CN1693476A - New determination method of semen fructose and glucose isomerase mutant used for said method - Google Patents
New determination method of semen fructose and glucose isomerase mutant used for said method Download PDFInfo
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Abstract
A process for measuring the content of pectose in sperm by use of high-activity glucose isomerase mutant, the amino acid sequences of the high-activity glucose isomerase mutant series, the nucleotide sequence for coding said enzyme, and the gene and protein engineering and the process for preparing said glucose isomerase mutants are disclosed.
Description
Technical field the present invention relates to biology field and field of genetic engineering, specifically, relates to a kind of new seminal fluid fructose measuring method; The engineering of a series of glucose isomerase mutants and these enzymes and preparation method.
(Glucose isomerase is called for short GI to the technical background glucose isomerase; E.C.5.3.1.5), claim that again (Xylose isomerase is called for short XI to xylose isomerase; E.C.5.3.1.5) the catalysis conversion of glucose is a fructose, and therefore, glucose isomerase can be used for producing high fructose syrup industrial; But glucose isomerase also catalysis wood sugar is converted into xylulose, is xylan is converted into key enzyme in the ethanol approach.Therefore, glucose isomerase can be used for producing alcohol again industrial.But glucose isomerase also catalysis fructose is converted into glucose.Utilize this characteristic, the present invention is used in glucose isomerase medically first, is used for measuring the content of fructose in the seminal fluid.But existing glucose isomerase is owing to active low, the good inadequately content that is difficult to be used for measuring fructose in the seminal fluid of stability.The present invention has produced the glucose isomerase mutant of a series of high reactivities and good stability through gene and protein engineering technology, and these mutant can be effective to measure the content of fructose in the seminal fluid.
Fructose in the seminal fluid is secreted by seminal vesicle, and it is the main glucide that utilizes for sperm.In normal human's seminal fluid the content of fructose be the 9.11-17.67 mmole/liter.Measure fructose content in the refining, its purpose is: 1. weigh the function of seminal vesicle, during the seminal vesicle inflammation, fructose content descends; As fructose is zero, should consider that seminal vesicle lacks as; 2. differentiate the cause of disease of azoospermia, be mainly used in the azoospermia of differentiating that simple property obstruction of vas deferen and vas deferens, seminal vesicle lack as cause at present; 3. the function that reflects interstitial glands secretion testosterone indirectly; 4. fructose is formed in the seminal vesicle transformation by glucose in the blood in the refining, apparently higher than normally, should notice then that getting rid of the patient has non-diabetic as fructose content in the refining.The method of measuring seminal fluid fructose at present mainly contains chromatography, chemical method and Fructose dehydrogenase method.Chromatography is subjected to the restriction of instrument, and the rarer high performance liquid chromatograph of clinical labororatory also lacks relevant technologies.Chemical method is to utilize compound reactions such as fructose and Resorcinol, indoles or carbazole to produce colour-change, measures fructose content by colorimetric then.But the chemical colour reaction reaction must be carried out in the strongly-acid medium, and is dangerous; In addition, because the colour developing unstable products, so color reaction repeatability is very poor.Though it is gentle that the dehydrogenation enzyme process is a reaction conditions, measurement result is more accurate,, the developer instability of using in Fructose dehydrogenase itself and the Fructose dehydrogenase reaction, both all need facing with preceding fresh preparation, are very much time-consuming therefore; And Fructose dehydrogenase reagent costs an arm and a leg, so its commercial exploitation is restricted.So, still do not have a kind of more satisfactory and practical seminal fluid fructose measuring method so far.
(ATCC 49915 from Thermoanaerobacterium sachcharolyticum B6A bacterium in the present invention, USA) be separated to glucose isomerase (Lee Y.E.etal. in, Journal of General Microbiology 1993,139:1227-1234), its nucleotide sequence such as sequence table SEQ .ID NO.1, aminoacid sequence is sequence table SEQ .IDNO.2.Then, the present invention by gene and protein engineering technique improvement the glucose isomerase that is separated to of the present invention, the activity that to make its catalyzed conversion fructose be glucose has had and has significantly improved, and the stability of this glucose isomerase mutant and the expression level in intestinal bacteria have had and significantly improve.The present invention's glucose isomerase mutant in measuring seminal fluid aspect the fructose content more aforesaid chromatography, chemical method and fructose Off hydrogen enzyme have following characteristics: (1) is easy and simple to handle; (2) result is accurate; (3) mild condition; (4) with low cost.
Glucose isomerase mutant of the present invention can also be used in the human body or a certain organ or other organism seminal fluid DETERMINATION OF FRUCTOSE IN in the body.
Summary of the invention the object of the present invention is to provide a kind of seminal fluid fructose measuring method.Order of the present invention also is to provide a kind of high catalytic activity and heat-stable glucose isomerase mutant.The present invention also aims to provide the glucose isomerase mutant that uses high catalytic activity of the present invention effectively to measure fructose content in the seminal fluid.The present invention also aims to provide and use glucose isomerase mutant of the present invention to measure other organ or other organism seminal fluid fructose content in the human body.
The present invention from Thermoanaerobacterium sachcharolyticum B6A bacterium, be separated to glucose isomerase (Lee Y.E.etal., Journal of General Microbiology 1993,139:1227-1234).Then, the present invention is according to the secondary structure of the glucose isomerase that is separated to and the characteristic of simulation tertiary structure, by gene and protein engineering technique improvement this glucose isomerase, the activity that to make its catalyzed conversion fructose be glucose has had and has significantly improved, and the stability of this glucose isomerase mutant and the expression level in intestinal bacteria have had and significantly improve.
One aspect of the present invention provides a kind of seminal fluid fructose measuring method, and this method may further comprise the steps:
1) human body (or other organism, as ox, sheep, horse, dog) seminal fluid sample was handled 30 minutes in 80 ℃, centrifugal under the room temperature, supernatant liquor is changed in the clean tube again;
2) (BIO-RAD USA), removes in the seminal fluid the noisy material of glucose isomerase provided by the invention, and is centrifugal under the room temperature, collects supernatant liquor to add Chelex 100 resins;
3) get supernatant liquor, add CoCl
2And MgCl
2, add a kind of glucose isomerase mutant provided by the invention again, in 80 ℃ of reactions 10 minutes;
4) reaction product D-glucose is by the D-determination of glucose oxidase;
5), ask and calculate fructose content in the seminal fluid according to the fructose typical curve.
The present invention provides the nucleotide sequence of a series of high temperature resistant glucose isomerase mutants of coding on the other hand, described nucleotide sequence has the nucleotide sequence of sequence table SEQ .ID NO.3 or SEQ.ID NO.5 or SEQ.ID NO.7 or SEQ.ID NO.9 or the mutant form (〉=75% homology) of described nucleotide sequence, described sudden change comprises: disappearance, nonsense, insertion, missense, described sudden change do not comprise known codon degeneracy variation.
The present invention further provides nucleotide sequence coded corresponding sequence table SEQ .ID NO.4 or the polypeptide of SEQ.ID NO.5 or SEQ.IDNO.6 or SEQ.ID NO.10 aminoacid sequence or the modified forms (〉=90% homology) of described polypeptide by sequence table SEQ .ID NO.3 or SEQ.ID NO.5 or SEQ.ID NO.7 or SEQ.IDNO.9, on this modified forms function quite or relevant with the high reactivity glucose isomerase.
The present invention further provides the engineering method of high temperature resistant glucose isomerase mutant, this method may further comprise the steps:
1) according to gene pool (L09699) gene order design primer TF:5 ' AGCCTAGGTTAATTAACTTTAAGAAGGAGATATACATATGAATAAATATTTTGAGA A 3 ' and TR:5 ' ATAAGCTCAGCGGCGCGCCTTATTCTGCAAACAAATACT 3 ', utilize primer that TF and TR and conventional PCR skill dog (available from ATCC 49915, are amplified parent's glucose isomerase gene (TS-F) in USA) from Thermoanaerobacterium saccharolyticum;
2) secondary structure and the tertiary structure of analysis parent glucose isomerase are selected site R81, W139, R182, V186, Q59 and single site is carried out in T299 six sites or/and multidigit point combinatorial mutagenesis;
3) produce mutator gene by round pcr sudden change, (Promega, USA) connection must contain the plasmid of mutator gene with the mutator gene and the carrier pGEMT-Easy that produce;
4) plasmid is changed over to competence bacterial cell HB101, (DIFCO USA) filters out the clone of tool glucose isomerase activity on dull and stereotyped (containing 1% D-wood sugar and 50mg/L penbritin) at 1% MacConkey;
5) from the clone, extract plasmid DNA, determine that through dna sequencing the point mutation of introducing is errorless.
The present invention further provides the preparation method of high temperature resistant glucose isomerase mutant, this method may further comprise the steps:
1) will contain the plasmid transformed competence colibacillus bacterial cell HB101 of glucose isomerase mutant gene, be coated in MacConkey (DIFCO, USA) on dull and stereotyped (containing 1%D-wood sugar and 50mg/L penbritin), select for 37 ℃ to cultivate 36 hours, produce clone with glucose isomerase activity;
2) the single clone in liquid LB substratum (containing the 50mg/L penbritin) of inoculation cultivated;
3) centrifugal collection thalline, and be suspended from the sodium phosphate buffer (pH 6.5), add CoCl
2And MgCl
2Be respectively 250 μ M and 5mM to final concentration.Use the ultrasonic treatment bacterial cell then;
4) centrifugal and collection supernatant liquor;
5) supernatant liquor is through 80 ℃ of thermal treatments after 10 minutes, and is centrifugal and further collect supernatant liquor.Supernatant liquor is partially purified glucose isomerase.
Prepare in the method for high temperature resistant glucose isomerase mutant in the present invention, described carrier can be selected various carrier known in the art for use, includes but not limited to prokaryotic expression carrier pGEMT-Easy, pRSET and pET21.When producing high temperature resistant glucose isomerase provided by the invention, high temperature resistant glucose isomerase gene order can be linked to each other with expression regulation sequence, and then form high temperature resistant glucose isomerase expression vector.Expression vector contain replication origin and expression regulation sequence, promotor or/and enhanser or/and necessary machining information site or/and signal coding sequence.Expression vector also must contain alternative marker gene, as ampicillin resistance gene, kalamycin resistance gene.These expression vectors can prepare with recombinant DNA technology well known in the art, can be with reference to Sambrook, and et al., Molecularcloning:A laboratory manual.New York:Cold Spring Harbor Laboratory Press, 1989).
Prepare in the method for glucose isomerase in the present invention, described glucose isomerase can expression in prokaryotic cell prokaryocyte (as HB101, BL21, DH5 α) or eukaryotic cell (as yeast saccharomyces cerevisiae, finish red saccharomyces pastorianus), also can adopt any other proper method known in the art to be implemented in prokaryotic cell prokaryocyte or eukaryotic cell is expressed outward.
The accompanying drawing table illustrates that following accompanying drawing table is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
The secondary structure of Fig. 1 parent glucose isomerase TS-F.
The simulation tertiary structure of Fig. 2 parent glucose isomerase TS-F.
Fig. 3 glucose isomerase mutant GI-1 polyacrylamide gel electrophoresis figure.
The specific activity of Fig. 4 parent glucose isomerase TS-F and glucose isomerase mutant.
The primer of table 1 parent glucose isomerase TS-F and glucose isomerase mutant amplified reaction.
Embodiment the following example only is used to the present invention is described and is not used in the scope of the present invention that limits.Implement unreceipted actual conditions person among the embodiment, the condition of conditioned disjunction manufacturers suggestion is carried out routinely.
Embodiment 1: the amplification of parental gene (TS-F) and the structure of carrier thereof
According to gene pool (L09699) gene order design primer TF and TR (seeing Table 1).Utilize primer to TF and the TR glucose isomerase coding parental gene (TS-F) that from T.saccharolyticum, increases.
Amplification reaction condition is: 20mM Tris-HCl, 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1% Triton X-100,0.2mM dNTP, 400nM primer TF and 400nM primer TR, (Promega, USA) archaeal dna polymerase with a little T.saccharolyticum thalline of transfering loop picking, are transferred reaction volume to 50 μ l with sterilized water to 1.5UPfu again.
The pcr amplification reaction program is: 95 ℃ 3 minutes, 40 circle circulations: 95 ℃ 30 seconds, 50 ℃ 30 seconds and 72 ℃ 3 minutes, last 72 ℃ 10 minutes.The amplification gene be connected to carrier pGEMT-Easy (Promega, USA) on, plasmid pGMT-TS-F.Utilize rapid plasmid to prepare test kit (Marligen Bioscience, USA) extract plasmid pGMT-TS-F, determine that through dna sequencing the nucleotides sequence of TS-F glucose isomerase classifies sequence table SEQ .ID NO.1 as, amino acid sequence corresponding is sequence table SEQ .ID NO.2.
Embodiment 2: glucose isomerase secondary structure and tertiary structure analysis
(referring to McGufGIn L.J.etal., Bioinformatics 2000,16:404-405) to utilize PSIPRED software to obtain the secondary structure of glucose isomerase of parental gene coding.The glucose isomerase secondary structure the results are shown in Figure 1.Utilize the SWISS-PROT software analysis obtain the glucose isomerase of parental gene coding the simulation tertiary structure (referring to Reutrakul S.etal., The Journal of Clinical Endocrinology ﹠amp; Metabolism2001,86 (10): 5039-5044).The simulation tertiary structure of glucose isomerase is seen Fig. 2.Sharp because of BLAST software (see network address:
Http:// www.ncbi.nlm.nih.gov/BLAST/) other glucose isomerase gene in parent's glucose isomerase gene and the gene pool (is particularly derived from the glucose isomerase gene of thermoduric bacteria, GI gene as gene pool A72225, P45687 and P29441) compares, obtain glucose isomerase gene conservative sequence information.On the basis of parent GI secondary structure and tertiary structure, select site R81, W139, R182, V186, Q59 and T182 four sites to carry out single site and multidigit point combinatorial mutagenesis (seeing embodiment 3,4 and 5) again.
Embodiment 3: Sites Combination sudden change GI-1 more than the glucose isomerase
Site-directed mutagenesis technique is with reference to HO S.N.etal., and Gene 1989,77 (1): the description of 51-59 one literary composition.With plasmid pGEMT-TS-F is template, and the design primer is to 81AF and 81AR, 139FF and 139FR, 182AF and 182AR, 186TF and 186TR and 299QF and 299QR (seeing Table 1).Primer TF and TR see embodiment 1.To TF and 81AR, the TFAR fragment increases with primer; Primer is to 81AF and 139FR, and the AFFR fragment increases; Primer is to 139FF and 182AR, and the FFAR fragment increases; Primer is to 182AF and 186TR, and the AFTR fragment increases; Primer is to 186TF and 299QR, and the TFQR fragment increases; Primer is to 299QF and TR, and the QFTR fragment increases.Amplification reaction condition is: 20mM Tris-HCl, 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1% Triton X-100,0.2mM dNTP, the single primer of 400nM (from a pair of primer), 1.5U Pfu archaeal dna polymerase, 40ngpGMT-TS-F transfers reaction volume to 50ul with sterilized water again.The pcr amplification reaction program is: 95 ℃ 3 minutes, 35 circle circulations: 95 ℃ 50 seconds, 52 ℃ 30 seconds and 72 ℃ 3 minutes, last 72 ℃ 5 minutes.(QIAGEN German) reclaims, and obtains TFAR fragment, AFFR fragment, FFAR fragment, AFTR fragment, TFQR and QFTR fragment respectively through the separation of 1% agarose gel electrophoresis and with test kit QIAquick DNA.Full-length gene then increases.Amplification reaction condition is: 20mM Tris-HCl, 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
40.1% Triton X-100,0.2mM dNTP, 400nM primer TF and 400nM TR, 1.5U Pfu archaeal dna polymerase, 40ng TFAR fragment, 40ng AFFR fragment, 40ng FFAR fragment, 40ng AFTR fragment, 40ng TFQR fragment and 40ng QFTR fragment are transferred reaction volume to 50 μ l with sterilized water again.The pcr amplification reaction program is: 95 ℃ 3 minutes, 35 circle circulations: 95 ℃ 50 seconds, 52 ℃ 30 seconds and 72 ℃ 3 minutes, last 72 ℃ 5 minutes.Reclaim through the separation of 1% agarose gel electrophoresis and with test kit QIAquick DNA, obtain total length mutator gene GI-1.GI-1 is connected with carrier pGEMT-Easy, gets plasmid pGEMT-GI-1.Change plasmid pGEMT-GI-1 over to competence bacterial cell HB101, (DIFCO USA) filters out the clone of tool glucose isomerase activity on dull and stereotyped (containing 1% D-wood sugar and 50mg/L penbritin) at 1%MacConkey.From the clone, extract plasmid pGEMT-GI-1 DNA, determine that through dna sequencing the point mutation of introducing is errorless.The G1-1 sequence is seen sequence table SEQ .ID NO.3-4.
Embodiment 4: Sites Combination sudden change GI-2 more than the glucose isomerase
Site-directed mutagenesis technique is with reference to HO S.N.etal., and Gene 1989,77 (1): the description of 51-59 one literary composition.With plasmid pGEMT-TS-F is template, and the design primer is to 81AF and 81AR, 139FF and 139FR, 182AF and 182AR, 187SF and 187SR and 299IF and 299IR (seeing Table 1).Primer TF and TR see embodiment 1.To TF and 81AR, the TFAR fragment increases with primer; Primer is to 81AF and 139FR, and the AFFR fragment increases; Primer is to 139FF and 182AR, and the FFAR fragment increases; Primer is to 182AF and 187SR, and the AFSR fragment increases; Primer is to 187SF and 299IR, and the SFIR fragment increases; Primer is to 299IF and TR, and the IFTR fragment increases.Amplification reaction condition is: 20mM Tris-HCl, 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1% Triton X-100,0.2mM dNTP, the single primer of 400nM (from a pair of primer), 1.5U Pfu archaeal dna polymerase, 20ngpGMT-TS-F transfers reaction volume to 50 μ l with sterilized water again.The pcr amplification reaction program is: 95 ℃ 3 minutes, 35 circle circulations: 95 ℃ 50 seconds, 52 ℃ 30 seconds and 72 ℃ 3 minutes, last 72 ℃ 5 minutes.Reclaim through the separation of 1% agarose gel electrophoresis and with QIAquick DNA, obtain TFAR fragment, AFFR fragment, FFAR fragment, AFSR fragment, SFIR and IFTR fragment respectively.Full-length gene then increases.Amplification reaction condition is: 20mM Tris-HCl, 10mMKCl, 10mM (NH
4)
2SO
4, 2mM MgSO
40.1% Triton X-100,0.2mM dNTP, 400nM primer TF and 400nM TR, 1.5U Pfu archaeal dna polymerase, 40ng TFAR fragment, 40ng AFFR fragment, 40ngFFAR fragment, 40ng AFSR fragment, 40ng SFIR fragment and 40ng IFTR fragment are transferred reaction volume to 50 μ l with sterilized water again.The pcr amplification reaction program is: 95 ℃ 3 minutes, 35 circle circulations: 95 ℃ 50 seconds, 52 ℃ 30 seconds and 72 ℃ 3 minutes, last 72 ℃ 5 minutes.Reclaim through the separation of 1% agarose gel electrophoresis and with QIAquick DNA, obtain total length mutator gene GI-2.GI-2 is connected with carrier pGEMT-Easy, gets plasmid pGEMT-GI-2.Change plasmid pGEMT-GI-2 over to competence bacterial cell HB101, (DIFCO USA) filters out the clone of tool glucose isomerase activity on dull and stereotyped (containing 1%D-wood sugar and 50mg/L penbritin) at 1% MacConkey.From the clone, extract plasmid pGEMT-G1-2DNA, determine that through dna sequencing the point mutation of introducing is errorless.The GI-2 sequence is seen sequence table SEQ .ID NO.5-6.
Embodiment 5: the single site mutation of glucose isomerase
Site-directed mutagenesis technique is with reference to HO S.N.etal., and Gene 1989,77 (1): the description of 51-59 one literary composition.With plasmid pGEMT-GI-1 (referring to embodiment 3) is template, and the design primer sports Asp (D) to 139DF and 139DR (seeing Table 1) with the Phe (F) in the 139th site in the GI-1 aminoacid sequence, obtains mutant GI-F139D.Primer TF and TR see embodiment 1.Utilize primer to TF and 139DR, amplification TFDR fragment; Primer is to 139DF and TR, and the DFTR fragment increases.Amplification reaction condition is: 20mM Tris-HCl, 10mM KCl, 10mM (NH
4)
2SO
4, 2mMMgSO
4, 0.1% Triton X-100,0.2mM dNTP, the single primer of 400nM (from a pair of primer), 1.5U PfuDNA polysaccharase, 40ng pGEMT-GI-1 transfers reaction volume to 50 μ l with sterilized water again.The pcr amplification reaction program is: 95 ℃ 3 minutes, 35 circle circulations: 95 ℃ 50 seconds, 52 ℃ 30 seconds and 72 ℃ 3 minutes, last 72 ℃ 5 minutes.Reclaim through the separation of 1% agarose gel electrophoresis and with QIAquick DNA, obtain TFDR fragment and DFTR fragment.Full-length gene then increases.Amplification reaction condition is: 20mM Tris-HCl, 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1% Triton X-100,0.2mM dNTP, 400nM primer TF and 400nMTR, 1.5U Pfu archaeal dna polymerase, 40ng TFDR and 40ng DFTR transfer reaction volume to 50 μ l with sterilized water again.The pcr amplification reaction program is: 95 ℃ 3 minutes, 35 circle circulations: 95 ℃ 50 seconds, 52 ℃ 30 seconds and 72 ℃ 3 minutes, last 72 ℃ 5 minutes.Reclaim through the separation of 1% agarose gel electrophoresis and with QIAquick DNA, obtain total length mutator gene GI-F139D.GI-F139D is connected with carrier pGEMT-Easy, gets plasmid pGEMT-GI-F139D.Change plasmid pGEMT-GI-F139D over to competence bacterial cell HB101, (DIFCO USA) filters out the clone of tool glucose isomerase activity on dull and stereotyped (containing 1% D-wood sugar and 50mg/L penbritin) at 1% MacConkey.From the clone, extract plasmid pGEMT-GI-F139D DNA, determine that through dna sequencing the point mutation of introducing is errorless.The GI-F139D sequence is seen sequence table SEQ .ID NO.7-8.
According to similar step, with plasmid pGEMT-GI-1 (referring to embodiment 3) is template, the design primer is to 299EF and 299ER (seeing Table 1), the Gln (Q) in the 299th site in the GI-1 aminoacid sequence is sported Glu (E), make up mutant GI-Q299E, mutant is connected with carrier pGEMT-Easy, gets plasmid pGEMT-GI-Q299E.The GI-Q299E sequence is seen sequence table SEQ .ID NO.9-10.
Embodiment 6: the extraction and purification of glucose isomerase TS-F
The extraction and purification main reference Lee Y.E.etal. of glucose isomerase, Journal of GeneralMicrobiology.1993,139:1227-1234.
The plasmid pGEMT-TS-F transformed competence colibacillus bacterial cell HB101 that will contain parent's glucose isomerase gene, (DIFCO USA) on dull and stereotyped (containing 1%D-wood sugar and 50mg/L penbritin), selects for 37 ℃ to cultivate 36 hours at MacConkey.Inoculate in the single 5ml of the being cloned in LB liquid nutrient medium (containing the 50mg/L penbritin) and cultivated 16 hours.Centrifugal collection thalline, and be suspended in the 1ml 20mM sodium phosphate buffer (pH 6.5), add CoCl
2And MgCl
2Be respectively 250 μ M and 5mM to final concentration.Use the ultrasonic treatment bacterial cell then.Centrifugal (10 ℃, 15,000 g, 15 minutes) also collect supernatant liquor.After 10 minutes, centrifugal (10 ℃, 15,000 g, 15 minutes) also collect supernatant liquor to supernatant liquor through 80 ℃ of thermal treatments.Supernatant liquor is partially purified glucose isomerase, and the mensuration and the seminal fluid fructose content that can be used for enzymic activity are measured.
Embodiment 7: the extraction and purification of glucose isomerase mutant
The extraction and purification of glucose isomerase mutant GI-1 and embodiment 5 with, be used plasmid be pGEMT-GI-1.The glucose isomerase of purifying is seen Fig. 3.The also described like this extraction and purification of other glucose isomerase mutant.
Embodiment 8: the active mensuration of parent's glucose isomerase TS-F
Get the glucose isomerase of 10 μ l, be added in the fructose and 20mM buffer solution of sodium phosphate of 90 μ l 1.0M (pH 6.5), and contain CoCl by embodiment 5 preparation
2And MgCl
2Final concentration is respectively 250 μ M and 5mM, reacts on 80 ℃ and carries out 10 minutes.Reactant is put on ice with termination reaction.Reaction product D-glucose is by the D-determination of glucose oxidase, and measuring method is referring to Trinder, P.Ann.Clin.Biochem.1981, and 18:64-67, and Shanghai Ke Hua-Dong water chestnut diagnosis is with the Reagent kit of glucose working instructions of product company limited.Use Coomassie
(PIERCE USA) measures zymoprotein concentration to Plus Protein AssayReagent Kit.One unit specific enzyme activity is defined as under these conditions per minute, and to transform micromole's fructose be the required enzyme amount of glucose.Fig. 4 shows the specific activity of parent TS-F glucose isomerase.
Embodiment 9: glucose isomerase mutant GI-1 determination of activity
Glucose isomerase mutant GI-1 determination of activity and embodiment 8 are together.The also described like this mensuration of the activity of other glucose isomerase mutant.Fig. 4 shows the difference of glucose isomerase mutant mutant and parent TS-F glucose isomerase specific enzyme activity.
Embodiment 10: measure the seminal fluid fructose content with glucose isomerase mutant
In 80 ℃ of processing 30 minutes, centrifugal under the room temperature (15,000g, 20 minutes) changed supernatant liquor in one clean tube over to again with human seminal fluid's sample.(B1O-RAD, USA) to final concentration 20%, in 80 ℃ of reactions 30 minutes, centrifugal under the room temperature (15,000g 15 minutes) collected supernatant liquor to add Chelex 100 resins.Get 180 μ l supernatant liquors, add CoCl
2And MgCl
2Be respectively 250 μ M and 5mM to final concentration, add that 20 μ l extract by embodiment 6 and the glucose isomerase mutant of purifying, in 80 ℃ of reactions 10 minutes.Reaction product D-glucose is by the D-glucose oxidase method and press embodiment 7 mensuration.According to fructose typical curve (be the fructose standardized solution of 0mM, 0.5mM, 1.0mM, 2mM, 4mM, 8mM and 10mM and formulate), ask and calculate fructose content in the seminal fluid by embodiment 7 by concentration.According to similar step, measure the fructose content in other organism refining.
The present invention is not subjected to the restriction of above-mentioned concrete text description, and the present invention can make various changes in the generalized scope of claims institute.These change all within the scope of the present invention.
The primer of table 1 parent glucose isomerase TS-F and glucose isomerase mutant pcr amplification reaction
| The parent | Primer is right |
| ????TS-F | TF：5′agcctaggttaattaactttaagaaggagatatacatatgaataaat attttgagaa?3′ TR：5′ataagctcagcggcgcgccttattctgcaaacaaatact?3′ |
| Mutant | Primer is right |
| ????GI-1 | 81AF：5′tagcgaaagcaagggtagaagcagcatttga?3′ 81AR：5′tctacccttgctttcgctatatccataggat?3′ 139FF：5′aagttttgtttggtaccgcaaatcttttctc?3′ 139FR：5′gcggtaccaaacaaaacttttgtcttgctgg?3′ 182AF：5′agcttggcgcggaaaactacgtattttgggg?3′ 182AR：5′tagttttccgcgccaagctccttagtaatct?3′ 186TF：5′aaaactacacattttggggtggaagagaagg?3′ 186TR：5′ccccaaaatgtgtagttttcgcggccaagct?3′ 299QF：5′acgcaaatcaaggcgacatgcttttgggatg?3′ 299QR：5′atgtcgccttgatttgcgtcaattgatccta?3′ |
| ????GI-2 | 81AF：5′tagcgaaagcaagggtagaagcagcatttga?3′ 81AR：5′tctacccttgctttcgctatatccataggat?3′ 139FF：5′aagttttgtttggtaccgcaaatcttttctc?3′ 139FR：5′gcggtaccaaacaaaacttttgtcttgctgg?3′ 182AF：5′agcttggcgcggaaaactacgtattttgggg?3′ 182AR：5′tagttttccgcgccaagctccttagtaatct?3′ 187SF：5′actacgtgagctggggtggaagagaagggt?3′ 187SR：5′ccaccccagctcacgtagttttcgcggccaa?3′ 299IF：5′acgcaaatattggcgacatgcttttgggatg?3′ 299IR：5′atgtcgccaatatttgcgtcaattgatccta?3′ |
| ????GI-F139D | 139DF：5′aagttttggatggtaccgcaaatcttttctc?3′ 139DR：5′gcggtaccatccaaaacttttgtcttgctgg?3′ |
| ????GI-D299E | 299EF：5′acgcaaatgaaggcgacatgcttttgggatg?3′ 299ER：5′atgtcgccttcatttgcgtcaattgatccta?3′ |
Sequence table
Sequence (SEQ.ID NO.) 1
(a) sequence signature:
* length: 1320 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: DNA
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
1?atgaataaat?attttgagaa?cgtatctaaa?ataaaatatg?aaggaccaaa?atcaaataat
61?ccttattcct?ttaaatttta?caatccagag?gaagtaatcg?atggcaagac?gatggaggag
121?catctccgct?tttctatagc?ttattggcac?acttttactg?ctgatggaac?agatcaattt
181?ggcaaggcta?ctatgcaaag?accatggaac?cactacacag?atcctatgga?tatagcgaaa
241?cgaagggtag?aagcagcatt?tgagtttttt?gataagataa?atgcaccttt?cttctgcttc
301?catgataggg?atattgcccc?tgaaggagat?actcttagag?agacaaacaa?aaacttagat
361?acaatagttg?ctatgataaa?ggattactta?aagaccagca?agacaaaagt?tttgtggggt
421?accgcaaatc?ttttctccaa?tccgagattt?gtacatggtg?catcaacatc?ctgcaatgct
481?gacgtttttg?catattctgc?agcgcaagtc?aaaaaagccc?ttgagattac?taaggagctt
541?ggccgcgaaa?actacgtatt?ttggggtgga?agagaagggt?acgagacgct?tctcaataca
601?gatatggagt?tagagcttga?taactttgca?agatttttgc?acatggctgt?tgactatgca
661?aaggaaatcg?gctttgaagg?tcagttcttg?attgagccga?agccaaagga?gcctacaaaa
721?catcaatacg?actttgacgt?ggcaaatgta?ttggcattct?tgagaaaata?cgaccttgac
781?aaatatttca?aagtaaatat?cgaagcaaac?catgcgacat?tggcattcca?cgacttccaa
841?catgagctaa?gatacgccag?aataaacggt?gtattaggat?caattgacgc?aaatacaggc
901?gacatgcttt?tgggatggga?tacggaccag?ttccctacag?atatacgcat?gacaacgctt
961?gctatgtatg?aagtcataaa?gatgggtgga?tttgacaaag?gtggccttaa?ctttgatgca
1021?aaagtaagac?gtgcttcatt?tgagccagaa?gatcttttct?taggtcacat?agcaggaatg
1081?gatgcttttg?caaaaggctt?taaagttgct?tacaagcttg?tgaaagatgg?cgtatttgac
1141?aagttcatcg?aagaaagata?cgcaagctac?aaagaaggca?ttggcgctga?tattgtaagc
1201?ggtaaagctg?acttcaagag?ccttgaaaag?tatgcattag?agcacagcca?gattgtaaac
1261?aaatcaggca?gacaagagct?attagaatca?atcctaaatc?agtatttgtt?tgcagaataa
Sequence (SEQ.ID NO.) 2
(a) sequence signature:
* length: 440 amino-acid residues
* type: polypeptide
* chain: strand
* topological framework: linearity
(b) molecule type: Protein
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
mnkyfenvsk?ikyegpksnn?pysfkfynpe?evidgktmee?hlrfsiaywh?tftadgtdqf
gkatmqrpwn?hytdpmdiak?rrveaafeff?dkinapffcf?hdrdiapegd?tlretnknld
tivamikdyl?ktsktkvlwg?tanlfsnprf?vhgastscna?dvfaysaaqv?kkaleitkel
grenyvfwgg?regyetllnt?dmeleldnfa?rflhmavdya?keigfegqfl?iepkpkeptk
hqydfdvanv?laflrkydld?kyfkvniean?hatlafhdfq?helryaring?vlgsidantg
dmllgwdtdq?fptdirmttl?amyevikmgg?fdkgglnfda?kvrrasfepe?dlflghiagm
dafakgfkva?yklvkdgvfd?kfieeryasy?kegigadivs?gkadfkslek?yalehsqivn
ksgrqelles?ilnqylfae
Sequence (SEQ.ID NO.) 3
(a) sequence signature:
* length: 1320 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: DNA
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
1?atgaataaat?attttgagaa?cgtatctaaa?ataaaatatg?aaggaccaaa?atcaaataat
61?ccttattcct?ttaaatttta?caatccagag?gaagtaatcg?atggcaagac?gatggaggag
121?catctccgct?tttctatagc?ttattggcac?acttttactg?ctgatggaac?agatcaattt
181?ggcaaggcta?ctatgcaaag?accatggaac?cactacacag?atcctatgga?tatagcgaaa
241?gcaagggtag?aagcagcatt?tgagtttttt?gataagataa?atgcaccttt?cttctgcttc
301?catgataggg?atattgcccc?tgaaggagat?actcttagag?agacaaacaa?aaacttagat
361?acaatagttg?ctatgataaa?ggattactta?aagaccagca?agacaaaagt?tttgtttggt
421?accgcaaatc?ttttctccaa?tccgagattt?gtacatggtg?catcaacatc?ctgcaatgct
481?gacgtttttg?catattctgc?agcgcaagtc?aaaaaagccc?ttgagattac?taaggagctt
541?ggcgcggaaa?actacacatt?ttggggtgga?agagaagggt?acgagacgct?tctcaataca
601?gatatggagt?tagagcttga?taactttgca?agatttttgc?acatggctgt?tgactatgca
661?aaggaaatcg?gctttgaagg?tcagttcttg?attgagccga?agccaaagga?gcctacaaaa
721?catcaatacg?actttgacgt?ggcaaatgta?ttggcattct?tgagaaaata?cgaccttgac
781?aaatatttca?aagtaaatat?cgaagcaaac?catgcgacat?tggcattcca?cgacttccaa
841?catgagctaa?gatacgccag?aataaacggt?gtattaggat?caattgacgc?aaatcaaggc
901?gacatgcttt?tgggatggga?tacggaccag?ttccctacag?atatacgcat?gacaacgctt
961?gctatgtatg?aagtcataaa?gatgggtgga?tttgacaaag?gtggccttaa?ctttgatgca
1021?aaagtaagac?gtgcttcatt?tgagccagaa?gatcttttct?taggtcacat?agcaggaatg
1081?gatgcttttg?caaaaggctt?taaagttgct?tacaagcttg?tgaaagatgg?cgtatttgac
1141?aagttcatcg?aagaaagata?cgcaagctac?aaagaaggca?ttggcgctga?tattgtaagc
1201?ggtaaagctg?acttcaagag?ccttgaaaag?tatgcattag?agcacagcca?gattgtaaac
1261?aaatcaggca?gacaagagct?attagaatca?atcctaaatc?agtatttgtt?tgcagaataa
Sequence (SEQ.ID NO.) 4
(a) sequence signature:
* length: 440 amino-acid residues
* type: polypeptide
* chain: strand
* topological framework: linearity
(b) molecule type: Protein
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
MNKYFENVSKIKYEGPKSNNPYSFKFYNPEEVIDGKTMEEHLRFSIAYWHTFTADGTDQF
GKATMQRPWNHYTDPMDIAKARVEAAFEFFDKINAPFFCFHDRDIAPEGDTLRETNKNLD
TIVAMIKDYLKTSKTKVLFGTANLFSNPRFVHGASTSCNADVFAYSAAQVKKALEITKEL
GAENYTFWGGREGYETLLNTDMELELDNFARFLHMAVDYAKEIGFEGQFLIEPKPKEPTK
HQYDFDVANVLAFLRKYDLDKYFKVNIEANHATLAFHDFQHELRYARINGVLGSIDANQG
DMLLGWDTDQFPTDIRMTTLAMYEVIKMGGFDKGGLNFDAKVRRASFEPEDLFLGHIAGM
DAFAKGFKVAYKLVKDGVFDKFIEERYASYKEGIGADIVSGKADFKSLEKYALEHSQIVN
KSGRQELLESILNQYLFAE*
Sequence (SEQ.ID NO.) 5
(a) sequence signature:
* length: 1320 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: DNA
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
1?atgaataaat?attttgagaa?cgtatctaaa?ataaaatatg?aaggaccaaa?atcaaataat
61?ccttattcct?ttaaatttta?caatccagag?gaagtaatcg?atggcaagac?gatggaggag
121?catctccgct?tttctatagc?ttattggcac?acttttactg?ctgatggaac?agatcaattt
181?ggtaaggcta?ctatgcaaag?accatggaac?cactacacag?atcctatgga?tatagcgaaa
241?gcaagggtag?aagcagcatt?tgagtttttt?gataagataa?atgcaccttt?cttctgcttc
301?catgataggg?atattgcccc?tgaaggagat?actcttagag?agacaaacaa?aaacttagat
361?acaatagttg?ctatgataaa?ggattactta?aagaccagca?agacaaaagt?tttgtttggt
421?accgcaaatc?ttttctccaa?tccgagattt?gtacatggtg?catcaacatc?ctgcaatgct
481?gacgtttttg?catattctgc?agcgcaagtc?aaaaaagccc?ttgagattac?taaggagctt
541?ggcgcggaaa?actacgtgag?ctggggtgga?agagaagggt?acgagacgct?tctcaataca
601?gatatggagt?tagagcttga?taactttgca?agatttttgc?acatggctgt?tgactatgca
661?aaggaaatcg?gctttgaagg?tcagttcttg?attgagccga?agccaaagga?gcctacaaaa
721?catcaatacg?actttgacgt?ggcaaatgta?ttggcattct?tgagaaaata?cgaccttgac
781?aaatatttca?aagtaaatat?cgaagcaaac?catgcgacat?tggcattcca?cgacttccaa
841?catgagctaa?gatacgccag?aataaacggt?gtattaggat?caattgacgc?aaatattggc
901?gacatgcttt?tgggatggga?tacggaccag?ttccctacag?atatacgcat?gacaacgctt
961?gctatgtatg?aagtcataaa?gatgggtgga?tttgacaaag?gtggccttaa?ctttgatgca
1021?aaagtaagac?gtgcttcatt?tgagccagaa?gatcttttct?taggtcacat?agcaggaatg
1081?gatgcttttg?caaaaggctt?taaagttgct?tacaagcttg?tgaaagatgg?cgtatttgac
1141?aagttcatcg?aagaaagata?cgcaagctac?aaagaaggca?ttggcgctga?tattgtaagc
1201?ggtaaagctg?acttcaagag?ccttgaaaag?tatgcattag?agcacagcca?gattgtaaac
1261?aaatcaggca?gacaagagct?attagaatca?atcctaaatc?agtatttgtt?tgcagaataa
Sequence (SEQ.ID NO.) 6
(a) sequence signature:
* length: 440 amino-acid residues
* type: polypeptide
* chain: strand
* topological framework: linearity
(b) molecule type: Protein
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
MNKYFENVSKIKYEGPKSNNPYSFKFYNPEEVIDGKTMEEHLRFSIAYWHTFTADGTDQF
GKATMQRPWNHYTDPMDIAKARVEAAFEFFDKINAPFFCFHDRDIAPEGDTLRETNKNLD
TIVAMIKDYLKTSKTKVLFGTANLFSNPRFVHGASTSCNADVFAYSAAQVKKALEITKEL
GAENYVSWGGREGYETLLNTDMELELDNFARFLHMAVDYAKEIGFEGQFLIEPKPKEPTK
HQYDFDVANVLAFLRKYDLDKYFKVNIEANHATLAFHDFQHELRYARINGVLGSIDANIG
DMLLGWDTDQFPTDIRMTTLAMYEVIKMGGFDKGGLNFDAKVRRASFEPEDLFLGHIAGM
DAFAKGFKVAYKLVKDGVFDKFIEERYASYKEGIGADIVSGKADFKSLEKYALEHSQIVN
KSGRQELLESILNQYLFAE*
Sequence (SEQ.ID NO.) 7
(a) sequence signature:
* length: 1320 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: DNA
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
1?atgaataaat?attttgagaa?cgtatctaaa?ataaaatatg?aaggaccaaa?atcaaataat
61?ccttattcct?ttaaatttta?caatccagag?gaagtaatcg?atggcaagac?gatggaggag
121?catctccgct?tttctatagc?ttattggcac?acttttactg?ctgatggaac?agatcaattt
181?ggcaaggcta?ctatgcaaag?accatggaac?cactacacag?atcctatgga?tatagcgaaa
241?gcaagggtag?aagcagcatt?tgagtttttt?gataagataa?atgcaccttt?cttctgcttc
301?catgataggg?atattgcccc?tgaaggagat?actcttagag?agacaaacaa?aaacttagat
361?acaatagttg?ctatgataaa?ggattactta?aagaccagca?agacaaaagt?tttggatggt
421?accgcaaatc?ttttctccaa?tccgagattt?gtacatggtg?catcaacatc?ctgcaatgct
481?gacgtttttg?catattctgc?agcgcaagtc?aaaaaagccc?ttgagattac?taaggagctt
541?ggcgcggaaa?actacacatt?ttggggtgga?agagaagggt?acgagacgct?tctcaataca
601?gatatggagt?tagagcttga?taactttgca?agatttttgc?acatggctgt?tgactatgca
661?aaggaaatcg?gctttgaagg?tcagttcttg?attgagccga?agccaaagga?gcctacaaaa
721?catcaatacg?actttgacgt?ggcaaatgta?ttggcattct?tgagaaaata?cgaccttgac
781?aaatatttca?aagtaaatat?cgaagcaaac?catgcgacat?tggcattcca?cgacttccaa
841?catgagctaa?gatacgccag?aataaacggt?gtattaggat?caattgacgc?aaatcaaggc
901?gacatgcttt?tgggatggga?tacggaccag?ttccctacag?atatacgcat?gacaacgctt
961?gctatgtatg?aagtcataaa?gatgggtgga?tttgacaaag?gtggccttaa?ctttgatgca
1021?aaagtaagac?gtgcttcatt?tgagccagaa?gatcttttct?taggtcacat?agcaggaatg
1081?gatgcttttg?caaaaggctt?taaagttgct?tacaagcttg?tgaaagatgg?cgtatttgac
1141?aagttcatcg?aagaaagata?cgcaagctac?aaagaaggca?ttggcgctga?tattgtaagc
1201?ggtaaagctg?acttcaagag?ccttgaaaag?tatgcattag?agcacagcca?gattgtaaac
1261?aaatcaggca?gacaagagct?attagaatca?atcctaaatc?agtatttgtt?tgcagaataa
Sequence (SEQ.ID NO.) 8
(a) sequence signature:
* length: 440 amino-acid residues
* type: polypeptide
* chain: strand
* topological framework: linearity
(b) molecule type: Protein
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
MNKYFENVSKIKYEGPKSNNPYSFKFYNPEEVIDGKTMEEHLRFSIAYWHTFTADGTDQF
GKATMQRPWNHYTDPMDIAKARVEAAFEFFDKINAPFFCFHDRDIAPEGDTLRETNKNLD
TIVAMIKDYLKTSKTKVLDGTANLFSNPRFVHGASTSCNADVFAYSAAQVKKALEITKEL
GAENYTFWGGREGYETLLNTDMELELDNFARFLHMAVDYAKEIGFEGQFLIEPKPKEPTK
HQYDFDVANVLAFLRKYDLDKYFKVNIEANHATLAFHDFQHELRYARINGVLGSIDANQG
DMLLGWDTDQFPTDIRMTTLAMYEVIKMGGFDKGGLNFDAKVRRASFEPEDLFLGHIAGM
DAFAKGFKVAYKLVKDGVFDKFIEERYASYKEGIGADIVSGKADFKSLEKYALEHSQIVN
KSGRQELLESILNQYLFAE*
Sequence (SEQ.ID NO.) 9
(a) sequence signature:
* length: 1320 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: DNA
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
1?atgaataaat?attttgagaa?cgtatctaaa?ataaaatatg?aaggaccaaa?atcaaataat
61?ccttattcct?ttaaatttta?caatccagag?gaagtaatcg?atggcaagac?gatggaggag
121?catctccgct?tttctatagc?ttattggcac?acttttactg?ctgatggaac?agatcaattt
181?ggcaaggcta?ctatgcaaag?accatggaac?cactacacag?atcctatgga?tatagcgaaa
241?gcaagggtag?aagcagcatt?tgagtttttt?gataagataa?atgcaccttt?cttctgcttc
301?catgataggg?atattgcccc?tgaaggagat?actcttagag?agacaaacaa?aaacttagat
361?acaatagttg?ctatgataaa?ggattactta?aagaccagca?agacaaaagt?tttgtttggt
421?accgcaaatc?ttttctccaa?tccgagattt?gtacatggtg?catcaacatc?ctgcaatgct
481?gacgtttttg?catattctgc?agcgcaagtc?aaaaaagccc?ttgagattac?taaggagctt
541?ggcgcggaaa?actacacatt?ttggggtgga?agagaagggt?acgagacgct?tctcaataca
601?gatatggagt?tagagcttga?taactttgca?agatttttgc?acatggctgt?tgactatgca
661?aaggaaatcg?gctttgaagg?tcagttcttg?attgagccga?agccaaagga?gcctacaaaa
721?catcaatacg?actttgacgt?ggcaaatgta?ttggcattct?tgagaaaata?cgaccttgac
781?aaatatttca?aagtaaatat?cgaagcaaac?catgcgacat?tggcattcca?cgacttccaa
841?catgagctAa?gatacgccag?aataaacggt?gtattaggat?caattgacgc?aaatgaaggc
901?gacatgcttt?tgggatggga?tacggaccag?ttccctacag?atatacgcat?gacaacgctt
961?gctatgtatg?aagtcataaa?gatgggtgga?tttgacaaag?gtggccttaa?ctttgatgca
1021?aaagtaagac?gtgcttcatt?tgagccagaa?gatcttttct?taggtcacat?agcaggaatg
1081?gatgcttttg?caaaaggctt?taaagttgct?tacaagcttg?tgaaagatgg?cgtatttgac
1141?aagttcatcg?aagaaagata?cgcaagctac?aaagaaggca?ttggcgctga?tattgtaagc
1201?ggtaaagctg?acttcaagag?ccttgaaaag?tatgcattag?agcacagcca?gattgtaaac
1261?aaatcaggca?gacaagagct?attagaatca?atcctaaatc?agtatttgtt?tgcagaataa
Sequence (SEQ.ID NO.) 10
(a) sequence signature:
* length: 440 amino-acid residues
* type: polypeptide
* chain: strand
* topological framework: linearity
(b) molecule type: Protein
(c) suppose: not
(d) antisense: not
(e) initial source: Thermoanaerobacterium sachcharolyticum B6A.
MNKYFENVSKIKYEGPKSNNPYSFKFYNPEEVIDGKTMEEHLRFSIAYWHTFTADGTDQF
GKATMQRPWNHYTDPMDIAKARVEAAFEFFDKINAPFFCFHDRDIAPEGDTLRETNKNLD
TIVAMIKDYLKTSKTKVLFGTANLFSNPRFVHGASTSCNADVFAYSAAQVKKALEITKEL
GAENYTFWGGREGYETLLNTDMELELDNFARFLHMAVDYAKEIGFEGQFLIEPKPKEPTK
HQYDFDVANVLAFLRKYDLDKYFKVNIEANHATLAFHDFQHELRYARINGVLGSIDANEG
DMLLGWDTDQFPTDIRMTTLAMYEVIKMGGFDKGGLNFDAKVRRASFEPEDLFLGHIAGM
DAFAKGFKVAYKLVKDGVFDKFIEERYASYKEGIGADIVSGKADFKSLEKYALEHSQIVN
KSGRQELLESILNQYLFAE*
Claims (16)
1. a seminal fluid fructose is measured novel method, it is characterized in that: this method has used a kind of energy efficient catalytic fructose to be converted into the high reactivity glucose isomerase of glucose.
2. claim 1 a described measuring method, its feature also is: use chelex 100 resins or its analogue to remove glucose isomerase enzyme reaction interfering substance in the seminal fluid.
3. isolated DNA molecule is characterized in that: it is the nucleotide sequence of coding claim 1 a described high reactivity glucose isomerase.
4. claim 3 a described dna molecular, it is characterized in that: described nucleotide sequence has the nucleotide sequence of sequence table SEQ .IDNO.3 or the mutant form of described nucleotide sequence (having 〉=75% homology), and described sudden change comprises: disappearance, nonsense, insertion, missense.
5. claim 4 a described dna molecular, it is characterized in that: the polypeptide of the aminoacid sequence among the described nucleotide sequence coded sequence table SEQ .IDNO.4 or the modified forms of described polypeptide (having 〉=90% homology), on this modified forms function quite or relevant with the high reactivity glucose isomerase.
6. claim 3 a described dna molecular, its feature also is: described nucleotide sequence has the nucleotide sequence of sequence table SEQ .IDNO.5 or the mutant form of described nucleotide sequence (having 〉=75% homology), and described sudden change comprises: disappearance, nonsense, insertion, missense.
7. claim 6 a described dna molecular, it is characterized in that: the polypeptide of the aminoacid sequence among the described nucleotide sequence coded sequence table SEQ .IDNO.6 or the modified forms of described polypeptide (having 〉=90% homology), on this modified forms function quite or relevant with the high reactivity glucose isomerase.
8. claim 3 a described dna molecular, its feature also is: described nucleotide sequence has the nucleotide sequence of sequence table SEQ .IDNO.7 or the mutant form of described nucleotide sequence (having 〉=75% homology), and described sudden change comprises: disappearance, nonsense, insertion, missense.
9. claim 8 a described dna molecular, it is characterized in that: the polypeptide of the aminoacid sequence among the described nucleotide sequence coded sequence table SEQ .IDNO.8 or the modified forms of described polypeptide (having 〉=90% homology), on this modified forms function quite or relevant with the high reactivity glucose isomerase.
10. claim 3 a described dna molecular, its feature also is: described nucleotide sequence has the nucleotide sequence of sequence table SEQ .ID NO.9 or the mutant form of described nucleotide sequence (having 〉=75% homology), and described sudden change comprises: disappearance, nonsense, insertion, missense.
11. claim 10 a described dna molecular, it is characterized in that: the polypeptide of the aminoacid sequence among the described nucleotide sequence coded sequence table SEQ .ID NO.10 or the modified forms of described polypeptide (having 〉=90% homology), on this modified forms function quite or relevant with the high reactivity glucose isomerase.
12. as claim 1 a described high reactivity glucose isomerase is the enzyme of partial purification or complete purifying.
13. as claim 1 a described high reactivity glucose isomerase is liquid phase enzyme or solid enzyme.
14. as claim 1 a described high reactivity glucose isomerase can be to be present in intestinal bacteria, yeast or other protokaryon or the eukaryotic cells with the solid phase cells form.
15. claim 1 a described seminal fluid fructose measuring method can be used for the male sterility diagnosis.
16. claim 1 a described seminal fluid fructose measuring method also can be used on the fructose content of measuring other organism seminal fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410037442 CN1693476A (en) | 2004-05-08 | 2004-05-08 | New determination method of semen fructose and glucose isomerase mutant used for said method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410037442 CN1693476A (en) | 2004-05-08 | 2004-05-08 | New determination method of semen fructose and glucose isomerase mutant used for said method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1693476A true CN1693476A (en) | 2005-11-09 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410037442 Withdrawn CN1693476A (en) | 2004-05-08 | 2004-05-08 | New determination method of semen fructose and glucose isomerase mutant used for said method |
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| Country | Link |
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| CN (1) | CN1693476A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1956083A1 (en) * | 2005-11-18 | 2008-08-13 | Bioright Worldwide Company Limited | Glucose isomerase mutants, the use thereof and the dnas encoding the same |
| CN102012374A (en) * | 2010-09-29 | 2011-04-13 | 南京欣迪生物药业工程有限责任公司 | Kit for detecting concentration of seminal plasma fructose and detection method |
| CN116121217A (en) * | 2023-02-24 | 2023-05-16 | 山东大学 | Glucose oxidase mutant with improved xylose oxidizing ability and application thereof |
-
2004
- 2004-05-08 CN CN 200410037442 patent/CN1693476A/en not_active Withdrawn
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1956083A1 (en) * | 2005-11-18 | 2008-08-13 | Bioright Worldwide Company Limited | Glucose isomerase mutants, the use thereof and the dnas encoding the same |
| EP1956083A4 (en) * | 2005-11-18 | 2009-02-25 | Geneharbor Hong Kong Technolog | Glucose isomerase mutants, the use thereof and the dnas encoding the same |
| US7704719B2 (en) | 2005-11-18 | 2010-04-27 | Geneharbor (Hk) Technologies Ltd. | Glucose isomerase mutants, DNA thereof and use thereof |
| US7923222B2 (en) | 2005-11-18 | 2011-04-12 | Geneharbor (Hk) Technologies Ltd. | Methods of using isolated glucose isomerase |
| US8067561B2 (en) | 2005-11-18 | 2011-11-29 | Bioright Worldwide Company Limited | Isolated DNA encoding recombinant glucose isomerase |
| CN102012374A (en) * | 2010-09-29 | 2011-04-13 | 南京欣迪生物药业工程有限责任公司 | Kit for detecting concentration of seminal plasma fructose and detection method |
| CN102012374B (en) * | 2010-09-29 | 2012-08-01 | 南京欣迪生物药业工程有限责任公司 | Kit for detecting concentration of seminal plasma fructose and detection method |
| CN116121217A (en) * | 2023-02-24 | 2023-05-16 | 山东大学 | Glucose oxidase mutant with improved xylose oxidizing ability and application thereof |
| CN116121217B (en) * | 2023-02-24 | 2023-09-08 | 山东大学 | Glucose oxidase mutant with improved xylose oxidizing ability and application thereof |
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