CN1884508A - Mutant xylose isomerase and its gene - Google Patents
Mutant xylose isomerase and its gene Download PDFInfo
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- CN1884508A CN1884508A CN200610085762.1A CN200610085762A CN1884508A CN 1884508 A CN1884508 A CN 1884508A CN 200610085762 A CN200610085762 A CN 200610085762A CN 1884508 A CN1884508 A CN 1884508A
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Abstract
The invention discloses a discontinuous xylo-pfan isomerase and its genes. The discontinuous xylo-pfan isomerase selects form Thermus thermophilus and its amino acid sequence is indicated by SEQ ID NO.2. The said discontinuous xylo-pfan isomerase has improved specificity to the substrate D-wood sugar without changing original heat endurance. The discontinuous xylo-pfan isomerase can be used in miscible liquids including wood sugar and amylaceum to specially detect the wood sugar content and can be used in constructing recombination bacteria with wood sugar.
Description
Technical field
The invention belongs to the genetically engineered and the enzyme biochemical engineering field of enzyme.Specifically, the present invention relates to a kind of discontinuous xylo-pfan isomerase, this discontinuous xylo-pfan isomerase can turn to the D-xylose isomerase D-xylulose, and can not generate D-fructose by catalysis D-glucose.The invention still further relates to the gene of this sudden change xylose isomerase of coding.
Background technology
Xylose isomerase (Xylose Isomerase, XI) (EC 5.3.1.5) claims glucose isomerase again, can catalysis D-wood sugar isomerization reaction to D-xylulose and D-glucose to D-fructose, playing an important role in the intravital carbohydrate metabolism process of microorganism, is the key enzyme of industrial production high fructose syrup.
According to sequence homology, xylose isomerase can be divided into two big classes, and the first kind is made up of 390 amino-acid residues approximately, and second class is made up of 440 amino-acid residues approximately.Xylose isomerase enzyme require Mg
2+, Mn
2+Or Co
2+As cofactor; Its active centre mainly comprises with the residue and the substrate of two metallic ion coordination and combines residue.
Utilization for wood sugar in the nature microorganism body mainly contains two approach, and one is that two steps effect by Xylose reductase and xylitol dehydrogenase is converted into xylulose with wood sugar; An other approach is directly wood sugar to be converted into xylulose by xylose isomerase; In recent years, along with increasingly sharpening of environmental pollution and energy dilemma, structure can become the research focus by xylose-fermenting production alcoholic acid recombinant Saccharomyces cerevisiae.The xylose isomerase enzymatic pathway becomes the first-selected convenient approach that makes up the wood-sugar fermentation bacterial classification because of not needing coenzyme.Recently, domestic production at xylose-fermenting also obtained gratifying progress aspect the ethanol recombination yeast structure, by in the yeast saccharomyces cerevisiae industrial strain, expressing Thermus thermophilus xylose isomerase, xylose metabolism approach (WANG Y., SHI W.L., LIU X.Y. have been set up, et al.Biotechnology Letters, 2004,26 (11): 885-890), but make that the reorganization bacterium is unsatisfactory to utilizing of wood sugar because the xylose isomerase enzymic activity is low.
United States Patent (USP) (US6475768) report passes through the xylose isomerase F163 to Thermus thermophilus, E372, and the locational amino acid of V379 suddenlys change, and has improved this enzyme activity at low temperatures.Though the lot of documents report is arranged, improved in the bacterial classifications such as deriving from Thermoanaerobacteriumthermosulfurigenes, Thermotoga neapolitana, Thermotoga neapolitana xylose isomerase to the catalytic activity of substrate D-glucose by orthogenesis or rite-directed mutagenesis method, but less for improving xylose isomerase to the specific research of substrate D-wood sugar.
Summary of the invention
The purpose of this invention is to provide a kind of discontinuous xylo-pfan isomerase, this discontinuous xylo-pfan isomerase is from Thermusthermophilus, on the basis that does not change the protoenzyme thermostability, improve specificity to substrate D-wood sugar, can not catalysis D-glucose generate the reaction of D-fructose.
Another object of the present invention provides the gene (nucleotide sequence) of coding said mutation xylose isomerase, and this gene can be used for making up the reorganization bacterium that utilizes wood sugar.
Purpose of the present invention can reach by following measures:
A kind of discontinuous xylo-pfan isomerase, its aminoacid sequence are shown in the SEQ ID NO.2.This sequence represents that 91 residue of log sugar isomerase aminoacid sequence (being the expressed aminoacid sequence of wild-type Thermus thermophilus xylA gene) is replaced by asparagicacid residue.
The nucleotide sequence of the described xylose isomerase of coding claim 1 (promptly undergoing mutation to expressing the codon of aspartic acid corresponding to the codon of 91 of wild-type xylose isomerase enzyme amino acid sequences) is as described in SEQ ID NO.1.
The expression vector that contains above-mentioned nucleotide sequence.As plasmid, phage etc.
Bacterium or yeast with above-mentioned expression vector conversion.What described bacterium was commonly used is intestinal bacteria.
The mixed solution (as mixed solutions such as wood sugar, glucose) that sudden change xylose isomerase provided by the present invention can be used for containing wood sugar detects the content of wood sugar single-mindedly.
Further will explain the present invention in detail:
(1) sudden change XI and sudden change xylA gene
Known 91 asparagine residue is positioned near the xylose isomerase enzyme active center.Replace asparagine residue corresponding to 91 with aspartic acid, what cause having high thermal stability is the expression of the mutain of suitable substrate with the D-wood sugar.
Can be by routine techniques with the codon mutation of coding Asn91 in the Thermus thermophilus HB8 xylose isomerase gene codon for coding Asp, and obtain the xylA gene of sudden change.
" corresponding to 91 amino-acid residue " expression is corresponding to 91 amino acids residues in the aminoacid sequence of SEQ ID NO.2 in the present invention.The position of amino-acid residue can change.For example, if insert amino-acid residue at the N end, the amino-acid residue that was positioned at 91 originally becomes 92.In this case, be designated as amino-acid residue of the present invention 91 corresponding to 91 original amino-acid residue.
Beneficial effect of the present invention:
The invention provides a kind of xylose isomerase and gene order of mutant, this discontinuous xylo-pfan isomerase has improved its specificity to substrate D-wood sugar on the basis that does not change the protoenzyme thermostability, makes it D-glucose isomerase can not be turned to D-fructose.Gene order provided by the present invention can be used for making up the reorganization bacterium that utilizes wood sugar.Sudden change xylose isomerase provided by the present invention also can be used for the content that mixed solutions such as wood sugar, glucose detect wood sugar single-mindedly.
Description of drawings
Fig. 1 is the recombinant expression design of graphics.
Fig. 2 is that the SDS-PAGE of expression product analyzes.
Embodiment
The invention will be further elaborated by the following examples, but do not limit the present invention.
The clone of embodiment 1:Thermus thermophilus xylA gene and Asn91 site mutation Thermus thermophilus HB8 are available from the ATCC27634 of American Type Culture Collecti (ATCC).
Medium preparation: yeast powder 4g/L, peptone 8g/L, NaCl 2.0g/L transfers to 7.0 with pH, 121 ℃ of autoclavings, 15min.
Thermus thermophilus HB8 is inoculated in the above-mentioned substratum, puts 75 ℃ of shaking tables, 200rpm cultivated 12-16 hour.8000rpm, 4 ℃, centrifugal 15min collects thalline.
The utilization genome extracts test kit (Shanghai China Shun biotechnology company limited), extracts Thermus Thermophilus genome.With this genome is template, uses following PCR primer (Bo Ya company in Shanghai is synthetic):
Forward primer 5 '-GGAATTCCATATGTACGAGCCCAAACCGGAGCACAGG-3 ' (SEQ IDNO.3)
Reverse primer 5 '-AAGGAAAAAAGCGGCCGCTCACCCCCGCACCCCCAG-3 ' (SEQ IDNO.4)
Pcr amplification xylose isomerase gene xylA.The PCR loop parameter is: 95 ℃, and 2min; 94 ℃, 30s; 70 ℃, 50s; 68 ℃, 1.5min carries out 35 circulations altogether.
Reclaim test kit (Takara company) with DNA the gene that amplifies is reclaimed purifying.The gene and the T-Vector of purifying are carried out conventional ligation, carry out the amplification of plasmid connecting liquid conversion DH5 α bacterial strain.Utilization plasmid extraction kit (Shanghai Bo Ya company) extracts plasmid.Use above-mentioned PCR primer to increase and identify and determine to insert the xylA gene, obtain positive reorganization T-vector-xylA plasmid.
Requirement according to multipoint mutation test kit (QuikChange Multi Site-Directed Mutagenesis Kit is available from STRATAGENE company) designs following primer, 5 '-ATGGTCACCGCC
GACCTCTTCTCCGAC-3 ' (SEQ ID NO.7) is that template is carried out PCR with the T-vector-xylA plasmid, and Asn91 is sported Asp91.Digest original plasmid template with restriction enzyme Dpn I, the PCR product is transformed high efficiency competent cell, cultivate back extraction plasmid and check order, can obtain containing the plasmid of mutator gene (SEQ ID NO.1).
Embodiment 2: the structure of recombinant expression, screening and evaluation
With forward primer 5 '-CCATATGTACGAACCAAAACCGGAACATCGCTTTACCTTT-3 ' (SEQ ID NO.5)
Reverse primer 5 '-AAGGAAAAAAGCGGCCGCTCAACCACGCACACCCAGGAG-3 ' (SEQ ID NO.6)
From embodiment 1, contain pcr amplification mutator gene xylA on the plasmid of mutator gene, with restriction enzyme NdeI and NotI double digestion mutator gene xylA and carrier pET-22b (+), mutator gene xylA that after the recovery above-mentioned double digestion is crossed and pET-22b (+) carrier connect, use conventional genetic engineering technique will connect liquid and be transformed into the bacillus coli DH 5 alpha competent cell, extract plasmid.Use above-mentioned primer to increase and identify that mutator gene xylA determines to be connected on the expression vector pET-22b (+).
The expression of embodiment 3:Thermus thermophilus xylose isomerase in intestinal bacteria Rosetta
PET-22b (+)-xylA recombinant plasmid transformed that will contain mutator gene xylA is in the business-like expressive host intestinal bacteria Rosetta of process (DE3), picking list bacterium colony is to the LB nutrient solution that contains 75 μ g/mL penbritins and 34 μ g/ml paraxin, 37 ℃ of shaking culture are spent the night, be inoculated in the LB nutrient solution that contains 75 μ g/mL penbritins and 34 μ g/mL paraxin by 2% (v/v) inoculum size then, 37 ℃ are cultured to OD
600Be about at 0.6 o'clock, add IPTG to final concentration 0.9mM, abduction delivering 7h gets certain volume bacterium liquid 8000rpm, and 4 ℃, centrifugal 15min collects thalline.
Embodiment 4: the xylose isomerase activity determination method
1. crude enzyme liquid preparation
Get 10mL and induce back nutrient solution 8000rpm, 4 ℃, centrifugal 15min collects thalline, after the sterilized water washed twice, thalline is resuspended in the 0.5mL pH7.5 50mM Tris-HCl damping fluid, ultrasonication cell in the ice bath, 12,000rpm, 4 ℃ of centrifugal 10min get supernatant and are crude enzyme liquid.Crude enzyme liquid is placed 80 ℃ of water-baths, heating 40min, 12,000rpm, 4 ℃ of centrifugal 10min can remove most albumen.After getting supernatant, carry out purifying by conventional method for purifying proteins again.(purified product meets SEQ ID NO.2 through identifying its sequence).
2. enzyme activity determination
The xylose isomerase enzyme activity determination adopts two-step approach, the first step: get the sudden change xylose isomerase 50 μ L of purifying, 0.1M D-wood sugar 50 μ L, 10mM MgCl
250 μ L, 85 ℃, reaction 15min, ice bath termination reaction; Second step: get the above-mentioned reaction solution of 10 μ L, add 1uL (0.08U/ μ L) sorbito dehy drogenase, 3 μ L 25mM NADH, 186 μ L pH7.550mM Tris-HCl damping fluids add the little culture plate in 96 holes.Use microplate reader to detect OD down at 25 ℃
340Photoabsorption change.Enzyme activity unit (U) is defined as per minute catalysis and produces the required enzyme amount of 1 μ mol xylulose.By Δ A
340The variation of/min is according to typical curve equation y=2805.81357X (Y:m Δ A wherein
340/ min; X: the concentration of xylulose, the mol/L of unit) determines the vigor of xylose isomerase.The result shows that xylose isomerase is about 160U/mg than living under above-mentioned reaction conditions.
＜110〉Nanjing University of Technology
＜120〉a kind of discontinuous xylo-pfan isomerase and gene thereof
<160>7
<210>1
<211>1164
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<213>artificial
<220>
＜223〉discontinuous xylo-pfan isomerase nucleotide sequence
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gtg?tac?gag?ccc?aaa?ccg?gag?cac?agg?ttt?acc?ttt?ggc?ctt?tgg?45
Met?Tyr?Glu?Pro?Lys?Pro?Glu?His?Arg?Phe?Thr?Phe?Gly?Leu?Trp
1 5 10 15
act?gtg?ggc?aat?gtg?ggc?cgt?gat?ccc?ttc?ggg?gac?gcg?gtt?cgg?90
Thr?Val?Gly?Asn?Val?Gly?Arg?Asp?Pro?Phe?Gly?Asp?Ala?Val?Arg
20 25 30
gag?agg?ctg?gac?ccg?gtt?tac?gtg?gtt?cat?aag?ctg?gcg?gag?ctt?135
Glu?Arg?Leu?Asp?Pro?Val?Tyr?Val?Val?His?Lys?Leu?Ala?Glu?Leu
35 40 45
ggg?gcc?tac?ggg?gta?aac?ctt?cac?gac?gag?gac?ctg?atc?ccg?cgg?180
Gly?Ala?Tyr?Gly?Val?Asn?Leu?His?Asp?Glu?Asp?Leu?Ile?Pro?Arg
50 55 60
ggc?acg?cct?cct?cag?gag?cgg?gac?cag?atc?gtg?agg?cgc?ttc?aag?225
Gly?Thr?Pro?Pro?Gln?Glu?Arg?Asp?Gln?Ile?Val?Arg?Arg?Phe?Lys
65 70 75
aag?gct?ctc?gat?gaa?acc?ggc?ctc?aag?gtc?ccc?atg?gtc?acc?gcc?270
Lys?Ala?Leu?Asp?Glu?Thr?Gly?Leu?Lys?Val?Pro?Met?Val?Thr?Ala
80 85 90
gac?ctc?ttc?tcc?gac?cct?gct?ttc?aag?gac?ggg?gcc?ttc?acg?agc?315
Asp?Leu?Phe?Ser?Asp?Pro?Ala?Phe?Lys?Asp?Gly?Ala?Phe?Thr?Ser
95 100 105
ccg?gac?cct?tgg?gtt?cgg?gcc?tat?gcc?ttg?cgg?aag?agc?ctg?gag?360
Pro?Asp?Pro?Trp?Val?Arg?Ala?Tyr?Ala?Leu?Arg?Lys?Ser?Leu?Glu
110 115 120
acc?atg?gac?ctg?ggg?gca?gag?ctt?ggg?gcc?gag?atc?tac?gtg?gtc?405
Thr?Met?Asp?Leu?Gly?Ala?Glu?Leu?Gly?Ala?Glu?Ile?Tyr?Val?Val
125 130 135
tgg?ccg?ggc?cgg?gag?gga?gct?gag?gtg?gag?gcc?acg?ggc?aag?gcc?450
Trp?Pro?Gly?Arg?Glu?Gly?Ala?Glu?Val?Glu?Ala?Thr?Gly?Lys?Ala
140 145 150
cgg?aag?gtc?tgg?gac?tgg?gtg?cgg?gag?gcg?ctg?aac?ttc?atg?gcc?495
Arg?Lys?Val?Trp?Asp?Trp?Val?Arg?Glu?Ala?Leu?Asn?Phe?Met?Ala
155 160 165
gcc?tac?gcc?gag?gac?cag?gga?tac?ggg?tac?cgg?ttt?gcc?ctc?gag?540
Ala?Tyr?Ala?Glu?Asp?Gln?Gly?Tyr?Gly?Tyr?Arg?Phe?Ala?Leu?Glu
170 175 180
ccc?aag?cct?aac?gag?ccc?cgg?ggg?gac?att?tac?ttc?gcc?acc?gtg?585
Pro?Lys?Pro?Asn?Glu?Pro?Arg?Gly?Asp?Ile?Tyr?Phe?Ala?Thr?Val
185 190 195
ggg?agc?atg?ctc?gcc?ttt?att?cat?acc?ctg?gac?cgg?ccc?gag?cgc?630
Gly?Ser?Met?Leu?Ala?Phe?Ile?His?Thr?Leu?Asp?Arg?Pro?Glu?Arg
200 205 210
ttc?ggc?ctg?aac?ccc?gag?ttc?gcc?cac?gag?acc?atg?gcc?ggg?ctt?675
Phe?Gly?Leu?Asn?Pro?Glu?Phe?Ala?His?Glu?Thr?Met?Ala?Gly?Leu
215 220 225
aac?ttt?gtc?cac?gcc?gtg?gcc?cag?gct?ctc?gac?gcc?ggg?aag?ctt?720
Asn?Phe?Val?His?Ala?Val?Ala?Gln?Ala?Leu?Asp?Ala?Gly?Lys?Leu
230 235 240
ttc?cac?att?gac?ctc?aac?gac?caa?cgg?atg?agc?cgg?ttt?gac?cag?765
Phe?His?Ile?Asp?Leu?Asn?Asp?Gln?Arg?Met?Ser?Arg?Phe?Asp?Gln
245 250 255
gac?ctc?cgc?ttc?ggc?tcg?gag?aac?ctc?aag?gcg?gcc?ttt?ttc?ctg?810
Asp?Leu?Arg?Phe?Gly?Ser?Glu?Asn?Leu?Lys?Ala?Ala?Phe?Phe?Leu
260 265 270
gtg?gac?ctc?ctg?gaa?agc?tcc?ggc?tac?cag?ggc?ccc?cgc?cac?ttt?855
Val?Asp?Leu?Leu?Glu?Ser?Ser?Gly?Tyr?Gln?Gly?Pro?Arg?His?Phe
275 280 285
gac?gcc?cac?gcc?ctg?cgt?acc?gag?gac?gaa?gaa?ggg?gtt?tgg?gcc?900
Asp?Ala?His?Ala?Leu?Arg?Thr?Glu?Asp?Glu?Glu?Gly?Val?Trp?Ala
290 295 300
ttc?gcc?cga?ggc?tgc?atg?cgt?acc?tac?ctg?atc?tta?aag?gaa?agg?945
Phe?Ala?Arg?Gly?Cys?Met?Arg?Thr?Tyr?Leu?Ile?Leu?Lys?Glu?Arg
305 310 315
gct?gaa?gcc?ttc?cgc?gag?gat?ccc?gag?gtc?aag?gag?ctt?ctt?gcc?990
Ala?Glu?Ala?Phe?Arg?Glu?Asp?Pro?Glu?Val?Lys?Glu?Leu?Leu?Ala
320 325 330
gct?tac?tat?caa?gaa?gat?cct?gcg?gcc?ttg?gcc?ctt?ttg?ggc?ccc?1035
Ala?Tyr?Tyr?Gln?Glu?Asp?Pro?Ala?Ala?Leu?Ala?Leu?Leu?Gly?Pro
335 340 345
tac?tcc?cgc?gag?aag?gcc?gaa?gcc?ctc?aag?cgg?gcg?gag?ctt?ccc?1080
Tyr?Ser?Arg?Glu?Lys?Ala?Glu?Ala?Leu?Lys?Arg?Ala?Glu?Leu?Pro
350 355 360
ctc?gag?gcc?aag?cgg?cgc?cgg?ggt?tat?gcc?ctg?gaa?cgc?ctg?gac?1125
Leu?Glu?Ala?Lys?Arg?Arg?Arg?Gly?Tyr?Ala?Leu?Glu?Arg?Leu?Asp
365 370 375
cag?ctg?gcg?gtg?gag?tac?ctc?ctg?ggg?gtg?cgg?ggg?tga 1164
Gln?Leu?Ala?Val?Glu?Tyr?Leu?Leu?Gly?Val?Arg?Gly
380 385
<210>2
<211>387
<212>PRT
<213>artificial
<220>
＜223〉discontinuous xylo-pfan isomerase aminoacid sequence
<400>
Met?Tyr?Glu?Pro?Lys?Pro?Glu?His?Arg?Phe?Thr?Phe?Gly?Leu?Trp
1 5 10 15
Thr?Val?Gly?Asn?Val?Gly?Arg?Asp?Pro?Phe?Gly?Asp?Ala?Val?Arg
20 25 30
Glu?Arg?Leu?Asp?Pro?Val?Tyr?Val?Val?His?Lys?Leu?Ala?Glu?Leu
35 40 45
Gly?Ala?Tyr?Gly?Val?Asn?Leu?His?Asp?Glu?Asp?Leu?Ile?Pro?Arg
50 55 60
Gly?Thr?Pro?Pro?Gln?Glu?Arg?Asp?Gln?Ile?Val?Arg?Arg?Phe?Lys
65 70 75
Lys?Ala?Leu?Asp?Glu?Thr?Gly?Leu?Lys?Val?Pro?Met?Val?Thr?Ala
80 85 90
Asp?Leu?Phe?Ser?Asp?Pro?Ala?Phe?Lys?Asp?Gly?Ala?Phe?Thr?Ser
95 100 105
Pro?Asp?Pro?Trp?Val?Arg?Ala?Tyr?Ala?Leu?Arg?Lys?Ser?Leu?Glu
110 115 120
Thr?Met?Asp?Leu?Gly?Ala?Glu?Leu?Gly?Ala?Glu?Ile?Tyr?Val?Val
125 130 135
Trp?Pro?Gly?Arg?Glu?Gly?Ala?Glu?Val?Glu?Ala?Thr?Gly?Lys?Ala
140 145 150
Arg?Lys?Val?Trp?Asp?Trp?Val?Arg?Glu?Ala?Leu?Asn?Phe?Met?Ala
155 160 165
Ala?Tyr?Ala?Glu?Asp?Gln?Gly?Tyr?Gly?Tyr?Arg?Phe?Ala?Leu?Glu
170 175 180
Pro?Lys?Pro?Asn?Glu?Pro?Arg?Gly?Asp?Ile?Tyr?Phe?Ala?Thr?Val
185 190 195
Gly?Ser?Met?Leu?Ala?Phe?Ile?His?Thr?Leu?Asp?Arg?Pro?Glu?Arg
200 205 210
Phe?Gly?Leu?Asn?Pro?Glu?Phe?Ala?His?Glu?Thr?Met?Ala?Gly?Leu
215 220 225
Asn?Phe?Val?His?Ala?Val?Ala?Gln?Ala?Leu?Asp?Ala?Gly?Lys?Leu
230 235 240
Phe?His?Ile?Asp?Leu?Asn?Asp?Gln?Arg?Met?Ser?Arg?Phe?Asp?Gln
245 250 255
Asp?Leu?Arg?Phe?Gly?Ser?Glu?Asn?Leu?Lys?Ala?Ala?Phe?Phe?Leu
260 265 270
Val?Asp?Leu?Leu?Glu?Ser?Ser?Gly?Tyr?Gln?Gly?Pro?Arg?His?Phe
275 280 285
Asp?Ala?His?Ala?Leu?Arg?Thr?Glu?Asp?Glu?Glu?Gly?Val?Trp?Ala
290 295 300
Phe?Ala?Arg?Gly?Cys?Met?Arg?Thr?Tyr?Leu?Ile?Leu?Lys?Glu?Arg
305 310 315
Ala?Glu?Ala?Phe?Arg?Glu?Asp?Pro?Glu?Val?Lys?Glu?Leu?Leu?Ala
320 325 330
Ala?Tyr?Tyr?Gln?Glu?Asp?Pro?Ala?Ala?Leu?Ala?Leu?Leu?Gly?Pro
335 340 345
Tyr?Ser?Arg?Glu?Lys?Ala?Glu?Ala?Leu?Lys?Arg?Ala?Glu?Leu?Pro
350 355 360
Leu?Glu?Ala?Lys?Arg?Arg?Arg?Gly?Tyr?Ala?Leu?Glu?Arg?Leu?Asp
365 370 375
Gln?Leu?Ala?Val?Glu?Tyr?Leu?Leu?Gly?Val?Arg?Gly
380 385
<210>3
<211>37
<212>DNA
<213>artificial
<220>
＜223〉upstream primer
<400>
ggaattccat?atgtacgagc?ccaaaccgga?gcacagg?37
<210>4
<211>36
<212>DNA
<213>artificial
<220>
＜223〉downstream primer
<400>
aaggaaaaaa?gcggccgctc?acccccgcac?ccccag?36
<210>5
<211>40
<212>DNA
<213>artificial
<220>
＜223〉upstream primer
<400>
ccatatgtac?gaaccaaaac?cggaacatcg?ctttaccttt?40
<210>6
<211>39
<212>DNA
<213>artificial
<220>
＜223〉downstream primer
<400>
aaggaaaaaa?gcggccgctc?aaccacgcac?acccaggag?39
<210>7
<211>27
<212>DNA
<213>artificial
<220>
＜223〉artificial primer
<400>
atggtcaccg?ccgacctctt?ctccgac?27
Claims (6)
1, a kind of discontinuous xylo-pfan isomerase, its aminoacid sequence are shown in the SEQ ID NO.2.
2, the nucleotide sequence of the described xylose isomerase of coding claim 1.
3, nucleotide sequence according to claim 2 is as described in SEQ ID NO.1.
4, the expression vector that contains the described nucleotide sequence of claim 2.
5, bacterium or the yeast that transforms with the described expression vector of claim 4.
6, the described xylose isomerase of claim 1 contains the application of wood sugar content in the mixed solution of wood sugar in detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610085762.1A CN1884508A (en) | 2006-06-29 | 2006-06-29 | Mutant xylose isomerase and its gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610085762.1A CN1884508A (en) | 2006-06-29 | 2006-06-29 | Mutant xylose isomerase and its gene |
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| Publication Number | Publication Date |
|---|---|
| CN1884508A true CN1884508A (en) | 2006-12-27 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200610085762.1A Pending CN1884508A (en) | 2006-06-29 | 2006-06-29 | Mutant xylose isomerase and its gene |
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| CN (1) | CN1884508A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445376C (en) * | 2006-11-27 | 2008-12-24 | 南京工业大学 | Mutant xylose isomerase and its gene and application |
| CN102399804A (en) * | 2010-09-15 | 2012-04-04 | 中国农业科学院作物科学研究所 | Function and application of D-xylose isomerase gene |
| CN109468305A (en) * | 2017-12-29 | 2019-03-15 | 吉林中粮生化有限公司 | Xylose isomerase enzyme mutant, the DNA molecular for encoding the enzyme, the recombinant bacterial strain for importing the DNA molecular and their application |
| CN110055184A (en) * | 2018-12-28 | 2019-07-26 | 吉林中粮生化有限公司 | Saccharomyces cerevisiae, comprising its microorganism formulation and using its produce ethyl alcohol method |
-
2006
- 2006-06-29 CN CN200610085762.1A patent/CN1884508A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445376C (en) * | 2006-11-27 | 2008-12-24 | 南京工业大学 | Mutant xylose isomerase and its gene and application |
| CN102399804A (en) * | 2010-09-15 | 2012-04-04 | 中国农业科学院作物科学研究所 | Function and application of D-xylose isomerase gene |
| CN109468305A (en) * | 2017-12-29 | 2019-03-15 | 吉林中粮生化有限公司 | Xylose isomerase enzyme mutant, the DNA molecular for encoding the enzyme, the recombinant bacterial strain for importing the DNA molecular and their application |
| CN109468305B (en) * | 2017-12-29 | 2021-11-16 | 吉林中粮生化有限公司 | Xylose isomerase mutant, DNA molecule encoding the enzyme, recombinant strain introduced with the DNA molecule, and uses thereof |
| CN110055184A (en) * | 2018-12-28 | 2019-07-26 | 吉林中粮生化有限公司 | Saccharomyces cerevisiae, comprising its microorganism formulation and using its produce ethyl alcohol method |
| CN110055184B (en) * | 2018-12-28 | 2022-06-28 | 吉林中粮生化有限公司 | Saccharomyces cerevisiae, microbial preparation comprising same, and method for producing ethanol using same |
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Open date: 20061227 |