CN102021156A - Mutant of cane sugar hydrolytic enzyme and application of mutant - Google Patents

Mutant of cane sugar hydrolytic enzyme and application of mutant Download PDF

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CN102021156A
CN102021156A CN2010105069077A CN201010506907A CN102021156A CN 102021156 A CN102021156 A CN 102021156A CN 2010105069077 A CN2010105069077 A CN 2010105069077A CN 201010506907 A CN201010506907 A CN 201010506907A CN 102021156 A CN102021156 A CN 102021156A
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gly
leu
asn
thr
phe
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CN102021156B (en
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庞浩
裴建新
左文朴
黄日波
黄志民
黎贞崇
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Guangxi Academy of Sciences
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Abstract

The invention relates to a mutant Sinv2 of cane sugar hydrolytic enzyme, which is characterized by comprising an amino acid sequence of SEQ ID NO: 1. Relative to the enzyme before mutation, the mutant enzyme has the characteristics of neutral optimum pH value and higher cane sugar hydrolysis function. The invention also relates to application of the cane sugar hydrolytic enzyme (SEQ ID NO: 1) in the aspect of degrading the cane sugar.

Description

The mutant of sucrose hydrolysis enzyme and application thereof
Technical field
The present invention relates to a kind of sucrose hydrolysis enzyme mutant body, and the application of this mutant enzyme in the sucrose degraded.
Background technology
Sucrose is the reproducible carbohydrate that photosynthesis produces in the plant, annual global output surpassed 500,000,000 tons (this savart of marc Ya Yake, the cooperation of sucrose alcoholic acid mini-bus, Business Watch, 2009,1:72).
Sucrose hydrolysis enzyme (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) is the lytic enzyme that a class becomes sucrose hydrolysis in glucose and fructose.The sucrose hydrolysis enzyme of identifying mainly is from plant and fungi such as yeast and aspergillus (Lammens W. at present, et al, 2008, Crystal structures of Arabidopsis thaliana cell-wall invertase mutants in complex with sucrose.J.Mol.Biol., 377:378-385.).The sucrose hydrolysis enzyme has different forms in organism, be divided into three classes according to the scope difference of optimum pH, acid, neutral, alkalescence (
Figure BSA00000303056600011
L.H.S., et al., Production and characterization of a thermostable extracellular [beta]-d-fructofuranosidase produced by Aspergillus ochraceus with agroindustrial residues as carbon sources.Enzyme and Microbial Technology, 2007.42 (1): 52-57.).On the origin of evolving, the sucrose hydrolysis enzyme can be divided into two classes, and acid sucrose hydrolysis enzyme is a class; Neutral and alkalescence be again a class (Bocock P.N., et al., 2008, Evolution and diversity of invertase genes in Populus trichocarpa.Planta, 227:565-576.).Acid sucrose hydrolysis enzyme is the sucrose hydrolysis enzyme of studying at most at present, wherein the acid sucrose hydrolysis enzyme from yeast saccharomyces cerevisiae is again (Goosen C. of greatest concern, et al., 2007, Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity.Eukaryot.Cell, 6:674-681.De Los Angeles Calixto-Romo M., etal., 2008), Expression, purification and immobilization of the intracellular invertase INVA, from Zymomonas mobilis on crystalline cellulose and Nylon-6.J.Ind.Microbiol.Biotechnol., 35:1455-1463.); And in, alkaline sucrose hydrolysis enzyme is because the difficulty and the zymoprotein instability of purifying, fewer (the Roitsch T. that is studied at present, et al., 2004, Function and regulation of plant invertases:sweet sensations.Trends Plant Sci., 9:606-613.).
In the production of the many products of foodstuffs industry, optimum pH value (the Serna-Saldivar S.R. in the neutral scope normally that is used for the sucrose hydrolysis enzyme of sucrose hydrolysis, et al., 2008, Production of invert syru p from sugarcane juice using immobilized invertase.US patent 7435564.).At present, the whole bag of tricks is being sought to improve the optimum pH of sucrose hydrolysis enzyme by some research groups.People's pass-through modes such as Sanjar are fixed sucrase, successfully the optimum pH of yeast sucrose hydrolysis enzyme is brought up to 6.0 (Sanjar G. from 5.0, et al., 2006, Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10.Food Chem., 94:573-579.).Searching is suitable for the new neutral sucrose hydrolysis enzyme that foodstuffs industry is produced, and improves the speed of sucrose hydrolysis, thereby shortens the fermentative production time, and the production cost that utilizes sucrose to produce various food for reduction has crucial meaning.
Summary of the invention
The objective of the invention is by the transformation to the sucrose hydrolysis enzyme, make it to obtain new sucrose hydrolysis enzyme mutant body, this mutant enzyme can be used for sucrose hydrolysis.
New sucrose hydrolysis enzyme mutant body Sinv2 (SEQ ID NO:1) of the present invention, it obtains by molecular modification sucrose hydrolysis enzyme gene inv2 (GenBank sequence number HQ267532), specifically be by preceding 360 bases on the disappearance inv2 molecule, transform and obtain a new sucrose hydrolysis enzyme mutant body.This sucrose hydrolysis enzyme mutant body is compared with protoenzyme, and the activity of sucrose hydrolysis has obtained 1 times raising, and the optimum pH of enzyme reaction simultaneously becomes neutral 6.0 by acid 4.5.
The protein of SEQ ID NO:1 is sucrose hydrolysis enzyme mutant body Sinv2, is made up of 466 amino acid.Aminoacid sequence is as follows:
Sequence signature:
Length: 466 amino acid
Type: polypeptide
Chain: strand
Geometry: solid
Molecule type: protein
Sequence description:
Phe?Arg?Pro?Gly?Phe?His?Phe?Thr?Pro?Glu?Tyr?Gly?Trp?Met?Asn
1 5 10 15
Asp?Pro?Asn?Gly?Leu?Val?Tyr?Leu?Asp?Gly?Glu?Tyr?His?Leu?Phe
16 20 25 30
Tyr?Gln?Tyr?Asn?Pro?Tyr?Gly?Asn?Arg?Trp?Gly?Asn?Met?His?Trp
31 35 40 45
Gly?His?Ala?Val?Ser?Thr?Asp?Leu?Thr?Ser?Trp?Thr?Tyr?Leu?Pro
46 50 55 60
Thr?Ala?Ile?Glu?Pro?Asp?Lys?Leu?Gly?Asp?Ile?Phe?Ser?Gly?Ser
61 65 70 75
Ala?Val?Val?Asp?Ser?Thr?Asn?Ser?Ala?Gly?Phe?Gly?Lys?Asn?Ala
76 80 85 90
Leu?Ile?Ala?Ile?Tyr?Thr?Ala?Asn?Gly?Ala?Thr?Gln?Gln?Gln?Cys
91 95 100 105
Ile?Ala?Tyr?Ser?Ile?Asp?Lys?Gly?Arg?Thr?Phe?Thr?Lys?Tyr?Glu
106 110 115 120
Lys?Asn?Pro?Val?Leu?Pro?Asn?Pro?Gly?Ile?Lys?Asp?Phe?Arg?Asp
121 125 130 135
Pro?Lys?Val?His?Trp?Asn?Glu?Gln?Ala?Lys?Gln?Trp?Val?Met?Ala
136 140 145 150
Leu?Ala?Thr?Gln?Gln?Thr?Ile?Thr?Phe?Phe?Gly?Ser?Pro?Asp?Leu
151 155 160 165
Lys?Asn?Trp?Thr?Arg?Leu?Ser?Glu?Phe?Gly?Lys?Asn?Tyr?Gly?Ala
166 170 175 180
His?Gly?Gly?Val?Trp?Glu?Cys?Pro?Asp?Leu?Phe?Pro?Leu?Glu?Phe
181 185 190 195
Glu?Gly?Lys?Thr?Lys?Trp?Val?Leu?Leu?Val?Ser?Ile?Asn?Pro?Gly
196 200 205 210
Gly?Pro?Asn?Gly?Gly?Ser?Ala?Thr?Gln?Tyr?Phe?Ile?Gly?Asp?Phe
211 215 220 225
Asp?Gly?Lys?Thr?Phe?Ser?Ala?Asp?Pro?Leu?Pro?Tyr?Pro?Leu?Trp
226 230 235 240
Val?Asp?Tyr?Gly?Arg?Asp?Asp?Tyr?Ala?Gly?Val?Thr?Phe?Ser?Asn
241 245 250 255
Ile?Gly?Lys?Asn?Asp?Gly?Arg?Arg?Ile?Phe?Met?Gly?Trp?Met?Ser
256 260 265 270
Asn?Trp?Asp?Tyr?Ala?Asn?Asp?Val?Pro?Thr?Lys?Ser?Phe?Arg?Asn
271 275 280 285
Ala?Met?Thr?Leu?Pro?Arg?Glu?Leu?Lys?Leu?Ala?Ser?Asn?Gly?Gln
286 290 295 300
His?Leu?Ile?Leu?Thr?Ser?Ala?Pro?Val?Ser?Glu?Val?Thr?Lys?Leu
301 305 310 315
Arg?Gly?Lys?Ser?Gly?Glu?Thr?Ile?Asn?Ile?Leu?Val?Asn?Ser?Glu
316 320 325 330
Lys?Ser?Ile?Thr?Asn?Val?Leu?Gln?Asp?Phe?Asn?Gly?Lys?Phe?Glu
331 335 340 345
Leu?Asn?Met?Thr?Ile?Gln?Arg?Lys?Asp?Ala?Lys?Val?Trp?Gly?Phe
346 350 355 360
Gly?Leu?Lys?Asn?Glu?Leu?Gly?Asp?Tyr?Leu?Asn?Phe?Thr?Phe?Asp
361 365 370 375
Trp?Glu?Gln?Lys?Ile?Leu?Lys?Val?Asp?Arg?Arg?Asn?Thr?Gly?Ile
376 380 385 390
Lys?Glu?Phe?Ser?Gln?Lys?Phe?Ala?Thr?Glu?Pro?Phe?Ala?Pro?Leu
391 395 400 405
Ala?Lys?His?Asp?Ser?Tyr?Thr?Ile?Arg?Leu?Phe?Met?Asp?Lys?Ala
406 410 415 420
Ser?Ser?Glu?Ile?Phe?Ile?Asn?Asp?Gly?Glu?Thr?Val?Leu?Thr?Asn
421 425 430 435
Leu?Val?Phe?Pro?Ser?Gln?Thr?Tyr?Lys?Ser?Leu?Thr?Phe?Phe?Thr
436 440 445 450
Ala?Asn?Lys?Pro?Trp?Asn?Val?Glu?Asn?Leu?Lys?Ile?Phe?Glu?Ile
451 455 460 465
Lys。
466
The invention still further relates to the host of containing sucrose hydrolysis enzyme mutant body of the present invention.
New sucrose hydrolysis enzyme mutant body of the present invention, this mutant enzyme has purposes widely in the degraded of sucrose.
The ability that the optimal pH that it is special to be it becomes neutral sucrose hydrolysis simultaneously improves 1 times, and this mutein can be used for the degraded of sucrose.
The preparation method of new sucrose hydrolysis enzyme mutant body of the present invention comprises the clone of sucrose hydrolysis enzyme gene, acquisition, sucrose hydrolysis enzyme mutant body expression of gene and the purifying of sucrose hydrolysis enzyme mutant body gene and the steps such as mensuration that sucrose hydrolysis enzyme mutant body sucrose hydrolysis enzyme is lived, and is specific as follows:
1) clone of sucrose hydrolysis enzyme gene inv2
Use upstream primer inv-1:5 '-CACTCATGATGCACCACCACCACCACCACAATAGACAGATGAATCCAGGTC-3 ' (comprising a PagI restriction enzyme site and a 6xHis label) and downstream primer inv-2:5 '-CACCTGCAGACGATGATTTCACAGATGCAAGC-3 ' (comprising a PstI restriction enzyme site) at 5 ' end, by polymerase chain reaction (PCR) amplification sucrose hydrolysis enzyme gene inv2, after cutting sucrose hydrolysis enzyme gene inv2 with restriction enzyme PagI and Pst I enzyme, be inserted into through NcoI and be connected with the expression vector pSE380 that Pst I enzyme is cut.The recombinant plasmid called after pSE-inv2 that obtains.
2) acquisition of sucrose hydrolysis enzyme mutant body gene Sinv2
The Sinv2 gene is to begin to design primer transformation according to inv2 gene order information from 361 bit bases of inv2 gene.With 1) in the pSE-inv2 that makes up be template, use upstream primer Sinv:5 '-CACTCATGATGCACCACCACCACCACCACTTTCGACCTGGATTTCATTTC-3 ' (comprising a PagI restriction enzyme site and a 6xHis label) and downstream primer inv-2 to carry out PCR, the PCR response procedures: 95 ℃ of the first steps 2 minutes at 5 ' end; Second step was carried out 30 circulations, circulation be 98 ℃ 10 seconds, 54 ℃ 15 seconds, 72 ℃ 1.5 minutes; The 3rd the step 72 ℃ 10 minutes.After the PCR product is cut with restriction enzyme PagI and Pst I enzyme, be inserted into the expression vector pSE380 that cuts through NcoI and Pst I enzyme, be transformed into XL1-Blue competent cell (conversion of CaCl2 chemical method).Carry out the dna sequencing analysis then and determine correct transformant.
3) expression of sucrose hydrolysis enzyme mutant body gene Sinv2 and the purifying of expression product
To contain the broken born of the same parents of recombinant escherichia coli strain inoculation, substratum, ultrasonic wave of plasmid pSE-Sinv2, centrifugal, purifying, chromatography, wash-out, and collect the protein soln of wash-out, checking finds to have the protein band of the size of clauses and subclauses.
4) sucrose hydrolysis enzyme mutant body Sinv2 sucrose hydrolysis enzyme mensuration alive
With sucrose hydrolysis enzyme mutant body Sinv2 purifying thing, add the dissolving of DNS solution, add reagent mix, to place in the boiling water and react, the room temperature cooling is with spectrophotometric instrumentation absorbancy OD 600Utilizing the absorbance measurement value to calculate enzyme lives.
The experimental data enzyme of the Sinv2 after Inv2 before the sudden change that obtains and the sudden change is alive and optimal pH is mapped.Found that: the optimum pH of sucrose hydrolysis enzyme mutant body Sinv2 brings up to 6.0 by 4.5, and the enzyme of sucrose hydrolysis is lived and improved 1 times simultaneously.
Embodiment
Following implementation method is for better explanation the present invention, and the purpose that should not be construed as limiting the invention.
Used in an embodiment of the present invention material comprises: intestinal bacteria (Escherichia coli) strain is XL1-Blue (available from a TaKaRa company); Carrier is the expression vector pSE380 available from Intrivogen company; Ni-NTA histidine protein purification media available from Intrivogen company; Restriction enzyme, modifying enzyme available from TaKaRa, MBI.
To be described in detail the present invention by embodiment below:
1) clone of sucrose hydrolysis enzyme gene inv2
Use upstream primer inv-1:5 '-CACTCATGA TGCACCACCACCACCACCACAATAGACAGATGAATCCAGGTC-3 ' (comprising a PagI restriction enzyme site and a 6xHis label) and downstream primer inv-2:5 '-CACCTGCAGACGATGATTTCACAGATGCAAGC-3 ' (comprising a PstI restriction enzyme site) at 5 ' end, by polymerase chain reaction (PCR) amplification sucrose hydrolysis enzyme gene inv2, after cutting sucrose hydrolysis enzyme gene inv2 with restriction enzyme PagI and Pst I enzyme, be inserted into through NcoI and be connected with the expression vector pSE380 that Pst I enzyme is cut.The recombinant plasmid called after pSE-inv2 that obtains.
2) acquisition of sucrose hydrolysis enzyme mutant body gene Sinv2
The Sinv2 gene is to begin to design primer transformation according to inv2 gene order information from 361 bit bases of inv2 gene.With 1) in the pSE-inv2 that makes up be template, use upstream primer Sinv:5 '-CACTCATGATGCACCACCACCACCACCACTTTCGACCTGGATTTCATTTC-3 ' (comprising a PagI restriction enzyme site and a 6xHis label) and downstream primer inv-2 to carry out PCR, the PCR response procedures: 95 ℃ of the first steps 2 minutes at 5 ' end; Second step was carried out 30 circulations, circulation be 98 ℃ 10 seconds, 54 ℃ 15 seconds, 72 ℃ 1.5 minutes; The 3rd the step 72 ℃ 10 minutes.After the PCR product is cut with restriction enzyme PagI and Pst I enzyme, be inserted into the expression vector pSE380 that cuts through NcoI and Pst I enzyme, be transformed into XL1-Blue competent cell (conversion of CaCl2 chemical method).Converted product clone delivers Shanghai biotechnology company limited and carries out the dna sequencing analysis and determine correct transformant.
3) expression of sucrose hydrolysis enzyme mutant body gene Sinv2 and the purifying of expression product
The recombination bacillus coli XL1-Blue inoculation that will contain plasmid pSE-Sinv2 contains in the LB substratum of penbritin (100 μ g/mL) to 20mL, and 37 ℃ of shaking culture are treated OD 600Be 0.6 o'clock, add IPTG (final concentration is 1mmol/L) 30 ℃ and induced 10 hours.11000 leave heart 3min, collect thalline, with 4mL lysis buffer (50mmol/LNaH 2PO 4, 300mmol/L NaCl, the 10mmol/L imidazoles, pH 8.0) resuspended thalline, ultrasonic wave is broken born of the same parents 9min.12000 leave heart 10min, get the protein purification that supernatant carries out the back.Nickel affinity chromatography colloid by every 4ml supernatant liquor adding 1mL 50% shook 60 minutes with 200 commentaries on classics at 4 ℃, and mixture is filled into pillar, collected effluent.Add 1ml dcq buffer liquid (50mmol/L NaH 2PO 4, 300mmol/L NaCl, the 20mmol/L imidazoles, pH 8.0) in pillar, slowly stir, collect effluent.Repeat rinse step 4 times.Add elution buffer (50mmol/L NaH 2PO 4, 300mmol/LNaCl, the 250mmol/L imidazoles, pH 8.0) elute protein.Collect the protein soln of wash-out, with polyacrylamide gel electrophoresis (SDS-PAGE) checking of sex change, discovery has the protein band of the size of clauses and subclauses.
4) sucrose hydrolysis enzyme mutant body Sinv2 sucrose hydrolysis enzyme mensuration alive
Get 5 μ L sucrose hydrolysis enzyme mutant body Sinv2 purifying things, adding 500 μ L 1% (w/v) sucrose (being dissolved in the phosphoric acid buffer of pH 7.0,100mmol/L) solution mixes, 45 ℃ the effect 30 minutes after, add [the DNS reagent preparation: take by weighing 1 gram sodium hydroxide and dissolve of 1mL DNS solution with about 40mL distilled water, take by weighing 1 gram dinitrosalicylic acid, 0.2 gram phenol, 0.05 gram sodium sulphite anhydrous 99.3,20 gram Rochelle salts again, it is dissolved in about 30mL distilled water, two kinds of solution mix, and constant volume is to 100mL.], to place in the boiling water and reacted 5 minutes, the room temperature cooling is with spectrophotometric instrumentation absorbancy OD600.Utilizing the absorbance measurement value to calculate enzyme lives.
The experimental data enzyme of the Sinv2 after Inv2 before the sudden change that obtains and the sudden change is alive and optimal pH is mapped.Found that: the optimum pH of sucrose hydrolysis enzyme mutant body Sinv2 brings up to 6.0 by 4.5, and the enzyme of sucrose hydrolysis is lived and improved 1 times simultaneously.
Description of drawings:
Fig. 1 is the SDS-PAGE figure of sucrose hydrolysis enzyme mutant body Sinv2 purifying thing.
Fig. 2 is the comparison diagram of (Inv2 and Sinv2) enzyme work of sucrose hydrolysis enzyme mutant front and back and optimal pH.
Recognize that from Fig. 1 the molecular weight of sucrose hydrolysis enzyme mutant Sinv2 purified is 51kDa.
Recognize that from Fig. 2 significant variation has taken place in (Inv2 and Sinv2) enzyme work before and after the sucrose hydrolysis enzyme mutant: the enzyme work of the rear Sinv2 of sudden change is 2 times of the front Inv2 of sudden change; Optimal pH is acidity 4.5 before the sudden change, and optimal pH is neutral 6 after the sudden change. Improve the speed of sucrose hydrolysis, thereby shorten the fermenting and producing time, utilize the production cost tool of the various food of sugar industry to be of great significance for reduction.
Sequence table
＜110〉Guangxi Academy Of Sciences
＜120〉mutant of sucrose hydrolysis enzyme and application thereof
<160>1
<170>PatentIn?Version?3.3
<210>1
<211>586
<212>PRT
＜213〉soil culturing micro-organisms not
<400>1
Phe?Arg?Pro?Gly?Phe?His?Phe?Thr?Pro?Glu?Tyr?Gly?Trp?Met?Asn
1 5 10 15
Asp?Pro?Asn?Gly?Leu?Val?Tyr?Leu?Asp?Gly?Glu?Tyr?His?Leu?Phe
16 20 25 30
Tyr?Gln?Tyr?Asn?Pro?Tyr?Gly?Asn?Arg?Trp?Gly?Asn?Met?His?Trp
31 35 40 45
Gly?His?Ala?Val?Ser?Thr?Asp?Leu?Thr?Ser?Trp?Thr?Tyr?Leu?Pro
46 50 55 60
Thr?Ala?Ile?Glu?Pro?Asp?Lys?Leu?Gly?Asp?Ile?Phe?Ser?Gly?Ser
61 65 70 75
Ala?Val?Val?Asp?Ser?Thr?Asn?Ser?Ala?Gly?Phe?Gly?Lys?Asn?Ala
76 80 85 90
Leu?Ile?Ala?Ile?Tyr?Thr?Ala?Asn?Gly?Ala?Thr?Gln?Gln?Gln?Cys
91 95 100 105
Ile?Ala?Tyr?Ser?Ile?Asp?Lys?Gly?Arg?Thr?Phe?Thr?Lys?Tyr?Glu
106 110 115 120
Lys?Asn?Pro?Val?Leu?Pro?Asn?Pro?Gly?Ile?Lys?Asp?Phe?Arg?Asp
121 125 130 135
Pro?Lys?Val?His?Trp?Asn?Glu?Gln?Ala?Lys?Gln?Trp?Val?Met?Ala
136 140 145 150
Leu?Ala?Thr?Gln?Gln?Thr?Ile?Thr?Phe?Phe?Gly?Ser?Pro?Asp?Leu
151 155 160 165
Lys?Asn?Trp?Thr?Arg?Leu?Ser?Glu?Phe?Gly?Lys?Asn?Tyr?Gly?Ala
166 170 175 180
His?Gly?Gly?Val?Trp?Glu?Cys?Pro?Asp?Leu?Phe?Pro?Leu?Glu?Phe
181 185 190 195
Glu?Gly?Lys?Thr?Lys?Trp?Val?Leu?Leu?Val?Ser?Ile?Asn?Pro?Gly
196 200 205 210
Gly?Pro?Asn?Gly?Gly?Ser?Ala?Thr?Gln?Tyr?Phe?Ile?Gly?Asp?Phe
211 215 220 225
Asp?Gly?Lys?Thr?Phe?Ser?Ala?Asp?Pro?Leu?Pro?Tyr?Pro?Leu?Trp
226 230 235 240
Val?Asp?Tyr?Gly?Arg?Asp?Asp?Tyr?Ala?Gly?Val?Thr?Phe?Ser?Asn
241 245 250 255
Ile?Gly?Lys?Asn?Asp?Gly?Arg?Arg?Ile?Phe?Met?Gly?Trp?Met?Ser
256 260 265 270
Asn?Trp?Asp?Tyr?Ala?Asn?Asp?Val?Pro?Thr?Lys?Ser?Phe?Arg?Asn
271 275 280 285
Ala?Met?Thr?Leu?Pro?Arg?Glu?Leu?Lys?Leu?Ala?Ser?Asn?Gly?Gln
286 290 295 300
His?Leu?Ile?Leu?Thr?Ser?Ala?Pro?Val?Ser?Glu?Val?Thr?Lys?Leu
301 305 310 315
Arg?Gly?Lys?Ser?Gly?Glu?Thr?Ile?Asn?Ile?Leu?Val?Asn?Ser?Glu
316 320 325 330
Lys?Ser?Ile?Thr?Asn?Val?Leu?Gln?Asp?Phe?Asn?Gly?Lys?Phe?Glu
331 335 340 345
Leu?Asn?Met?Thr?Ile?Gln?Arg?Lys?Asp?Ala?Lys?Val?Trp?Gly?Phe
346 350 355 360
Gly?Leu?Lys?Asn?Glu?Leu?Gly?Asp?Tyr?Leu?Asn?Phe?Thr?Phe?Asp
361 365 370 375
Trp?Glu?Gln?Lys?Ile?Leu?Lys?Val?Asp?Arg?Arg?Asn?Thr?Gly?Ile
376 380 385 390
Lys?Glu?Phe?Ser?Gln?Lys?Phe?Ala?Thr?Glu?Pro?Phe?Ala?Pro?Leu
391 395 400 405
Ala?Lys?His?Asp?Ser?Tyr?Thr?Ile?Arg?Leu?Phe?Met?Asp?Lys?Ala
406 410 415 420
Ser?Ser?Glu?Ile?Phe?Ile?Asn?Asp?Gly?Glu?Thr?Val?Leu?Thr?Asn
421 425 430 435
Leu?Val?Phe?Pro?Ser?Gln?Thr?Tyr?Lys Ser?Leu?Thr?Phe?Phe?Thr
436 440 445 450
Ala?Asn?Lys?Pro?Trp?Asn?Val?Glu?Asn?Leu?Lys Ile?Phe?Glu?Ile
451 455 460 465
Lys
466

Claims (3)

1. sucrose hydrolysis enzyme mutant body is characterized in that: have SEQ ID NO:1 aminoacid sequence:
(1) sequence signature:
I. length: 466 amino acid
Ii. type: polypeptide
Iii. chain: strand
Iv. geometry: solid
(2) molecule type: protein
(3) sequence description:
Phe?Arg?Pro?Gly?Phe?His?Phe?Thr?Pro?Glu?Tyr?Gly?Trp?Met?Asn
1 5 10 15
Asp?Pro?Asn?Gly?Leu?Val?Tyr?Leu?Asp?Gly?Glu?Tyr?His?Leu?Phe
16 20 25 30
Tyr?Gln?Tyr?Asn?Pro?Tyr?Gly?Asn?Arg?Trp?Gly?Asn?Met?His?Trp
31 35 40 45
Gly?His?Ala?Val?Ser?Thr?Asp?Leu?Thr?Ser?Trp?Thr?Tyr?Leu?Pro
46 50 55 60
Thr?Ala?Ile?Glu?Pro?Asp?Lys?Leu?Gly?Asp?Ile?Phe?Ser?Gly?Ser
61 65 70 75
Ala?Val?Val?Asp?Ser?Thr?Asn?Ser?Ala?Gly?Phe?Gly?Lys?Asn?Ala
76 80 85 90
Leu?Ile?Ala?Ile?Tyr?Thr?Ala?Asn?Gly?Ala?Thr?Gln?Gln?Gln?Cys
91 95 100 105
Ile?Ala?Tyr?Ser?Ile?Asp?Lys?Gly?Arg?Thr?Phe?Thr?Lys?Tyr?Glu
106 110 115 120
Lys?Asn?Pro?Val?Leu?Pro?Asn?Pro?Gly?Ile?Lys?Asp?Phe?Arg?Asp
121 125 130 135
Pro?Lys?Val?His?Trp?Asn?Glu?Gln?Ala?Lys?Gln?Trp?Val?Met?Ala
136 140 145 150
Leu?Ala?Thr?Gln?Gln?Thr?Ile?Thr?Phe?Phe?Gly?Ser?Pro?Asp?Leu
151 155 160 165
Lys?Asn?Trp?Thr?Arg?Leu?Ser?Glu?Phe?Gly?Lys?Asn?Tyr?Gly?Ala
166 170 175 180
His?Gly?Gly?Val?Trp?Glu?Cys?Pro?Asp?Leu?Phe?Pro?Leu?Glu?Phe
181 185 190 195
Glu?Gly?Lys?Thr?Lys?Trp?Val?Leu?Leu?Val?Ser?Ile?Asn?Pro?Gly
196 200 205 210
Gly?Pro?Asn?Gly?Gly?Ser?Ala?Thr?Gln?Tyr?Phe?Ile?Gly?Asp?Phe
211 215 220 225
Asp?Gly?Lys?Thr?Phe?Ser?Ala?Asp?Pro?Leu?Pro?Tyr?Pro?Leu?Trp
226 230 235 240
Val?Asp?Tyr?Gly?Arg?Asp?Asp?Tyr?Ala?Gly?Val?Thr?Phe?Ser?Asn
241 245 250 255
Ile?Gly?Lys?Asn?Asp?Gly?Arg?Arg?Ile?Phe?Met?Gly?Trp?Met?Ser
256 260 265 270
Asn?Trp?Asp?Tyr?Ala?Asn?Asp?Val?Pro?Thr?Lys?Ser?Phe?Arg?Asn
271 275 280 285
Ala?Met?Thr?Leu?Pro?Arg?Glu?Leu?Lys?Leu?Ala?Ser?Asn?Gly?Gln
286 290 295 300
His?Leu?Ile?Leu?Thr?Ser?Ala?Pro?Val?Ser?Glu?Val?Thr?Lys?Leu
301 305 310 315
Arg?Gly?Lys?Ser?Gly?Glu?Thr?Ile?Asn?Ile?Leu?Val?Asn?Ser?Glu
316 320 325 330
Lys?Ser?Ile?Thr?Asn?Val?Leu?Gln?Asp?Phe?Asn?Gly?Lys?Phe?Glu
331 335 340 345
Leu?Asn?Met?Thr?Ile?Gln?Arg?Lys?Asp?Ala?Lys?Val?Trp?Gly?Phe
346 350 355 360
Gly?Leu?Lys?Asn?Glu?Leu?Gly?Asp?Tyr?Leu?Asn?Phe?Thr?Phe?Asp
361 365 370 375
Trp?Glu?Gln?Lys?Ile?Leu?Lys?Val?Asp?Arg?Arg?Asn?Thr?Gly?Ile
376 380 385 390
Lys?Glu?Phe?Ser?Gln?Lys?Phe?Ala?Thr?Glu?Pro?Phe?Ala?Pro?Leu
391 395 400 405
Ala?Lys?His?Asp?Ser?Tyr?Thr?Ile?Arg?Leu?Phe?Met?Asp?Lys?Ala
406 410 415 420
Ser?Ser?Glu?Ile?Phe?Ile?Asn?Asp?Gly?Glu?Thr?Val?Leu?Thr?Asn
421 425 430 435
Leu?Val?Phe?Pro?Ser?Gln?Thr?Tyr?Lys?Ser?Leu?Thr?Phe?Phe?Thr
436 440 445 450
Ala?Asn?Lys?Pro?Trp?Asn?Val?Glu?Asn?Leu?Lys?Ile?Phe?Glu?Ile
451 455 460 465
Lys。
466
2. host cell, it is prokaryotic cell prokaryocyte or the eukaryotic cell that contains the described sucrose hydrolysis enzyme mutant of claim 1 body.
3. the described mutein of claim 1 is in the sucrose degraded with to the application in the processing that contains the sucrose material.
CN2010105069077A 2010-10-14 2010-10-14 Mutant of cane sugar hydrolytic enzyme and application of mutant Expired - Fee Related CN102021156B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109486793A (en) * 2018-11-27 2019-03-19 江南大学 A kind of sucrose hydrolysis enzyme mutant and the preparation method and application thereof
CN109576240A (en) * 2018-12-18 2019-04-05 江南大学 A kind of amylosucrase mutant and the preparation method and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101407820A (en) * 2008-09-26 2009-04-15 广西大学 Gene of encoding glycosyl hydrolase family 32 sucrase and use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101407820A (en) * 2008-09-26 2009-04-15 广西大学 Gene of encoding glycosyl hydrolase family 32 sucrase and use thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109486793A (en) * 2018-11-27 2019-03-19 江南大学 A kind of sucrose hydrolysis enzyme mutant and the preparation method and application thereof
CN109576240A (en) * 2018-12-18 2019-04-05 江南大学 A kind of amylosucrase mutant and the preparation method and application thereof

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