DNA of the about 5 μ g of DNAaesI processing total amount of 0.01U/ μ L with final concentration, place 80 ℃ of following deactivation 10min immediately behind 15 ℃ of processing 4-5min, get the DNA small segment.
(3) DNA small segment electrophoresis in the sepharose of 2wt% that step (2) is obtained reclaims the dna fragmentation between the 100-200bp, and puts into and do not have the PCR of primer system, carries out the dna fragmentation recombining reaction.
(4) the recombining reaction product that obtains with step (3) is a template, and with the primer 1 that step (2) provides, primer 2 is a primer, carries out pcr amplification, and the PCR reaction conditions is: 94 ℃ of sex change 2min; 94 ℃ of sex change 1min; 62 ℃ of annealing 1min; 72 ℃ are extended 90sec; 30 circulations are carried out in reaction, and last circulation extension time is 5min.Reclaim near the band of 1.4kb behind the PCR product electrophoresis that obtains, get goal gene.
The expression of ii, goal gene:
(1) goal gene that obtains in the extraction step with goal gene is cut with BglII, SacI enzyme, take directed connect skill wood and directed connection of plasmid vector pYPX251 (GenBank AY178046) of cutting processing through BglII, SacI enzyme enzyme, make up xylose isomerase gene sudden change library;
(2) the xylose isomerase gene sudden change library transformed into escherichia coli HB101 (ATCC 33694) that step (1) is obtained; And to be coated on the wood sugar be on the M9 flat board that is added with 50mg/L ammonia benzyl element of sole carbon source, through 20～40 hours cultivation, occurs single bacterium colony on the flat board; Carry out primary dcreening operation according to the bacterium colony size, select bacterium colony than macrocolony; The concrete prescription of described solid M9 substratum is: NH
4Cl 5g/L, Na
2HPO
430g/L, KH
2PO
415g/L, NaCl 2.5g/L, MgSO
4.7H
2O 0.25g/L, wood sugar 20g/L, agar 15g/L.
(3) from step (2) screen than the macrocolony, carry out the extraction of enzyme liquid in the following way, under 37 ℃ at LB substratum (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L, pH 7.0) in cultivate than macrocolony 10～12 hours to logarithmic phase, 8000 leave heart 5min collects thalline, use the 0.9%NaCl washed twice, be dissolved in lysis buffer (1mM PMSF, the 50mmol/L Tris-HCl of new preparation then, pH7.0), add an amount of granulated glass sphere (diameter 0.5mm), under cytoclasis instrument top speed, handle 30～50s, ice bath 5min, the broken repetition 3～5 times, the centrifugal 10min of 10000g collects supernatant, is enzyme liquid.
Enzyme work to above-mentioned enzyme liquid is measured:
700mmol/L D-wood sugar, the 0.01-0.025U xylose isomerase contains 10mmol/L MnCl
250mmol/L Tris-HClpH7.0 damping fluid in, respectively 45 ℃ the reaction 20min, add 150 μ L, 50% trichoroacetic acid(TCA) termination reaction and 185 μ L 2MNa
2CO
3The neutralization reaction damping fluid.Get 0.5mL and add 0.04U SDH (sorbito dehy drogenase, sigma company) and 0.15mM NADH (reduced form cigarette amino acid adenine dinucleotide), mixing detects the oxidized amount (promptly doing time scan at 340nm) of NADH under 340nm immediately.Try to achieve the xylulose amount according to typical curve.One of xylose isomerase (XI) is defined as than unit of activity (U/mg): the μ mol/L number of every mg zymoprotein per minute conversion of substrate.
Carry out multiple sieve by enzyme activity determination and obtain a plant mutant body, extract its plasmid, xylA is partly checked order, the result of order-checking is the sequence shown in the SEQ NO.1 (mxylA), and the polypeptide shown in the coding SEQ NO.2 promptly gets xylose isomerase.
Xylose isomerase gene mxylA provided by the invention is that the clone obtains xylose isomerase gene xylA (Genebank EU643621) from sorangium cellulosum (Sorangium cellulosum) 157-2, introduce catastrophe point by fallibility PCR with the external molecular orientation evolvement technology that DNA reorganization combines then, screening obtains.It has the nucleotide sequence shown in the SEQ NO.1, and ORF is long to be 1329bp, 443 the amino acid whose albumen (SEQ NO.2) of encoding.Compare with Genebank EU643621, it comprises: 53 C → T; 219 C → A; 243 G → A; 265 A → G; 437 G → T; 503 T → A; 596 G → A; 635 A → T; 755 A → G; 914 G → T; 930 C → T; 1006 G → A; 1175 G → A; 1255 A → G.The dna sequence dna that the present invention relates to also should comprise except the nucleotide sequence shown in the SEQ NO.1: the various combinations of other dna sequence dnas of the discontinuous xylo-pfan isomerase (SEQ NO.2) that the present invention relates to SEQ NO.1 complementary sequence, because of can encoding of causing of the degeneracy of codon and the point mutation of above each position.
The invention provides new polypeptide, it contains the coded aminoacid sequence of above-mentioned nucleotide sequence, promptly contains the sequence shown in the SEQ NO.2, and proteins encoded is sorangium cellulosum (Sorangium cellulosum) xylose isomerase (S1) of sudden change.This polypeptide and gene xylA encoded polypeptides (Genebank ACC99633) are relatively, have the amino-acid residue in 12 sites that variation has taken place, it comprises: P18-L, F73-L, T89-A, G146-V, V168-E, G199-D, K212-M, D252-G, R305-L, G336-R, R392-H and N419-D.The peptide sequence that the present invention relates to also comprises the various combinations of the point mutation of above each position.
Xylose isomerase of the present invention is as follows than the advantage of traditional xylose isomerase: temperature can give expression to active xylose isomerase in a kind of in yeast saccharomyces cerevisiae, under the growth temperature condition that yeast saccharomyces cerevisiae can tolerate, still can show higher activity, and, through the sudden change that the present invention relates to, 2.8 times have been improved before its specific activity sudden change in yeast saccharomyces cerevisiae.
Description of drawings
The structure schema in Fig. 1 xylose isomerase gene sudden change of the present invention library;
The optimal pH of Fig. 2 xylose isomerase of the present invention; The design of graphics of Fig. 3 recombinant vectors pHX-S1/pHX-SXI.
Embodiment
Xylose isomerase gene mxylA provided by the invention is that the clone obtains xylose isomerase gene xylA (Genebank EU643621) from sorangium cellulosum (Sorangium cellulosum) 157-2, introduce catastrophe point by fallibility PCR with the external molecular orientation evolvement technology that DNA reorganization combines then, screening obtains, and concrete steps are as follows:
(1) from sorangium cellulosum (Sorangium cellulosum) 157-2, extract total DNA:
Collect the 0.1g thalline, adding 0.75mL GTE damping fluid (25% sucrose, 50mmol/L Tris-Cl, 100mmol/LEDTA, PH 8.0) and an amount of granulated glass sphere, vortex oscillation, fully mixing thalline.Shift thalline to the centrifuge tube of 5mL, add 0.5mL GTE, add 10%SDS 150 μ L behind the vortex oscillation device mixing, the Proteinase K 15 μ L of 20mg/mL, abundant mixing, 37 ℃ of insulation 1h.Put upside down mixing during this time at regular intervals.Add 0.25mL 5mol/L NaCl, leave standstill 10min behind the mixing, add 0.25mL CTAB/NaCl (10%CTAB/0.7mol/L NaCl) then, mixing, 65 ℃ of insulation 20min (CTAB puts into 65 ℃ of water-baths in advance and is incubated).Isopyknic phenol/chloroform/primary isoamyl alcohol extracting twice is gently mixed the centrifugal 10min of 30min left and right sides 5000g, and supernatant is transferred to new pipe.Add the dehydrated alcohol of 2 times of volumes, put upside down mixing, room temperature leaves standstill 10min, and the centrifugal 10min of 10000g adds 70% washing with alcohol, and drying at room temperature 20min adds 200 μ L TE ,-20 ℃ of preservations.
(2) according to sorangium cellulosum 157-2 xylose isomerase gene ORF design of amplification primers:
Primer 1:5 '-ATCG
AGATCTATGACCGTCGTGATTGGA-3 '
Primer 2: 5 '-TTCG
GAGCTCTTACCGGATCCACCGATT-3 '.
(3) utilize the primer 1 that provides in the step (2), primer 2 and Taq DNA synthetic enzyme, total DNA is a template with the sorangium cellulosum (Sorangium cellulosum) that obtains in the step (1), containing the dATP of 0.2mmol/L, 0.2mmol/L dGTP, the dTTP of 1mmol/L, the dCTP of 1mmol/L, the MgCl of 7mmol/L
2, the MnCl of 0.3mmol/L
2, other are under the PCR reaction system of normal condition and carry out pcr amplification.
(4) the PCR product that reaction system in a plurality of independent step (3) is obtained reclaims purifying respectively, equivalent mixing then, with final concentration is the DNA of the about 5 μ g of DNAaesI processing total amount of 0.01U/ μ L, places 80 ℃ of following deactivation 10min immediately behind 15 ℃ of processing 4m30s.
(5) DNA small segment electrophoresis in 2% sepharose that step (4) is obtained reclaims small segment between the 100-200bp, is the dna fragmentation recombining reaction that template is carried out similar PCR sample with dissolved DNA small segment in not having the system of primer.
(6) the recombining reaction product that obtains with step (5) is a template, and with the primer 1 that step (2) provides, primer 2 is a primer, carries out pcr amplification.
(7) reclaim near the band of 1.4kb behind the PCR product electrophoresis that step (6) obtains.
(8) fragment that step (7) is obtained is cut with BglII, SacI enzyme, takes directed connection strategy and same enzyme to cut directed connection of plasmid vector pYPX251 (GenBank AY178046) of processing, makes up xylose isomerase gene sudden change library (Fig. 1).
(9) the xylose isomerase gene sudden change library transformed into escherichia coli HB101 (ATCC 33694) of step (7) acquisition, intestinal bacteria HB101 hereditary property is supE44, hsdS20 (r
B -m
B -), recA13, ara-14, proA2, lacY1, galK2, rpsL20, xyl-5, mtl-1, leuB6, thi-1.
(10) it is on the M9 flat board that is added with 50mg/L ammonia benzyl element of sole carbon source that the bacterium liquid after step (9) transforms is coated on the wood sugar, through 20～40 hours cultivation, occurs single bacterium colony on the flat board.Carry out primary dcreening operation according to the bacterium colony size, select the bigger next round of carrying out of bacterium colony to sieve again.The concrete prescription of used solid M9 substratum is: NH
4Cl 5g/L, Na
2HPO
430g/L, KH
2PO
415g/L, NaCl 2.5g/L, MgSO
4.7H
2O 0.25g/L, wood sugar 20g/L, agar 15g/L.
(11) from step (10) screening mutant, under 37 ℃ at LB substratum (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L, pH 7.0) 8000 leave heart 5min collected thalline to middle cultivation reorganization bacterium, used the 0.9%NaCl washed twice to logarithmic phase in 10～12 hours, be dissolved in lysis buffer (the 1mM PMSF of new preparation then, 50mmol/LTris-Cl pH7.0), adds an amount of granulated glass sphere (diameter 0.5mm), under cytoclasis instrument top speed, handle 30～50s, ice bath 5min.The broken repetition 3～5 times.The centrifugal 10min of 10000g collects supernatant, is crude enzyme liquid;
Adopt the XI-SDII method to measure enzyme activity, step is as follows:
With 700mmol/L D-wood sugar, the 0.01-0.025U xylose isomerase contains 10mmol/L MnCl
250mmol/LTris-HCl pH7.0 damping fluid in, respectively 45 ℃ the reaction 20min, add 150 μ L, 50% trichoroacetic acid(TCA) termination reaction and 185 μ L 2M Na
2CO
3The neutralization reaction damping fluid.Get 0.5mL and add 0.04U SDH (sigma) and 0.15mM NADH, mixing detects the oxidized amount (promptly doing time scan at 340nm) of NADH under 340nm immediately.Try to achieve the xylulose amount according to typical curve.One of xylose isomerase (XI) is defined as than unit of activity (U/mg): the μ mol/L number of every mg zymoprotein per minute conversion of substrate.
Carry out multiple sieve by enzyme activity determination and obtain a plant mutant body, extract its plasmid, xylA is partly checked order, the result of order-checking is the sequence shown in the SEQ NO.1 (mxylA), polypeptide shown in the coding SEQ NO.2, this polypeptide is the evolution enzyme of xylose isomerase, called after S1, the corresponding recombinant vectors that has the S1 encoding gene is pYPX251-S1, and the recombinant bacterial strain that contains recombinant vectors is HB101-S1.
Embodiment 2
The enzyme activity determination of evolution enzyme S1:
(1) the total DNA with sorangium cellulosum (Sorangium cellulosum) 157-2 is a template, utilize primer 1 and primer 2 amplification wild-type xylose isomerase gene xylA, cut with BglII, SacI enzyme, take directed connection strategy and same enzyme to cut directed connection of plasmid vector pYPX251 (GenBank AY178046) of processing, obtain recombinant plasmid pYPX251-SXI.
(2) the recombinant plasmid transformed intestinal bacteria HB101 in the step (1) obtains recombinant bacterial strain HR101-SXI.
(3) according to the enzyme activity determination mode of describing in the step (11) among the embodiment 1, under differing temps, measure different strains
The ratio vigor of the xylose isomerase that contains in the crude enzyme liquid in source sees Table 1.
Table 1 is the xylose isomerase specific activity of enzyme in the different strains under differing temps
(4), adjust the pH difference of measuring damping fluid according to the enzyme activity determination mode of describing in the step (11) among the embodiment 1
The optimal pH that equals to measure under 5,6,7,8,9 and 10 the condition evolution enzyme S1 at pH is 7.0 (accompanying drawings 2).
The activity expression of embodiment 3 sudden change xylose isomerase genes in yeast saccharomyces cerevisiae
(1) according to HXT7 upstream region of gene in the genes of brewing yeast group database-375bp--1bp sequences Design primer:
Primer 3:5 '-GATC
CCCGGGCGGGCCCCTGCGTG-3 '
Primer 4:5 '-TTGC
GGTACCTTTTTGATTAAAATTAAAAAAAC-3 '
With yeast saccharomyces cerevisiae karyomit(e) is template, pcr amplification HXT7 promotor (HXT7p) fragment, and with SmaI and KpnI double digestion PCR product.
(2) according to the design of the nucleotide sequence shown in SEQ NO.1 primer:
Primer 5:5 '-AGCT
GGTACCATGACCGTCGTGATTGGAAAC-3 '
Primer 6:5 '-TTGC
AGATCTTTACCGGATCCACCGATTGAC-3 '
With pYPX251-S1 is template, pcr amplification mxylA, and with KpnI and BglII double digestion PCR product.
(3) according to the design of the nucleotide sequence shown in SEQ NO.1 primer:
Primer 5:5 '-AGCT
GGTACCATGACCGTCGTGATTGGAAAC-3 '
Primer 6:5 '-TTGC
AGATCTTTACCGGATCCACCGATTGAC-3 '
With sorangium cellulosum karyomit(e) is template, pcr amplification xylA, and with KpnI and BglII double digestion PCR product.
(4) according to PGK1 gene downstream 1bp-279bp sequences Design primer in the genes of brewing yeast group database:
Primer 7:5 '-GCT
AGATCTTAACGAACGCAGAATTTTCG-3 '
Primer 8:5 '-TTG
AAGCTTTAAATTGAATTGAATTGAAAT-3 '
With yeast saccharomyces cerevisiae karyomit(e) is template, and pcr amplification PGK1 terminator (PGK1t) is with HindIII and BglII double digestion PCR product.
(5) with step (1), the product equivalent that obtains in (2), (4) connects mixture as template, with primer 3 and primer 8 amplifications, reclaims the fragment at 2000bp place, with HindIII and SmaI double digestion.
(6) plasmid YEp24 is with HindIII and SmaI double digestion, and and the product that obtains of step (5) is connected transformed into escherichia coli, called after pHX-S1 after the recombinant vectors after the amplification in intestinal bacteria is verified correctly.
(7) the recombinant vectors pHX-SXI transformed saccharomyces cerevisiae H158 of step (6) acquisition, recombinant bacterial strain called after H158-S1.
(8) with step (1), the product equivalent that obtains in (3), (4) connects mixture as template, with primer 3 and primer 8 amplifications, reclaims the fragment at 2000bp place, with HindIII and SmaI double digestion.
(9) plasmid YEp24 is with HindIII and SmaI double digestion, and and the product that obtains of step (8) is connected transformed into escherichia coli, called after pHX-SXI after the recombinant vectors after the amplification in intestinal bacteria is verified correctly.
(10) the recombinant vectors pHX-SXI transformed saccharomyces cerevisiae H158 of step (9) acquisition, recombinant bacterial strain called after H158-SXI.
(11) prepare the crude enzyme liquid of H158-SXI and H158-S1 according to the employed method of step (11) among the embodiment 1 and measure the work of XI enzyme, two kinds of XI have activity expression in yeast saccharomyces cerevisiae, XI enzyme work among the H158-SXI is 0.11, the enzyme work of XI among the H158-S1 is 0.039, and the activity of the evolution enzyme S1 that process is transformed is 2.8 times of wild-type SXI.
Sequence table
＜110〉Shandong University
＜120〉a kind of xylose isomerase and encoding gene thereof and application
<160>2
<170>PatentIn?Version?3.3
<210>1
<211>1329
<212>DNA
＜213〉sorangium cellulosum (Sorangium cellulosum)
<400>1
atgaccgtcg?tgattggaaa?caaagggtac?ttccccggtg?tcggcaagat?cctctacgag 60
gggcccgagt?ccgacaaccc?cgtcgccttc?aagtggtacg?acgagagccg?cgtcgtcgcc 120
ggcaagccca?tgaaggacca?cttcaagttc?gccgtgtgtt?actggcacac?cttctgtggt 180
cgtgggcacg?accccttcgg?tccggggacg?atcaacttac?cctgggacga?ggggaaggac 240
ccactgaccc?gcgctcgcat?gaaggcggac?gcgaacttcg?agttcatcac?caagctgggc 300
gcgacgcact?actgtttcca?cgacatcgac?ctggtggagg?agggcgacag?ccgcgccgag 360
acgtcgcgcc?gccttcaggg?catcgtcggg?tacgccaagc?agaagcaagc?gcagtcgggc 420
gtcaagctgc?tgtgggtcac?ggcgaacttg?ttctccaacc?cgcgctacat?gaacggcgcc 480
tccacgaacc?cggatttctc?cgaggtggcc?tacgccggcg?cccagctgaa?ggacgccctc 540
gacgccacca?tcgcgctgaa?cggtgagaac?tacgtgttct?ggggcggccg?cgaggattac 600
atgagcctgc?tgaacacgga?catgaagcgc?gagatggagc?acttcgcgcg?cttcttgacc 660
atggcgcgcg?actacgcgcg?cgcccggggc?ttcaagggga?ccttcttcat?cgagcccaag 720
ccgatggagc?cgtcgaagca?ccagtacgac?ttcggcgccg?agaccgtgat?cgggttcctg 780
cgccagcacg?gcctggacaa?ggacttcaag?ctcaacctgg?agacgaacca?cgcgacgctc 840
gctggccaca?ccatggagca?cgagatgcag?gtggccgcgg?acgccggcat?gctcggcagc 900
atcgacgcga?acctcggcga?ctaccagaat?gcgtgggaca?ccgatcagtt?cccctacaac 960
atcaacgaga?cggtggagat?gatgctcatc?ctcctccgca?gcggcaggtt?ccagggtggc 1020
ggcgtcaact?tcgacgcgaa?gcgccgccgc?aactcgacgg?acctgatcga?caccttccac 1080
ggccacatcg?gcggcatgga?cgtgttcgcc?cgcgcgctca?tcatcgccgg?cgacctgatc 1140
cgcaagtcgc?cgctcgagtc?gatgcgcaag?gctcattacg?cgtcgttcga?cggcggcaag 1200
ggcgcggagt?tcgagcaggg?gaagctcacg?ctggagcagc?tggccgagat?cggcgacgcc 1260
ggcggcgagg?tcaagctcac?gagcggtcag?caagagctgt?acgagaacat?cgtcaatcgg 1320
tggatccgg 1329
<210>2
<211>443
<212>PRT
＜213〉sorangium cellulosum (Sorangium cellulosum)
<400>2
MTVVIGNKGY?FPGVGKILYE?GPESDNPVAF?KWYDESRVVA?GKPMKDHFKF?AVCYWHTFCG 60
RGHDPFGPGT?INLPWDEGKD?PLTRARMKAD?ANFEFITKLG?ATHYCFHDID?LVEEGDSRAE 120
TSRRLQGIVG?YAKQKQAQSG?VKLLWVTANL?FSNPRYMNGA?STNPDFSEVA?YAGAQLKDAL 180
DATIALNGEN?YVFWGGREDY?MSLLNTDMKR?EMEHFARFLT?MARDYARARG?FKGTFFIEPK 240
PMEPSKHQYD?FGAETVIGFL?RQHGLDKDFK?LNLETNHATL?AGHTMEHEMQ?VAADAGMLGS 300
IDANLGDYQN?AWDTDQFPYN?INETVEMMLI?LLRSGRFQGG?GVNFDAKRRR?NSTDLIDTFH 360
GHIGGMDVFA?RALIIAGDLI?RKSPLESMRK?AHYASFDGGK?GAEFEQGKLT?LEQLAEIGDA 420
GGEVKLTSGQ?QELYENIVNR?WIR 443
<210>3
<211>28
<212>DNA
＜213〉synthetic
<400>3
ATCGAGATCT?ATGACCGTCG?TGATTGGA 28
<210>4
<211>28
<212>DNA
＜213〉synthetic
<400>4
TTCGGAGCTC?TTACCGGATC?CACCGATT 28
<210>5
<211>24
<212>DNA
＜213〉synthetic
<400>5
GATCCCCGGG?CGGGCCCCTG?CGTG 24
<210>6
<211>33
<212>DNA
＜213〉synthetic
<400>6
TTGCGGTACC?TTTTTGATTA?AAATTAAAAA?AAC 33
<210>7
<211>31
<212>DNA
＜213〉synthetic
<400>7
AGCTGGTACC?ATGACCGTCG?TGATTGGAAA?C 31
<210>8
<211>31
<212>DNA
＜213〉synthetic
<400>8
TTGCAGATCT?TTACCGGATC?CACCGATTGA?C 31
<210>9
<211>29
<212>DNA
＜213〉synthetic
<400>9
GCTAGATCTT?AACGAACGCA?GAATTTTCG 29
<210>10
<211>30
<212>DNA
＜213〉synthetic
<400>10
TTGAAGCTTT?AAATTGAATT?GAATTGAAAT 30
Claims (7)
1, a kind of dna sequence dna of the xylose isomerase of encoding is characterized in that, it is that 53 of xylose isomerase gene xylA in sorangium cellulosum (Sorangium cellulosum) 157-2 sport T by C; 219 sport A by C; 243 sport A by G; 265 sport G by A; 437 sport T by G; 503 sport A by T; 596 sport A by G; 635 sport T by A; 755 sport G by A; 914 sport T by G; 930 sport T by C; 1006 sport A by G; 1175 sport A by G; 1255 sport G by A.
2, dna sequence dna as claimed in claim 1 is characterized in that, gene order is as follows:
atgaccgtcg?tgattggaaa?caaagggtac?ttccccggtg?tcggcaagat?cctctacgag 60
gggcccgagt?ccgacaaccc?cgtcgccttc?aagtggtacg?acgagagccg?cgtcgtcgcc 120
ggcaagccca?tgaaggacca?cttcaagttc?gccgtgtgtt?actggcacac?cttctgtggt 180
cgtgggcacg?accccttcgg?tccggggacg?atcaacttac?cctgggacga?ggggaaggac 240
ccactgaccc?gcgctcgcat?gaaggcggac?gcgaacttcg?agttcatcac?caagctgggc 300
gcgacgcact?actgtttcca?cgacatcgac?ctggtggagg?agggcgacag?ccgcgccgag 360
acgtcgcgcc?gccttcaggg?catcgtcggg?tacgccaagc?agaagcaagc?gcagtcgggc 420
gtcaagctgc?tgtgggtcac?ggcgaacttg?ttctccaacc?cgcgctacat?gaacggcgcc 480
tccacgaacc?cggatttctc?cgaggtggcc?tacgccggcg?cccagctgaa?ggacgccctc 540
gacgccacca?tcgcgctgaa?cggtgagaac?tacgtgttct?ggggcggccg?cgaggattac 600
atgagcctgc?tgaacacgga?catgaagcgc?gagatggagc?acttcgcgcg?cttcttgacc 660
atggcgcgcg?actacgcgcg?cgcccggggc?ttcaagggga?ccttcttcat?cgagcccaag 720
ccgatggagc?cgtcgaagca?ccagtacgac?ttcggcgccg?agaccgtgat?cgggttcctg 780
cgccagcacg?gcctggacaa?ggacttcaag?ctcaacctgg?agacgaacca?cgcgacgctc 840
gctggccaca?ccatggagca?cgagatgcag?gtggccgcgg?acgccggcat?gctcggcagc 900
atcgacgcga?acctcggcga?ctaccagaat?gcgtgggaca?ccgatcagtt?cccctacaac 960
atcaacgaga?cggtggagat?gatgctcatc?ctcctccgca?gcggcaggtt?ccagggtggc 1020
ggcgtcaact?tcgacgcgaa?gcgccgccgc?aactcgacgg?acctgatcga?caccttccac 1080
ggccacatcg?gcggcatgga?cgtgttcgcc?cgcgcgctca?tcatcgccgg?cgacctgatc 1140
cgcaagtcgc?cgctcgagtc?gatgcgcaag?gctcattacg?cgtcgttcga?cggcggcaag 1200
ggcgcggagt?tcgagcaggg?gaagctcacg?ctggagcagc?tggccgagat?cggcgacgcc 1260
ggcggcgagg?tcaagctcac?gagcggtcag?caagagctgt?acgagaacat?cgtcaatcgg 1320
tggatccgg 1329。
3, a kind of xylose isomerase by the described dna sequence encoding of claim 1, it is the mutant enzyme of the xylose isomerase gene xylA encoded polypeptides (Genebank ACC99633) in sorangium cellulosum (Sorangiumcellulosum) 157-2, relatively preceding with sudden change, have the amino-acid residue in 12 sites that variation has taken place, it comprises: 18 sport L by P, 73 sport L by F, 89 sport A by T, 146 sport V by G, 168 sport E by V, 199 sport D by G, 212 sport M by K, 252 sport G by D, 305 sport L by R, 336 sport R by G, 392 sport H and 419 by R and sport D by N.
4, xylose isomerase as claimed in claim 3 is characterized in that, aminoacid sequence is as follows:
MTVVIGNKGY?FPGVGKILYE?GPESDNPVAF?KWYDESRVVA?GKPMKDHFKF?AVCYWHTFCG 60
RGHDPFGPGT?INLPWDEGKD?PLTRARMKAD?ANFEFITKLG?ATHYCFHDID?LVEEGDSRAE 120
TSRRLQGIVG?YAKQKQAQSG?VKLLWVTANL?FSNPRYMNGA?STNPDFSEVA?YAGAQLKDAL 180
DATIALNGEN?YVFWGGREDY?MSLLNTDMKR?EMEHFARFLT?MARDYARARG?FKGTFFIEPK 240
PMEPSKHQYD?FGAETVIGFL?RQHGLDKDFK?LNLETNHATL?AGHTMEHEMQ?VAADAGMLGS 300
IDANLGDYQN?AWDTDQFPYN?INETVEMMLI?LLRSGRFQGG?GVNFDAKRRR?NSTDLIDTFH 360
GHIGGMDVFA?RALIIAGDLI?RKSPLESMRK?AHYASFDGGK?GAEFEQGKLT?LEQLAEIGDA 420
GGEVKLTSGQ?QELYENIVNR?WIR?443。
5, a kind of genophore that contains the described dna fragmentation of claim 1.
6, a kind of reconstitution cell that contains the described genophore of claim 5.
7, claim 1,3,5 and 6 described dna sequence dnas, xylose isomerase, genophore and transformant are in the application of chemical industry, food, field of medicaments.
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Cited By (7)
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| CN102174549A (en) * | 2011-02-22 | 2011-09-07 | 山东大学 | Nucleic acid molecules for coding xylose isomerase and xylose isomerase coded by same |
| CN102443578A (en) * | 2011-12-08 | 2012-05-09 | 江南大学 | Glucose isomerase mutant and application thereof |
| CN101698839B (en) * | 2008-12-26 | 2012-05-23 | 北京大学 | Uridine diphosphate xylose isomerase, coding gene thereof and use thereof |
| CN102888420A (en) * | 2012-10-12 | 2013-01-23 | 广西大学 | Gene for coding xylose isomerase and application thereof |
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| CN111235139A (en) * | 2018-11-29 | 2020-06-05 | 国投生物科技投资有限公司 | Xylose isomerase, encoding gene and preparation method thereof, vector and host cell and application thereof |
| CN111235138A (en) * | 2018-11-29 | 2020-06-05 | 国投生物科技投资有限公司 | Xylose isomerase, encoding gene and preparation method thereof, vector and host cell and application thereof |
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2008
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| CN101698839B (en) * | 2008-12-26 | 2012-05-23 | 北京大学 | Uridine diphosphate xylose isomerase, coding gene thereof and use thereof |
| US8586336B2 (en) | 2011-02-22 | 2013-11-19 | Shandong University | Nucleic acid molecule encoding xylose isomerase and xylose isomerase encoded by the nucleic acid molecule |
| WO2012113120A1 (en) * | 2011-02-22 | 2012-08-30 | 山东大学 | Nucleic acid molecule encoding xylose isomerase and xylose isomerase encoded thereof |
| CN102174549B (en) * | 2011-02-22 | 2012-10-10 | 山东大学 | Nucleic acid molecules for coding xylose isomerase and xylose isomerase coded by same |
| CN102174549A (en) * | 2011-02-22 | 2011-09-07 | 山东大学 | Nucleic acid molecules for coding xylose isomerase and xylose isomerase coded by same |
| CN102443578A (en) * | 2011-12-08 | 2012-05-09 | 江南大学 | Glucose isomerase mutant and application thereof |
| CN102888420A (en) * | 2012-10-12 | 2013-01-23 | 广西大学 | Gene for coding xylose isomerase and application thereof |
| CN102888420B (en) * | 2012-10-12 | 2014-04-09 | 广西大学 | Gene for coding xylose isomerase and application thereof |
| US9951326B2 (en) | 2015-07-13 | 2018-04-24 | MARA Renewables Corporation | Enhancing microbial metabolism of C5 organic carbon |
| US10662418B2 (en) | 2015-07-13 | 2020-05-26 | MARA Renewables Corporation | Enhancing microbial metabolism of C5 organic carbon |
| CN111235139A (en) * | 2018-11-29 | 2020-06-05 | 国投生物科技投资有限公司 | Xylose isomerase, encoding gene and preparation method thereof, vector and host cell and application thereof |
| CN111235138A (en) * | 2018-11-29 | 2020-06-05 | 国投生物科技投资有限公司 | Xylose isomerase, encoding gene and preparation method thereof, vector and host cell and application thereof |
| CN111235139B (en) * | 2018-11-29 | 2021-08-20 | 国投生物科技投资有限公司 | Xylose isomerase, encoding gene and preparation method thereof, vector and host cell and application thereof |
| CN111235138B (en) * | 2018-11-29 | 2021-08-24 | 国投生物科技投资有限公司 | Xylose isomerase, encoding gene and preparation method thereof, vector and host cell and application thereof |
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