CN101289669A - High-activity mutant gene of xylanase and point mutation process thereof - Google Patents
High-activity mutant gene of xylanase and point mutation process thereof Download PDFInfo
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Abstract
The invention relates to a high-activity xylanase mutant gene and a point mutation method for the same. The invention is to utilize the point mutation method to mutate the fifty-eighth amino acid of a xylanase gene from asparagines to asparagus amino acids so as to form the high-activity xylanase mutant gene. The method has the advantages of effective improvement of the activity of ferment and reduction of the use cost.
Description
Technical field
The invention relates to a kind of mutator gene and point mutation process thereof of highly active xylanase, it is to utilize this point mutation process that the 58th amino acid of the gene of one xylanase is sported acid, aspartic (aspartic acid) by asparagine (asparagine), to form the mutator gene of this highly active xylanase.
Background technology
Generally speaking, poly-wooden candy (xylan) is present in the structural polysaccharide of plant mostly, and it is the cellulosic function with protective plant, is considered as a kind of obstacle when therefore gathering wooden candy for the human development plant resources.For example, in the formed paper pulp, poly-wooden candy will adhere to the surface of fibre of plant matter with the xylogen of plant in paper-making process, must utilize monochloride that paper pulp is carried out a bleaching operation usually.After operation finished, the chemical by-product that muriate produced was to have toxicity and carinogenicity in bleaching, and with the accumulation (bioaccumulating) of persistence (persistent) in physical environment, and then physical environment and ecology caused have a strong impact on.
In livestock industry, when a feed enters the Digestive tract of animal, because feed has contained cellulosic and half cellulosic, simultaneously many nutrients that are utilized of feed are often being coated by itself cellulosic and half cellulosic, thereby block nutrient and absorbed by other ferment effect or intestines and stomach, and then influence the growth of animal, and without the nutrient that utilizes, in case after the discharge animal body is outer, must forms source of pollution, and cause the low of environmental quality.Therefore, how to decompose that to remove poly-wooden candy be to be the considerable problem of industry.
Generally speaking, because the traditional xylanase that separates from a rumen microorganism (rumenmicroorganisms) is the ability with branch depolymerization wood candy, therefore can effectively improve the problems referred to above.With regard to paper-making industry, xylanase can decompose the hemicellulose in the paper pulp and disintegrate the binding of xylogen and Mierocrystalline cellulose and hemicellulose, and so, paper pulp can discharge xylogen, and then finishes bleaching action.
In food-processing industry, except that half cellulosic that can decompose in the fruit juice, more can be in order to make a wood oligose as various food raw materials.
If when being applied in livestock industry, then wood oligose can be added in the feed, utilize this xylanase to decompose poly-wooden candy in this feed, absorbed by the Digestive tract of animal easily to help nutrient, and then improve or improve the dietetic alimentation amount of animal.
Yet, active limited based on above-mentioned traditional xylanase, so that often need to improve the usage quantity of xylanase, and then cause a large amount of uses and the higher shortcoming of cost.
Moreover the conventional point mutation method causes the enzyme activity deficiency of xylanase owing to can't improve the enzyme activity of xylanase.
Point mutation is a kind of mode that is commonly used to improve the ferment characteristic, one conventional point mutation method is to be published in J.Mol.Biol. (2000) 299 as Joshi et al., shown in the Hydrogen Bonding and Catalysis:A NovelExplanation for How a Single Amino Acid Substitution CanChange the pH Optimum of a Glycosidase of 255-279, it is that the 35th amino acid with the xylanase of a Bacillus circulans is when sporting acid, aspartic by asparagine, make the pKa value of xylanase descend, and then improve the acid resistance of xylanase.The conventional point mutation method only can improve the acid-resistant property of xylanase, still has the insufficient shortcoming of enzyme activity that can't effectively improve xylanase.
In view of this, the present invention utilizes a point mutation process that the 58th amino acid of the gene of one xylanase is sported acid, aspartic by asparagine, to form the mutator gene of a highly active xylanase.By this, the present invention can effectively improve the enzyme activity of xylanase really, and then reduces use cost.
Summary of the invention
Main purpose of the present invention provides a kind of mutator gene of highly active xylanase, and the 58th amino acid of the mutator gene of highly active xylanase is to be acid, aspartic, makes the present invention have the effect that improves enzyme activity.
Secondary objective of the present invention provides a kind of point mutation process, it is that at least one amino acid in the gene of a ferment is sported acid, aspartic by asparagine, with a mutator gene of formation ferment, and then make the present invention have the effect of raising enzyme activity and reduction use cost.
The object of the present invention is achieved like this: a kind of mutator gene of highly active xylanase is characterized in that: the 58th amino acid of the mutator gene of this xylanase is to be acid, aspartic.(aspartic acid) (gene order of the mutator gene of this active xylanase is to contain just like recognition sequence to number: the full sequence of 3 (SEQ ID NO:3)), or the 38th amino acid of the mutator gene of this xylanase is acid, aspartic (aspartic acid) (gene order of the mutator gene of this xylanase is to number as recognition sequence: shown in 4 (the SEQ ID NO:4)).
The gene of this xylanase is to utilize a carrier to produce, to form one first reorganization plastid.This carrier is to be a pET system.This first reorganization plastid mix a dNTP, buffered soln, one forward desire sudden change end introduction, a reverse desire sudden change end introduction and a polysaccharase carry out this polymerase chain reaction, contain one second reorganization plastid of the mutator gene of this highly active xylanase with formation.This forward desire sudden change end introduction be to have the recognition sequence numbering: 1 gene order, and should reverse desire sudden change end introduction be to have the recognition sequence numbering: 2 gene order, wherein, recognition sequence is numbered: 1 is 5 ' CTGGTTAGAT
GACACCGGTGGTAGC3 ', the recognition sequence numbering: 2 is 5 ' GCTACCACCGGT
GTCATCTAACCAG3 '.
The mutator gene of this highly active xylanase is to be integrated in a plastid or the karyomit(e) via gene recombination technology.The gene of this xylanase is a hybrid bacterial strain that separates from a rumen microorganism, and the gene order of the gene of this xylanase is to login in gene database, and the login number is AY941119.
The present invention also provides a kind of point mutation process, it is characterized in that: be that at least one amino acid in the gene of a ferment is sported acid, aspartic by asparagine, to form a mutator gene of this ferment.
This ferment is an xylanase, and this amino acid is the 58th amino acid of the gene of this xylanase.The gene order of the mutator gene of this highly active xylanase is to contain the recognition sequence numbering: 3 sequence.
Major advantage of the present invention is: according to the mutator gene and the point mutation process thereof of highly active xylanase of the present invention, it is that the 58th amino acid that belongs to the gene of an xylanase sports acid, aspartic by asparagine, to form the mutator gene of a highly active xylanase.Utilize this point mutation process to transform the 58th amino acid of the gene of xylanase, sport acid, aspartic, so can effectively improve the enzyme activity of xylanase to reach by asparagine.
Further specify below in conjunction with preferred embodiment and accompanying drawing.
Description of drawings
Fig. 1: the preparation flow synoptic diagram of the mutator gene of the highly active xylanase of the embodiment of the invention 1 and point mutation process thereof.
Fig. 2: the gene order synoptic diagram of the mutator gene of the highly active xylanase of the embodiment of the invention 1 (the recognition sequence numbering: 3, S EQ ID NO:3).
Fig. 3: the S DS-PAGE of the wild-type xylanase of the embodiment of the invention 1 analyzes synoptic diagram.
Fig. 4: the SDS-PAGE of the mutant xylanase of the embodiment of the invention 1 analyzes synoptic diagram.
Fig. 5: the relative enzyme activity (%) of the wild-type xylanase of the embodiment of the invention 1 and mutant wood candy enzyme is to the variation synoptic diagram of pH value.
Fig. 6: the gene order synoptic diagram of the mutator gene of the highly active xylanase of the embodiment of the invention 2 (the recognition sequence numbering: 4, S EQ ID NO:4).
Embodiment
For above-mentioned purpose of the present invention, feature and advantage can be become apparent, preferred embodiment of the present invention cited below particularly, and conjunction with figs. are described in detail below:
See also shown in Figure 1ly, embodiments of the invention 1 mainly are to utilize a point mutation process that the 58th amino acid of the gene of one xylanase is sported acid, aspartic by asparagine, to form the mutator gene of a highly active xylanase.More in detail, the present invention further forms the mutator gene of highly active xylanase via the following step to utilize point mutation process:
First step S1 is that the gene with xylanase utilizes a carrier to produce, and to form one first reorganization plastid, again the first reorganization plastid is changeed shape and goes in the competent cell (competent cell), further forms a wild-type expression vector;
The second step S2 mixes a dNTP, a buffered soln (reaction buffer), one with the first reorganization plastid forward desire sudden change end introduction (forward primer), a reverse desire sudden change holds an introduction (reverse primer) and a polysaccharase (polymerase) to carry out a polymerase chain reaction (polymerase chainreaction), the one second reorganization plastid that contains the mutator gene of highly active xylanase with formation, again the second reorganization plastid is changeed shape and go into to be competent in the cell, to form a mutant expression vector.But because the gene of polymerase chain reaction massive duplication xylanase, and forward reach in the reverse desire sudden change end introduction and respectively have a sudden change position, make polymerase chain reaction when duplicating the gene of xylanase, can produce the mutator gene of highly active xylanase.Therefore, forward reach reverse desire sudden change end introduction, just can further make the 58th amino acid of the gene of xylanase sport acid, aspartic, to form the mutator gene of highly active xylanase by asparagine by control.
Please consult shown in Figure 1ly again, the first step S1 of embodiments of the invention 1 is that the gene with xylanase utilizes carrier production, to form the first reorganization plastid, again the first reorganization plastid is changeed shape and goes into to be competent in the cell, further forms the wild-type expression vector.More in detail, the gene order of the gene of the xylanase of wild-type is to login in gene database (the login number is AY941119), and its source is to select to separate certainly the not rumen microorganism of separation and purification (rumen microorganisms).Carrier is selection but is not subject to a pET system.The operating method of this pET system is as follows:
The gene of the xylanase of wild-type is former to be stored in the plastid (plasmid), contains one of gene reorganization plastid of xylanase with formation, and plastid is to be chosen as a DGEX5X-1 (Amersham Pharmacia, Sweden).The plastid of will recombinating again changes shape and goes into one first microorganism, and is inoculated in and contains in the antibiotic nutrient solution.First microorganism is to be chosen as a bacillus coli DH 5 alpha (E.coli DH5 α), nutrient solution is to be chosen as one to contain antibiotic Luria-Bertani broth nutrient solution, microbiotic is to be chosen as an Ampicillin Trihydrate (ampicillin), and the concentration of Ampicillin Trihydrate is to be chosen as 100 μ g/mL.Then, select after cultivating 16 hours under 37 ℃, be preferably selection with an a small amount of plastid purifying cover group (mini-M
TMPlasmid DNAExtraction System, Viogene, Taiwan) carry out the plastid purifying, with the reorganization plastid behind the acquisition purifying.After reorganization plastid after utilizing two restriction enzymes to purifying again cuts off, the promptly recyclable dna fragmentation that contains the gene of xylanase.Two restriction enzymes are to be chosen as a BamHI and a NotI.After one first plastid utilizes two restriction enzymes to cut off, again the dna fragmentation that makes the gene that contains this xylanase via a ligase enzyme (ligase) with cut off after first plastid carry out a bonding reaction (ligation), contain the first reorganization plastid of the gene of xylanase with formation.First plastid is to be chosen as a pET21C (Novagen, USA), and ligase enzyme is to be chosen as a T4 ligase enzyme (T4 ligase, Roche, Germany).So, can finish the operation of pET system.Again the first reorganization plastid is changeed shape and go into to be competent in the cell, and confirm dna fragmentation with certain sequential mode, competent cell is to be chosen as bacillus coli DH 5 alpha, so just finishes first step S1.
Please consult shown in Figure 1 again, the second step S 2 of embodiments of the invention 1 be with first reorganization plastid the mixings dNTP, buffered soln, forward desire sudden change end introduction, oppositely the desire sudden change holds introduction and polysaccharase to carry out polymerase chain reaction, the second reorganization plastid that contains the mutator gene of highly active xylanase with formation changes shape with the second reorganization plastid again and goes into to be competent in the cell to form mutant expression vector.More in detail, select in the thin-walled centrifuge tube of one 200 μ L to add the first reorganization plastid, dNTP, buffered soln, forward desire sudden change end introduction, oppositely desire sudden change end introduction and polysaccharase.The add-on of the first reorganization plastid is to be 50ng, dNTP is by a dATP, one dTTP, one dCTP and a dGTP form, and dATP, dTTP, the concentration of dCTP and dGTP all is to be chosen as 360 μ M, buffered soln is the 10X reaction buffer that is chosen as 5 μ L, the ultimate density that forward reaches reverse desire sudden change end introduction is to be chosen as 300nM, and forward the gene order of desire sudden change end introduction (number: 1 by recognition sequence, SEQ ID NO:1) and the gene order of reverse desire sudden change end introduction (recognition sequence numbering: 2, S EQ ID NO:2) as shown in Table 1, to indicate the position that bottom line is arranged be to be the sudden change position to the gene order that wherein forward reaches reverse desire sudden change end introduction.Polysaccharase is the Expand long template DNA polymerase (Roche, Germany) that is chosen as 0.75 μ L (3.75units).At last, select to add a distilled water to adjust cumulative volume to 50 μ L.Through of short duration centrifugal after, polysaccharase is put into a polymerase chain reaction machine, the polymerase chain reaction machine is to be chosen as Applied Biosystems, 2700, USA.
Table one, forward reach the gene order of reverse desire sudden change end introduction
| Forward desire sudden change end introduction recognition sequence is numbered: 1 | 5′CTGGTTAGAT GACACCGGTGGTAGC 3′ |
| Oppositely desire sudden change end introduction recognition sequence is numbered: 2 | 5′GCTACCACCGGT GTCATCTAACCAG 3′ |
Please consult Figure 1 and Figure 2 again, polymerase chain reaction is earlier to make two stocks of gene of xylanase from (denature) with a high temperature, forward reach reverse desire sudden change end introduction will with the gene slow cooling pairing (annealing) of the xylanase of sub-thread, forward reach reverse desire sudden change and hold introduction to define the segmental two ends of a desire repetition DNA separately, synthesize this desire repetition DNA fragment (extension) via polysaccharase from forward reaching the extension of reverse desire sudden change end introduction again.Therefore, the polymerase chain reaction machine at first is to select to be set at 95 ℃, 3 minutes; Again with 95 ℃, 45 seconds (denature); 55 ℃, 1 minute (annealing); 68 ℃, 9 minutes (extension) as 1 circulation, repeat to finish 20 circulations after, then be 55 ℃, 1 minute; 68 ℃, 15 minutes, lower the temperature at last in 4 ℃ to form a polymerase chain reaction product, so promptly finish polymerase chain reaction.Just can form the second reorganization plastid of the mutator gene that contains this highly active xylanase via polymerase chain reaction.Then take out polymerase chain reaction product 20 μ L and add a Restriction enzyme to cut in the polymerase chain reaction product without the first reorganization plastid that suddenlys change.Restriction enzyme is the DpnI that is chosen as 1 μ L, and selects in effect under 37 ℃ to select to react 10 minutes down in 65 ℃ after 1 hour again, with second reorganization plastid commentaries on classics shape to the first microorganism, to form mutant expression vector.Mutant expression vector is incubated at contains in the antibiotic nutrient solution, select to select 3 at last and change the bacterium colonies that form merits, and confirm the second reorganization plastid, so just finish the second step S2 in the sequencing mode to screen.
Respectively has the sudden change position owing to forward reach reverse desire sudden change end introduction, therefore duplicate via polymerase chain reaction, in process, just can produce the second reorganization plastid of the mutator gene that contains highly active xylanase, so just can make the 58th amino acid of the gene of xylanase sport acid, aspartic, to form the mutator gene of highly active xylanase by asparagine.The gene order of the mutator gene of xylanase is the sequence that contains as shown in Figure 2 (recognition sequence numbering: 3, SEQ ID NO:3).Be the 58th amino acid of the mutator gene of xylanase among Fig. 2 with the zone of frame wire tag, it is an acid, aspartic.
The present invention is a difference of verifying the mutator gene of the gene of xylanase and highly active xylanase, present embodiment is produced a wild-type xylanase and a mutant xylanase respectively via the gene of xylanase and the mutator gene of highly active xylanase in addition, go forward side by side its enzyme activity of pacing amount and ferment optimal reaction pH-value have the effect of the enzyme activity that improves xylanase really with the mutator gene of verifying highly active xylanase of the present invention.
See also Fig. 3 and shown in Figure 4, at first extract the reorganization of first in wild-type expression vector plastid out, and change shape and go into a growing carrier that contains the gene of xylanase in one second microorganism with formation, second microorganism is to be chosen as an e. coli bl21 (DE3).
Then, growing carrier is selected to be inoculated in 5mL contain in the antibiotic nutrient solution, forming a bacterium liquid, and select under 37 ℃ and 225rpm condition concussion to cultivate 16 hours.In bacterium liquid cultivate finish after, the bacterium liquid of selecting again every 5mL is inoculated in the antibiotic nutrient solution of containing of 500mL, and selects to cultivate with concussion under 37 ℃ and the 180rpm condition, as the OD of bacterium liquid
600When value reaches 0.6-0.8, select to add an IPTG (isopropyl-β-D-thiogalactoside) in substratum with as an inductor, the ultimate density of IPTG is to be chosen as 1mM.After IPTG induces 4 hours, just can generate the wild-type xylanase, then select after centrifugal 20 minutes, the thalline Shen in the bacterium liquid to be fallen with the condition of 4000g.With microorganism collection and select back to be dissolved in the citric acid solution (citric acid buffer), citric acid solution be for pH6 and its concentration be 50mM.Then select to add a PMSF (phenylmethylsulfonyl fluoride) and a leupeptin as a proteinase inhibitor, formed wild-type xylanase is decomposed to avoid the proteolytic enzyme in second microorganism.The ultimate density of PMSF is to be chosen as 0.5mM, and the ultimate density of leupeptin is to be chosen as 1ug/mL.Then, just form a thick enzyme liquid after utilizing ultrasound with the broken bacterium of thalline, select under the condition of 10000g centrifugal 30 minutes with this thick enzyme liquid of separation.Further thick enzyme liquid is selected to carry out purifying with a CM positively charged ion tubing string (CM-Sepharose column) and a Ni-NTA affinity tubing string in regular turn, to obtain the wild-type xylanase behind the purifying, at last the wild-type xylanase behind the purifying is dialysed, removing unnecessary salt, and the displacement citric acid solution.So just can obtain this wild-type xylanase to carry out follow-up test.
The preparation method of mutant xylanase is identical with the preparation method of above-mentioned wild-type xylanase, its difference only is " extracting the first reorganization plastid in the wild-type expression vector out " changed into " extracting the second reorganization plastid in the mutant expression vector out ", can obtain the poly-wooden candy of mutant after the aforesaid way operation.
Please consult Fig. 3 and shown in Figure 4 again, wild-type and mutant xylanase are done further to analyze with SDS-PAGE, to confirm the purified state and the molecular weight of wild-type and mutant xylanase.Wherein 1a and 1b road are the molecular weight markers for a standard protein; The 2a road is the formed thick enzyme liquid of the first reorganization plastid; The 3a road is through the wild-type xylanase behind the CM positively charged ion tubing string purifying; The 4a road is the wild-type xylanase through this Ni-NTA tubing string purifying; The 2b road is the formed thick enzyme liquid of this second reorganization plastid; The 3b road is through the mutant xylanase behind this CM positively charged ion tubing string purifying; The 4b road is the mutant xylanase through this Ni-NTA tubing string purifying.Can be learnt by 4a and 4b road: the molecular weight of wild-type and mutant xylanase all is about 34KDa.
See also shown in the table two, measure wild-type and mutant xylanase respectively in the enzyme activity of different purification phase, with enzyme activity difference more between the two.
At first, the wild-type xylanase of getting 5ng adds one to be subjected in the matter liquid, being subjected to matter liquid is a damping fluid of poly-wooden candy (soluble oatspelt xylan) of selecting to contain the solubility reorganization wheat of 20mg/mL, and this damping fluid is the citric acid solution that is chosen as 50mM, and its pH value is 6.5.After mixing, select to be placed in 50 ℃, reacted 10 minutes, make the wild-type xylanase decompose the poly-wooden candy that is subjected in the matter liquid.
Then, utilize a DNS (dinitrosalicylic acid) standard measure reduction to be subjected to the amount of remaining poly-wooden candy in the matter liquid, further to try to achieve enzyme activity (U/mg).Per unit activity (U) but be the activity of the matrix of per minute catalysis 1 μ mole.The concentration of wild-type xylanase is to select to carry out quantitatively with a BCA quantification of protein cover group (PierceLtd.USA).The working method of the enzyme activity test of mutant xylanase is identical with the working method of the enzyme activity test of above-mentioned wild-type xylanase.Can learn: the enzyme activity of the mutant xylanase behind the purifying is about 2.4 times of enzyme activity of the wild-type xylanase behind the purifying.So, can verify that the mutator gene of highly active xylanase of the present invention has the effect of lifting enzyme activity really.
The enzyme activity of table two, this wild-type and mutant xylanase
| Purification phase | The enzyme activity of wild-type xylanase (U/mg) | The enzyme activity of mutant xylanase (U/mg) |
| Thick enzyme liquid | 2568.27 | 11264.39 |
| CM positively charged ion tubing string | 14568.65 | 45396.16 |
| Ni-NTA affinity tubing string | 23244.85 | 57496.61 |
See also shown in Figure 5ly, change, therefore wild-type and mutant xylanase are done a ferment optimal reaction pH-value and test for understanding wild-type and the mutant xylanase relative enzyme activity in different pH-value environment under.Selection adds being subjected in the matter liquid of 295 μ L with the wild-type xylanase of 5ng, and being subjected to matter liquid is the damping fluid of poly-wooden candy that contains the solubility reorganization wheat of 20mg/mL.Buffered soln is to may be selected to be a glycine damping fluid (glycine buffer), and the pH value is between in 2 to 3.5; Damping fluid is to be chosen as citrate buffer solution, and the pH value is between in 3 to 6.5; Damping fluid is to be chosen as monophosphate damping fluid (phosphate buffer), and the pH value is between in 6 to 7.5; Damping fluid is to be chosen as a tris buffer (Trisbuffer), and the pH value is between in 7 to 10; Damping fluid is to be chosen as a CAPS damping fluid, and the pH value is between in 10 to 11.After mixing, the wild-type xylanase is selected respectively to react 10 minutes down in a proper temperature, measures the amount that reduction is subjected to remaining poly-wooden candy in the matter liquid with the DNS method again, and the estimation enzyme activity.Wherein, the test of ferment optimal reaction pH-value is to be benchmark with pH6.5, with the relative enzyme activity of further other pH-value of estimation.The working method of the ferment optimal reaction pH-value test of mutant xylanase is identical with the wild-type xylanase.No matter can learn the mutant xylanase in sour environment or alkaline environment, its relative enzyme activity is all than the relative enzyme activity height of wild-type xylanase.Can verify that so the present invention has the effect of the enzyme activity that improves xylanase really.
See also Fig. 2 and shown in Figure 6, the gene order of the mutator gene of the xylanase of embodiments of the invention 2 is the sequences that contain as shown in Figure 6 (recognition sequence numbering: 4, SEQ ID NO:4).Zone with the frame wire tag among Fig. 6 is the position of undergoing mutation, and it is the recognition sequence numbering: 4 the 38th amino acid, and be acid, aspartic.Wherein, because the recognition sequence numbering: 4 are the recognition sequence numbering: the 21st to 200 formed gene order of amino acid in 3.And then can understand, though number with respect to recognition sequence the position of sudden change: 3 and recognition sequence numbering: 4 position difference, however the amino acid whose position of sudden change is that essence is identical.Also therefore can find to contain just like recognition sequence and number: the mutator gene of the xylanase of 4 gene order also has the effect of the enzyme activity that improves xylanase.
Therefore, can learn that utilizing point mutation process of the present invention is at least one amino acid in the gene of a ferment can be sported acid, aspartic by asparagine, with the mutator gene of formation ferment, and the enzyme activity of raising ferment.Ferment is to may be selected to be a redox ferment, a transferase, a hydrolytic enzyme, a steatolysis ferment, an isomery ferment or a NOS.Hydrolytic enzyme is to may be selected to be xylanase.
In addition, the mutator gene of highly active xylanase of the present invention also can be integrated in via gene recombination technology and form a recombinant chou in a plastid or the karyomit(e), to use as further; Also recombinant chou can be handled entering in the cell via genetically engineered, to use as further.
As mentioned above, compared to traditional xylanase, because traditional xylanase is active limited, consequently the usage quantity of traditional xylanase is bigger, and then causes a large amount of uses and the higher shortcoming of cost.Moreover the conventional point mutation method can't promote the enzyme activity of xylanase.
Review, the present invention utilizes a point mutation process that the 58th amino acid of wild-type xylanase gene is sported acid, aspartic by asparagine, to form high reactivity xylanase mutator gene.By this, the present invention can effectively improve enzyme activity really, and reduces use cost.
Claims (12)
1, a kind of mutator gene of highly active xylanase is characterized in that: the 58th amino acid of the mutator gene of this xylanase is to be acid, aspartic.
2, the mutator gene of highly active xylanase according to claim 1 is characterized in that: the gene order of the mutator gene of this highly active xylanase is to contain the recognition sequence numbering: 3 full sequence.
3, a kind of mutator gene of highly active xylanase is characterized in that: the 38th amino acid of the mutator gene of this xylanase is to be acid, aspartic, and the gene order of the mutator gene of this xylanase is the recognition sequence numbering: shown in 4.
4, according to the mutator gene of claim 1, one of them described highly active xylanase of 2 or 3, it is characterized in that: the gene of this xylanase is to utilize a carrier to produce, to form one first reorganization plastid.
5, the mutator gene of highly active xylanase according to claim 4 is characterized in that: this carrier is a pET system.
6, the mutator gene of highly active xylanase according to claim 4, it is characterized in that: this first reorganization plastid mix a dNTP, buffered soln, one forward desire sudden change end introduction, a reverse desire sudden change end introduction and a polysaccharase carry out this polymerase chain reaction, contain one second reorganization plastid of the mutator gene of this highly active xylanase with formation.
7, the mutator gene of highly active xylanase according to claim 6, it is characterized in that: this forward desire sudden change end introduction be to have the recognition sequence numbering: 1 gene order, and this reverse desire sudden change end introduction is to have the recognition sequence numbering: 2 gene order, wherein
The recognition sequence numbering: 1 is 5 ' CTGGTTAGAT
GACACCGGTGGTAGC 3 '
The recognition sequence numbering: 2 is 5 ' GCTACCACCGGT
GTCATCTAACCAG 3 '.
8, according to the mutator gene of claim 1, one of them described highly active xylanase of 2 or 3, it is characterized in that: the mutator gene of this highly active xylanase is to be integrated in a plastid or the karyomit(e) via gene recombination technology.
9, the mutator gene of highly active xylanase according to claim 1, it is characterized in that: the gene of this xylanase is a hybrid bacterial strain that separates from a rumen microorganism, and the gene order of the gene of this xylanase is to login in gene database, and the login number is AY941119.
10, a kind of point mutation process of mutator gene of highly active xylanase is characterized in that: be that at least one amino acid in the gene of a ferment is sported acid, aspartic by asparagine, to form a mutator gene of this ferment.
11, the point mutation process of the mutator gene of highly active xylanase according to claim 10 is characterized in that: this ferment is an xylanase, and this amino acid is the 58th amino acid of the gene of this xylanase.
12, the point mutation process of the mutator gene of highly active xylanase according to claim 11 is characterized in that: the gene order of the mutator gene of this highly active xylanase is to contain the recognition sequence numbering: 3 sequence.
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| CN101289668B (en) * | 2007-04-16 | 2010-09-08 | 屏东科技大学 | Mutant gene of xylanase in high acid-base reaction range and point mutation process thereof |
| CN109355272A (en) * | 2018-12-28 | 2019-02-19 | 江南大学 | A kind of xylanase mutant that catalytic efficiency improves |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101289668B (en) * | 2007-04-16 | 2010-09-08 | 屏东科技大学 | Mutant gene of xylanase in high acid-base reaction range and point mutation process thereof |
| CN109355272A (en) * | 2018-12-28 | 2019-02-19 | 江南大学 | A kind of xylanase mutant that catalytic efficiency improves |
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