CN110669748A - Beta-mannase mutant with improved heat resistance and specific activity and coding gene thereof - Google Patents
Beta-mannase mutant with improved heat resistance and specific activity and coding gene thereof Download PDFInfo
- Publication number
- CN110669748A CN110669748A CN201810711310.2A CN201810711310A CN110669748A CN 110669748 A CN110669748 A CN 110669748A CN 201810711310 A CN201810711310 A CN 201810711310A CN 110669748 A CN110669748 A CN 110669748A
- Authority
- CN
- China
- Prior art keywords
- gly
- mutant
- asn
- ala
- lys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the field of biotechnology and enzyme engineering, and particularly discloses a beta-mannase mutant Man5AS11R with improved heat resistance and specific activity and a coding gene thereof. The mutant Man5AS can be obtained by carrying out double mutation of K16C and D296C on beta-mannase Man5A with an amino acid sequence shown as SEQ ID NO.1, and the heat resistance of the mutant is improved by 5 compared with that of Man5AoC, i.e. Man5AS at 80oC can retain more than 80% of enzyme activity after being kept for 3min at high temperature, while the Man5A can only be kept at 75%oKeeping the enzyme activity for 3min and maintaining 80 percent of the enzyme activity; on the basis, the simultaneous mutation of three points R21V, I80D and I218E is continuously carried out on the Man5AS amino acid sequence shown AS SEQ ID NO.2 to obtain the mutant Man5AS11R with the amino acid sequence shown AS SEQ ID NO.4, and the specific activity of the mutant Man5AS11 is improved by 22.5% and 31.4% respectively compared with Man5A and Man5AS on the premise that the heat resistance of the mutant Man5AS11 is not changed compared with Man5 AS. Showing better development and application prospect.
Description
Technical Field
The invention belongs to the field of biotechnology and enzyme engineering, and relates to a beta-mannase mutant with improved heat resistance and specific activity and a coding gene thereof.
Background
At present, beta-mannase is one of 12 feed-grade enzyme preparations approved by China and widely applied to the feed industry as an efficient feed additive. Since the 90 s of the last century, 90% of poultry feed and 60% of pig diet are added with enzyme preparations in European and American countries. China starts late, but the research is always the key point of feed production and application research, and in recent years, mannase is more and more regarded as an important enzyme for feed. The mannase is added into the feed for chickens (poultry), so that the growth of the chickens can be effectively improved, the laying rate of the layers can be improved, the utilization rate of the feed can be improved, the excretion of excrement of the chickens can be reduced, and the environmental stress can be relieved. The mannase is applied to the normal feed of pigs, can enhance the disease resistance of the pigs, reduce the application of antibiotics and the like, and is beneficial to producing high-quality pork and series meat products. However, most mannanases in the current market are not ideal in heat resistance, enzyme activity and the like, and become a bottleneck for application of mannanases in the feed industry, and a solution is needed. In addition, the beta-mannase in the domestic and foreign markets has low enzyme activity, small yield and high relative price of products, so that the application range of the beta-mannase is greatly reduced.
Disclosure of Invention
The invention aims to provide a heat-resistant acidic beta-mannase mutant Man5AS11R with improved heat resistance and specific activity, and has better application potential in the field of feed.
Specifically, the invention aims at the heat-resistant acidic beta-mannase Man5A developed earlier to carry out necessary enzymological characteristic improvement, the Man5A is most suitable for reaction at the pH value of 3, and has better stability when the pH value is in the range of 2.5-7.0, and better enzyme activity can be maintained in animal gastric juice (pH 2.5) and intestinal tract (pH 5.5). The optimum action temperature is 60oC, at 75oAnd C, the enzyme activity can be kept 80% at high temperature for 3 min. However, the heat resistance and enzyme activity of the feed additive are still to be further improved.
In order to improve the heat resistance of Man5A, the invention carries out modeling analysis on the spatial structure of the enzyme, improves the heat resistance of the enzyme by introducing a pair of intramolecular disulfide bonds, carries out double-point mutation of K16C and D296C on the enzyme, and obtains a mutant Man5AS with the mutation rate of 80oThe enzyme activity can be maintained at 80% at high temperature for 3min, and the product is more resistant than wild typeHigh temperature degree is improved by 5oAnd C, the amino acid sequence of which is shown as SEQ ID NO. 2.
On the basis of the Man5AS mutant, aiming at the computational analysis of a spatial structure of Man5AS, the invention locates 4 key amino acid sites near a catalytic activity center to carry out site-specific saturation mutation, designs a fluorescent substrate for achieving the purpose of screening the mutant at high flux, screens a plurality of mutants with improved specific activity in a short time by applying advanced technologies and equipment such AS droplet microfluidics and high flux automatic screening, wherein the highest specific activity of Man5AS11R is improved by 22.5 percent compared with that of Man5A, three-point mutation with mutation sites of R21V, I80D and I218E is found by sequencing, and site-specific mutation verification is carried out to find that any one point or any two points of R21V, I80D and I218E can not achieve the purpose of improving the specific activity of the enzyme, and the amino acid sequence of the mutant is shown AS SEQ ID No. 4.
In another aspect, the invention also provides nucleic acid sequences SEQ ID NO.3 and SEQ ID NO.5 encoding sequences SEQ ID NO.2 and SEQ ID NO.4, respectively.
On the other hand, the recombinant expression vector used in the present invention is a pET series expression vector including pET22b, pET26b, pET32a and the like, and the expression host bacterium used isE.coliBL21(DE3)。
Drawings
FIG. 1 optimal pH for recombinant beta-mannanase mutants
FIG. 2 pH stability of recombinant beta-mannanase mutants
FIG. 3 optimal temperature for recombinant beta-mannanase mutants
FIG. 4 temperature stability of recombinant beta-mannanase mutants
FIG. 5 specific activity of recombinant β -mannanase mutants.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1Optimum action pH and pH stability of heat-resistant acidic beta-mannase mutant
The optimal action pH of the mutants of Man5AS (K16C and D296C), Man5A11R (R21V, I80D and I218E) and Man5AS11R (K16C, R21V, I80D, I218E and D296C) is respectively determined and analyzed by comparison with the wild-type thermotolerant acidic beta-mannanase Man5A, and AS a result, AS shown in a figure 1, the optimal action pH of each mutant is 3, the relative enzyme activity change trend between pH2.5 and 7.5 is consistent with that of Man5A, and the mutation sites of each mutant can not change the optimal action pH of the enzyme.
Compared with wild-type thermostable beta-mannase Man5A, mutants of Man5AS (K16C and D296C), Man5A11R (R21V, I80D and I218E) and Man5AS11R (K16C, R21V, I80D, I218E and D296C) are respectively tested and analyzed to keep the pH stability for 1h under the condition of pH2.5-7, and AS a result, AS shown in FIG. 2, the change trend of the residual enzyme activity of each mutant enzyme is consistent with that of Man5A, and each mutant can keep the acid resistance of the wild-type enzyme and the stability of wide-range pH.
Example 2Optimum action temperature and temperature stability of heat-resistant acidic beta-mannase mutant
The control wild-type hot-acid-resistant beta-mannanase Man5A was subjected to assay for the optimal action temperature of its mutants Man5AS (K16C and D296C), Man5A11R (R21V, I80D and I218E) and Man5AS11R (K16C, R21V, I80D, I218E and D296C), respectively, and the results are shown in FIG. 3, wherein the optimal action temperature of each mutant is 60oC, wherein mutants Man5AS and Man5AS11R are at 80oThe relative enzyme activity of C is improved, while the mutant Man5A11R is consistent with Man5A, so that the introduction of intramolecular disulfide bond can improve the relative enzyme activity of the enzyme under high temperature.
Mutants of Man5AS (K16C and D296C), Man5A11R (R21V, I80D and I218E) and Man5AS11R (K16C, R21) were assayed against the wild-type thermotolerant acidic beta-mannanase Man5A, respectivelyV, I80D, I218E and D296C) at 40-80oThe residual enzyme activity after incubation for 3min under the condition C is shown in FIG. 4, and the results are shown in FIG. 4, where the mutant enzymes Man5AS and Man5AS11R are at 80oAnd C, the residual enzyme activity of more than 80 percent can be maintained after the heat preservation for 3min, while the residual enzyme activity of 55 percent and 60 percent can be maintained by Man5A11R and Man5A respectively, so that the stability of the enzyme under the high-temperature condition is obviously improved by introducing intramolecular disulfide bonds.
Example 3Specific activity improvement of thermostable acidic beta-mannase mutant
According to the invention, three point mutations of R21V, I80D and I218E are introduced on the basis of a mutant enzyme Man5AS to further improve the specific activity of the mutant enzyme, and simultaneously, in order to investigate the influence of the three point mutations on the enzyme activity, the specific activity of each mutant is obtained through fixed point mutation AS shown in Table 1, the specific activity change of each mutant is shown in figure 5, compared with a wild enzyme Man5A, the specific activities of Man5A11R and Man5AS11R in the mutant are respectively improved by 25.6% and 22.5%, but AS shown in figures 3 and 4, the heat resistance of Man5A11R is not obviously improved, and the heat resistance and the specific activity of Man5AS11R are improved. Meanwhile, the invention also discovers that on the basis of Man5AS, only one or two mutation sites in Man5A11R are mutated, the specific activity of the enzyme is not improved, and the three sites have synergistic effect on the aspect of improving the specific activity of the enzyme.
TABLE 1 Man5A respective mutant enzymes and mutation sites
The foregoing description is intended to be illustrative rather than limiting, and it will be apparent to those of ordinary skill in the art that numerous modifications, variations or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> beta-mannase mutant with improved heat resistance and specific activity and coding gene thereof
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>405
<212>PRT
<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
<400>1
Thr Gly Phe Tyr Val Asn Gly Gly Lys Leu Tyr Asp Ser Thr Gly Lys
1 5 10 15
Pro Phe Tyr Ile Arg Gly Ile Asn His Gly His Ser Trp Phe Lys Asn
20 25 30
Asp Thr Ala Thr Ala Ile Pro Ala Ile Ala Lys Thr Gly Ala Asn Thr
35 40 45
Val Arg Ile Val Leu Ser Asn Gly Thr Gln Tyr Thr Lys Asp Asp Leu
50 55 60
Asn Ser Val Lys Asn Ile Ile Asn Leu Ala Glu Glu Asn Lys Ile Ile
65 70 75 80
Ala Val Leu Glu Val His Asp Ala Thr Gly Lys Asp Asp Phe Asn Ser
85 90 95
Leu Asp Ala Ala Val Asn Tyr Trp Ile Ser Ile Lys Glu Ala Leu Ile
100 105 110
Gly Lys Glu Asp Arg Val Ile Val Asn Ile Ala Asn Glu Trp Tyr Gly
115 120 125
Thr Trp Asn Gly Ser Ala Trp Ala Asp Gly Tyr Lys Lys Ala Ile Pro
130 135 140
Lys Leu Arg Asp Ala Gly Ile Lys Asn Thr Leu Ile Val Asp Ala Ala
145 150 155 160
Gly Trp Gly Gln Tyr Pro Gln Ser Ile Val Asp Tyr Gly Gln Ser Val
165 170 175
Phe Ala Ala Asp Ser Gln Lys Asn Thr Ala Phe Ser Ile His Ile Tyr
180 185 190
Glu Tyr Ala Gly Lys Asp Ala Ala Thr Val Lys Ser Asn Ile Glu Asn
195 200 205
Val Leu Asn Lys Gly Leu Ala Leu Ile Ile Gly Glu Phe Gly Gly Tyr
210 215 220
His Thr Asn GlyAsp Val Asp Glu Tyr Ala Ile Ile Lys Tyr Gly Leu
225 230 235 240
Glu Lys Gly Val Gly Trp Leu Ala Trp Ser Trp Tyr Gly Asn Gly Ile
245 250 255
Lys Trp Asn Tyr Leu Asp Leu Ala Thr Gly Pro Asn Gly Ser Leu Thr
260 265 270
Ser Tyr Gly Asn Thr Val Val Asn Asp Thr Tyr Gly Ile Lys Asn Thr
275 280 285
Ser Gln Lys Ala Gly Ile Phe Asp Gly Asp Asp Gly Val Gly Asp Gly
290 295 300
Gly Pro Gly Asp Ser Asn Gly Thr Lys Thr Thr Leu Tyr Asn Phe Glu
305 310 315 320
Thr Gly Thr Glu Gly Trp Ser Gly Lys Asn Ile Glu Thr Gly Pro Trp
325 330 335
Ser Val Asn Glu Trp Ala Ala Lys Gly Asn His Ser Leu Lys Ala Asp
340 345 350
Val Asn Leu Gly Asp Asn Ser Glu His Tyr Leu Lys Leu Thr Gln Asn
355 360 365
Leu Asn Phe Ser Gly Lys Ser Gln Leu Thr Ala Thr Val Lys His Ala
370 375 380
Asp Trp Gly Asn Phe GlyAsp Glu Ile Asn Ala Lys Leu Tyr Val Lys
385 390 395 400
Thr Glu Ser Asp Trp
405
<210>2
<211>405
<212>PRT
<213>Artificial sequence
<400>2
Thr Gly Phe Tyr Val Asn Gly Gly Lys Leu Tyr Asp Ser Thr Gly Cys
1 5 10 15
Pro Phe Tyr Ile Arg Gly Ile Asn His Gly His Ser Trp Phe Lys Asn
20 25 30
Asp Thr Ala Thr Ala Ile Pro Ala Ile Ala Lys Thr Gly Ala Asn Thr
35 40 45
Val Arg Ile Val Leu Ser Asn Gly Thr Gln Tyr Thr Lys Asp Asp Leu
50 55 60
Asn Ser Val Lys Asn Ile Ile Asn Leu Ala Glu Glu Asn Lys Ile Ile
65 70 75 80
Ala Val Leu Glu Val His Asp Ala Thr Gly Lys Asp Asp Phe Asn Ser
85 90 95
Leu Asp Ala Ala Val Asn Tyr Trp Ile Ser Ile Lys Glu Ala Leu Ile
100 105 110
Gly LysGlu Asp Arg Val Ile Val Asn Ile Ala Asn Glu Trp Tyr Gly
115 120 125
Thr Trp Asn Gly Ser Ala Trp Ala Asp Gly Tyr Lys Lys Ala Ile Pro
130 135 140
Lys Leu Arg Asp Ala Gly Ile Lys Asn Thr Leu Ile Val Asp Ala Ala
145 150 155 160
Gly Trp Gly Gln Tyr Pro Gln Ser Ile Val Asp Tyr Gly Gln Ser Val
165 170 175
Phe Ala Ala Asp Ser Gln Lys Asn Thr Ala Phe Ser Ile His Ile Tyr
180 185 190
Glu Tyr Ala Gly Lys Asp Ala Ala Thr Val Lys Ser Asn Ile Glu Asn
195 200 205
Val Leu Asn Lys Gly Leu Ala Leu Ile Ile Gly Glu Phe Gly Gly Tyr
210 215 220
His Thr Asn Gly Asp Val Asp Glu Tyr Ala Ile Ile Lys Tyr Gly Leu
225 230 235 240
Glu Lys Gly Val Gly Trp Leu Ala Trp Ser Trp Tyr Gly Asn Gly Ile
245 250 255
Lys Trp Asn Tyr Leu Asp Leu Ala Thr Gly Pro Asn Gly Ser Leu Thr
260 265 270
Ser Tyr Gly AsnThr Val Val Asn Asp Thr Tyr Gly Ile Lys Asn Thr
275 280 285
Ser Gln Lys Ala Gly Ile Phe Cys Gly Asp Asp Gly Val Gly Asp Gly
290 295 300
Gly Pro Gly Asp Ser Asn Gly Thr Lys Thr Thr Leu Tyr Asn Phe Glu
305 310 315 320
Thr Gly Thr Glu Gly Trp Ser Gly Lys Asn Ile Glu Thr Gly Pro Trp
325 330 335
Ser Val Asn Glu Trp Ala Ala Lys Gly Asn His Ser Leu Lys Ala Asp
340 345 350
Val Asn Leu Gly Asp Asn Ser Glu His Tyr Leu Lys Leu Thr Gln Asn
355 360 365
Leu Asn Phe Ser Gly Lys Ser Gln Leu Thr Ala Thr Val Lys His Ala
370 375 380
Asp Trp Gly Asn Phe Gly Asp Glu Ile Asn Ala Lys Leu Tyr Val Lys
385 390 395 400
Thr Glu Ser Asp Trp
405
<210>3
<211>1218
<212>DNA
<213>Artificial Sequence
<400>3
acaggtttct acgtgaatgg aggcaaattg tacgattcta cgggttgtcc attttacata 60
aggggtatca atcatgggca ctcctggttt aagaatgata cagcaacggc tatccctgcg 120
atcgcaaaga cgggtgccaa tacggtacga attgtattat caaacggtac acaatacacc 180
aaggatgatc tgaattccgt aaaaaacatc attaatttgg cagaagaaaa caagattatt 240
gctgtgcttg aagtacacga tgccactggg aaagatgact tcaactcgtt ggatgcagcg 300
gtcaactact ggataagcat caaagaagca ctgatcggga aggaagatcg ggttattgta 360
aacattgcaa acgagtggta cggaacatgg aacggaagcg cgtgggctga cgggtacaag 420
aaggctattc cgaaattaag agatgcgggt attaagaata ccttgattgt agatgcagca 480
ggctggggtc agtaccctca atcgatcgtc gattacggac aaagcgtatt cgccgcggat 540
tcacagaaga atacggcgtt ttccattcac atttatgagt atgcaggcaa ggatgcggcc 600
accgtcaaat ccaatatcga aaatgtgctg aataaagggc tggccttaat cattggtgag 660
ttcggaggat atcacaccaa tggagatgtc gatgaatatg caatcatcaa atatggtctg 720
gagaaaggag taggatggct tgcatggtct tggtacggta atggtatcaa atggaactat 780
cttgatttgg caacaggacc taacggcagt ttgacgagct atggtaatac ggttgtcaat 840
gatacttacg gaattaaaaa tacgtcccag aaagcgggaa tcttttgtgg agatgatggt 900
gtcggtgatg gtggacccgg tgatagcaat ggtacaaaaa cgacgttgta caattttgaa 960
acgggaacgg aaggatggtc tggaaaaaat atcgaaacgg gaccctggag cgtcaatgaa 1020
tgggcagcaa agggtaatca tagcttgaag gcagatgtca atttgggtga taattctgaa 1080
cattacttga aattgacaca aaatttgaat tttagcggaa agtctcaatt gacggcaaca 1140
gtcaagcacg cagattgggg aaattttgga gatgaaatca atgcaaagtt gtacgtgaag 1200
acagaaagcg attggtaa 1218
<210>4
<211>405
<212>PRT
<213>Artificial sequence
<400>4
Thr Gly Phe Tyr Val Asn Gly Gly Lys Leu Tyr Asp Ser Thr Gly Cys
1 5 10 15
Pro Phe Tyr Ile Val Gly Ile Asn His Gly His Ser Trp Phe Lys Asn
20 25 30
Asp Thr Ala Thr Ala Ile Pro Ala Ile Ala Lys Thr Gly Ala Asn Thr
35 40 45
Val Arg Ile Val Leu Ser Asn Gly Thr Gln Tyr Thr Lys Asp Asp Leu
50 55 60
Asn Ser Val Lys Asn Ile Ile Asn Leu Ala Glu Glu Asn Lys Ile Asp
65 70 75 80
Ala Val Leu Glu Val His Asp Ala Thr Gly Lys Asp Asp Phe Asn Ser
85 90 95
Leu Asp Ala Ala Val Asn Tyr Trp Ile Ser Ile Lys Glu Ala Leu Ile
100 105 110
Gly Lys Glu Asp Arg Val Ile Val Asn Ile Ala Asn Glu Trp Tyr Gly
115 120 125
Thr Trp Asn Gly Ser Ala Trp Ala Asp Gly Tyr Lys Lys Ala Ile Pro
130 135 140
Lys Leu Arg Asp Ala Gly Ile Lys Asn Thr Leu Ile Val Asp Ala Ala
145 150 155 160
Gly Trp Gly Gln Tyr Pro Gln Ser Ile Val Asp Tyr Gly Gln Ser Val
165 170 175
Phe Ala Ala Asp Ser Gln Lys Asn Thr Ala Phe Ser Ile His Ile Tyr
180 185 190
Glu Tyr Ala Gly Lys Asp Ala Ala Thr Val Lys Ser Asn Ile Glu Asn
195 200 205
Val Leu Asn Lys Gly Leu Ala Leu Ile Glu Gly Glu Phe Gly Gly Tyr
210 215 220
His Thr Asn Gly Asp Val Asp Glu Tyr Ala Ile Ile Lys Tyr Gly Leu
225 230 235 240
Glu Lys Gly Val Gly Trp Leu Ala Trp Ser Trp Tyr Gly Asn Gly Ile
245 250 255
Lys Trp Asn Tyr Leu Asp Leu Ala Thr Gly Pro Asn Gly Ser Leu Thr
260 265 270
Ser Tyr Gly Asn Thr Val Val Asn Asp Thr Tyr Gly Ile Lys Asn Thr
275 280 285
Ser Gln Lys Ala Gly Ile Phe Cys Gly Asp Asp Gly Val Gly Asp Gly
290 295 300
Gly Pro Gly Asp Ser Asn Gly Thr Lys Thr Thr Leu Tyr Asn Phe Glu
305 310 315 320
Thr Gly Thr Glu Gly Trp Ser Gly Lys Asn Ile Glu Thr Gly Pro Trp
325 330 335
Ser Val Asn Glu Trp Ala Ala Lys Gly Asn His Ser Leu Lys Ala Asp
340 345 350
Val Asn Leu Gly Asp Asn Ser Glu His Tyr Leu Lys Leu Thr Gln Asn
355 360 365
Leu Asn Phe Ser Gly Lys Ser Gln Leu Thr Ala Thr Val Lys His Ala
370 375 380
Asp Trp Gly Asn Phe Gly Asp Glu Ile Asn Ala Lys Leu Tyr Val Lys
385 390 395 400
Thr Glu Ser Asp Trp
405
<210>5
<211>1218
<212>DNA
<213>Artificial Sequence
<400>5
acaggtttct acgtgaatgg aggcaaattg tacgattcta cgggttgtcc attttacata 60
gtaggtatca atcatgggca ctcctggttt aagaatgata cagcaacggc tatccctgcg 120
atcgcaaaga cgggtgccaa tacggtacga attgtattat caaacggtac acaatacacc 180
aaggatgatc tgaattccgt aaaaaacatc attaatttgg cagaagaaaa caagattgat 240
gctgtgcttg aagtacacga tgccactggg aaagatgact tcaactcgtt ggatgcagcg 300
gtcaactact ggataagcat caaagaagca ctgatcggga aggaagatcg ggttattgta 360
aacattgcaa acgagtggta cggaacatgg aacggaagcg cgtgggctga cgggtacaag 420
aaggctattc cgaaattaag agatgcgggt attaagaata ccttgattgt agatgcagca 480
ggctggggtc agtaccctca atcgatcgtc gattacggac aaagcgtatt cgccgcggat 540
tcacagaaga atacggcgtt ttccattcac atttatgagt atgcaggcaa ggatgcggcc 600
accgtcaaat ccaatatcga aaatgtgctg aataaagggc tggccttaat cgaaggtgag 660
ttcggaggat atcacaccaa tggagatgtc gatgaatatg caatcatcaa atatggtctg 720
gagaaaggag taggatggct tgcatggtct tggtacggta atggtatcaa atggaactat 780
cttgatttgg caacaggacc taacggcagt ttgacgagct atggtaatac ggttgtcaat 840
gatacttacg gaattaaaaa tacgtcccag aaagcgggaa tcttttgtgg agatgatggt 900
gtcggtgatg gtggacccgg tgatagcaat ggtacaaaaa cgacgttgta caattttgaa 960
acgggaacgg aaggatggtc tggaaaaaat atcgaaacgg gaccctggag cgtcaatgaa 1020
tgggcagcaa agggtaatca tagcttgaag gcagatgtca atttgggtga taattctgaa 1080
cattacttga aattgacaca aaatttgaat tttagcggaa agtctcaatt gacggcaaca 1140
gtcaagcacg cagattgggg aaattttgga gatgaaatca atgcaaagtt gtacgtgaag 1200
acagaaagcg attggtaa 1218
Claims (7)
1. A beta-mannase mutant Man5AS11R with improved heat resistance and specific activity is characterized in that the amino acid sequence is shown AS SEQ ID NO. 4.
2. The amino acid sequence as claimed in claim 1, characterized in that the amino acid sequence shown in SEQ ID No.1 is subjected to five-site mutation of K16C, R21V, I80D, I218E and D296C.
3. A gene encoding the mutant β -mannanase according to claim 1, which is represented by SEQ id No. 5.
4. The beta-mannase mutant Man5AS11R, which is characterized in that the mutant enzyme has improved heat resistance and specific activity of original enzyme Man5A, can keep more than 80% of residual enzyme activity at 80 ℃ for 3min, and has improved specific activity by 22.5% compared with wild-type Man 5A.
5. The mutant enzyme of claim 2, which has only two mutations of K16C and D296C in the mutation site, and is characterized in that the mutant enzyme can keep more than 80% of residual enzyme activity at 80 ℃ for 3min, and the specific activity has no obvious change.
6. The mutant site of claim 2, wherein only R21V, I80D and I218E are subjected to three-point mutation, and the mutant enzyme is characterized in that the heat resistance of the mutant enzyme is not improved, and the specific activity is improved by 25.6 percent compared with that of Man 5A.
7. On the basis of the mutant as claimed in claim 5, mutation at any one or two of R21V, I80D and I218E can not improve the specific activity of the enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810711310.2A CN110669748A (en) | 2018-07-03 | 2018-07-03 | Beta-mannase mutant with improved heat resistance and specific activity and coding gene thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810711310.2A CN110669748A (en) | 2018-07-03 | 2018-07-03 | Beta-mannase mutant with improved heat resistance and specific activity and coding gene thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110669748A true CN110669748A (en) | 2020-01-10 |
Family
ID=69065432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810711310.2A Pending CN110669748A (en) | 2018-07-03 | 2018-07-03 | Beta-mannase mutant with improved heat resistance and specific activity and coding gene thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110669748A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454928A (en) * | 2020-05-18 | 2020-07-28 | 中国海洋大学 | Heat-resistant β -mannase mutant and coding gene and application thereof |
| CN114107258A (en) * | 2021-12-09 | 2022-03-01 | 河北农业大学 | Thermophilic mannase ManBK mutant and application thereof |
| WO2023041040A1 (en) * | 2021-09-18 | 2023-03-23 | 青岛蔚蓝生物集团有限公司 | High temperature resistant mannanase mutant |
-
2018
- 2018-07-03 CN CN201810711310.2A patent/CN110669748A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454928A (en) * | 2020-05-18 | 2020-07-28 | 中国海洋大学 | Heat-resistant β -mannase mutant and coding gene and application thereof |
| CN111454928B (en) * | 2020-05-18 | 2021-04-27 | 中国海洋大学 | Heat-resistant beta-mannase mutant and coding gene and application thereof |
| WO2023041040A1 (en) * | 2021-09-18 | 2023-03-23 | 青岛蔚蓝生物集团有限公司 | High temperature resistant mannanase mutant |
| CN114107258A (en) * | 2021-12-09 | 2022-03-01 | 河北农业大学 | Thermophilic mannase ManBK mutant and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110669748A (en) | Beta-mannase mutant with improved heat resistance and specific activity and coding gene thereof | |
| CN112204136B (en) | Phytase mutants | |
| CN104017791B (en) | Keratinase mutant with improved thermal stability and preparation method thereof | |
| CN104726436B (en) | Keratinase mutant that a kind of substrate specificity improves and preparation method thereof | |
| US10932481B2 (en) | Thermostable protease and methods of making and using the same | |
| CN110042092B (en) | Xylanase mutant with improved stability and coding gene and application thereof | |
| CN105950577A (en) | Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof | |
| CN114517192B (en) | Protease mutant BLAPR1 with improved thermal stability, and encoding gene and application thereof | |
| CN114107266B (en) | Protease mutant with improved heat resistance, encoding gene and application thereof | |
| WO2015070372A1 (en) | A method for producing a phytase variant with improved thermal stability, and a phytase variant and the use thereof | |
| CN107400666A (en) | A kind of aminopeptidase and its encoding gene and application | |
| CN109576249A (en) | A kind of acid-tolerant mutant of low temperature resistant chitinase and its application | |
| CN112877269B (en) | Microorganism producing lysine and method for producing lysine | |
| CN111575265B (en) | Keratinase mutant with improved thermal stability | |
| CN109997970A (en) | The acidic xylan enzyme mutant and its encoding gene and application that a kind of enzyme activity and heat resistance improve | |
| CN112725315B (en) | Application of chitosanase and mutant thereof in preparation of chitosan oligosaccharide | |
| CN112852764B (en) | Mutant protein of ketonizing enzyme and application | |
| CN109943553A (en) | A kind of low temperature resistant mutant of chitinase and its application | |
| CN103898081A (en) | Keratinase mutant and application thereof | |
| WO2016175202A1 (en) | Heat-resistant xylanase | |
| KR100874873B1 (en) | Novel xylanase enzyme and gene encoding the same | |
| CN109517811A (en) | A kind of β -one acyl-ACP synthase mutant | |
| CN110724676B (en) | Phytase mutant and carrier and application thereof | |
| CN117384891B (en) | Acidic xylanase with improved thermal stability, and gene and application thereof | |
| CN114438057B (en) | Heat-resistant alkali-resistant xylanase and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200110 |
|
| WD01 | Invention patent application deemed withdrawn after publication |