CN111621489B

CN111621489B - Thermostable inulase exonuclease mutant MutQ23 delta 6 and preparation and application thereof

Info

Publication number: CN111621489B
Application number: CN202010538023.3A
Authority: CN
Inventors: 周峻沛; 张蕊; 黄遵锡; 何丽梅; 韩楠玉; 唐湘华; 吴倩; 慕跃林
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2022-06-24
Anticipated expiration: 2040-06-12
Also published as: CN111621489A

Abstract

The invention discloses a thermolabile exoinulase mutant MutQ23 delta 6 and preparation and application thereof, wherein the mutant MutQ23 delta 6 has an amino acid sequence shown as SEQ ID NO.1, and compared with a wild enzyme InuAGN25DVS and a mutant enzyme MutQ23 delta 3, the mutant MutQ23 delta 6 has low activity at 50 ℃ and poor thermal stability at 55 ℃. After being treated at 55 ℃ for 5-30min, the enzyme activity of MutQ23 delta 6 is reduced from 62% to 8%, the enzyme activity of a wild enzyme InuAGN25DVS is reduced from 71% to 20%, and the enzyme activity of a mutant enzyme MutQ23 delta 3 is reduced from 90% to 22%. The thermolabile exoinulase mutant MutQ23 delta 6 can be applied to industries of food, wine brewing, biological energy and the like.

Description

Thermostable inulase exonuclease mutant MutQ23 delta 6 and preparation and application thereof

Technical Field

The invention relates to a heat-labile inulase exonuclease mutant MutQ23 delta 6 and preparation and application thereof.

Background

Inulin is widely present in various plants such as Jerusalem artichoke, burdock, chicory and the like. The jerusalem artichoke belongs to non-grain crops, has low price, higher yield, barren resistance, cold resistance, drought resistance, salt and alkali resistance and the like, and can be planted in saline-alkali soil, intertidal zones and desert lands. Inulin is therefore a renewable resource with a rich source.

Inulin is formed by polymerization of fructose molecules through beta-2, 1 glycosidic bonds, and the tail end of the inulin is connected with a molecule of glucose residue. The exoinulase hydrolyzes beta-2, 1 glycosidic bonds in the inulin one by one, and finally the inulin is completely degraded into fructose and a small amount of glucose, so as to generate fructose syrup with the content of 95 percent, and the fructose can also be used for producing bioethanol, 2, 3-butanediol and the like. Therefore, the inulase exonuclease can be applied to industries such as food, wine brewing and bioenergy (Singh RS et al. International Journal of Biological Macromolecules,2017,96: 312-.

The enzyme which is more sensitive to heat is easier to denature, the enzyme is easier to degrade after heat denaturation, the catalytic reaction of the enzyme can be easily controlled due to the characteristic, and meanwhile, the use of the enzyme is safer, so that the enzyme has application value in the field of low-temperature biotechnology, particularly food. The low-temperature enzyme has the characteristic of thermolabile property, and can be applied to the field of biotechnology under the requirement of low-temperature environment, such as the fermentation temperature of sake and wine is generally less than 25 ℃. In addition, treatment at low temperature (if juice is clarified) can prevent microbial contamination, nutrient loss and food quality degradation, and conversion of medium temperature or high temperature treatment mode to low temperature treatment mode can also serve to reduce energy consumption (Cavicchiali et al microbiological Biotechnology,2011,4(4): 449-460.). Therefore, development of low-temperature or heat-unstable inulinase is required.

Disclosure of Invention

The invention aims to provide a thermolabile exoinulase mutant MutQ23 delta 6, and preparation and application thereof, wherein the thermolabile exoinulase mutant MutQ23 delta 6 has thermolabile performance and can be applied to industries such as food, wine brewing, biological energy and the like.

In order to achieve the above object, the present invention provides a heat-labile inulase exonuclease mutant MutQ23 Δ 6, wherein the mutant MutQ23 Δ 6 has an amino acid sequence as shown in SEQ ID NO. 1. The mutant MutQ23 delta 6 is compared with the low-temperature inulinase sequence AGC01503(SEQ ID NO.3) recorded by GenBank: MutQ23 Δ 6 does not contain the signal peptide sequence "MIKLKKYGVLMLLLGVFGTSLA" at the N-terminus of AGC 01503; furthermore, at the N-terminus, MutQ23 Δ 6 has more amino acids "GP" than AGC01503, but lacks 6 amino acids, namely lacks amino acids "QTGQHK" from position 23 to 28 of AGC 01503.

Another purpose of the invention is to provide a gene mutQ23 delta 6 of the mutant mutQ23 delta 6.

Preferably, the coding gene mutQ23 delta 6 has the nucleotide sequence shown in SEQ ID NO. 2.

Another objective of the invention is to provide a recombinant vector containing the encoding gene mutQ23 delta 6.

Another purpose of the invention is to provide a recombinant bacterium containing the encoding gene mutQ23 delta 6.

Another object of the present invention is to provide a method for preparing the mutant MutQ23 Δ 6, which comprises:

the 5' end of mutQ23 delta 6 is added with HRV3C protease enzyme cutting site coding sequence;

connecting the nucleotide sequence of the mutQ23 delta 6 with an expression vector pET-28a (+), and transforming the connection product into Escherichia coli BL21(DE3) to obtain a recombinant strain containing mutQ23 delta 6;

culturing the recombinant strain, inducing the expression of the recombinant exoinulase mutant MutQ23 delta 6, and carrying out protease digestion on the obtained recombinant exoinulase mutant MutQ23 delta 6 by using HRV3C to obtain the exoinulase mutant MutQ23 delta 6.

Preferably, the recombinant strain is cultured in a medium containing 50. mu.g.mL^-1LB broth of kanamycin.

Preferably, the recombinant strain is cultured at 37 ℃ in a medium containing 50. mu.g.mL^-1The culture was performed with shaking in LB medium containing kanamycin.

Preferably, the induction is when OD₆₀₀When the concentration reaches 0.6-1.0, adding IPTG for induction.

The invention also aims to provide application of the mutant MutQ23 delta 6 in food or wine brewing.

The thermolabile exoinulase mutant MutQ23 delta 6 and the preparation and application thereof have the following advantages:

compared with the wild enzyme InuAGN25DVS and the mutant enzyme MutQ23 delta 3, MutQ23 delta 6 has low activity at 50 ℃ and poor thermal stability at 55 ℃. The enzyme activity of MutQ23 delta 6 at 20 ℃ and 50 ℃ is 41 percent and 26 percent respectively; the enzyme activity of wild enzyme InuAGN25DVS at 20 ℃ and 50 ℃ is 36 percent and 89 percent respectively; the mutant enzyme MutQ23 delta 3 has 46 percent and 77 percent of enzyme activity at 20 ℃ and 50 ℃ respectively. After being treated at 55 ℃ for 5-30min, the enzyme activity of MutQ23 delta 6 is reduced from 62% to 8%, the enzyme activity of a wild enzyme InuAGN25DVS is reduced from 71% to 20%, and the enzyme activity of a mutant enzyme MutQ23 delta 3 is reduced from 90% to 22%. The thermolabile exoinulase mutant MutQ23 delta 6 can be applied to industries of food, wine brewing, biological energy and the like.

Drawings

FIG. 1 shows the thermal activity of purified wild enzyme InuAGN25DVS and mutant enzymes MutQ23 Δ 3 and MutQ23 Δ 6.

FIG. 2 shows the thermostability of the purified wild enzyme InuAGN25DVS and the mutant enzymes MutQ23 Δ 3 and MutQ23 Δ 6.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention relates to experimental materials and reagents in the following experimental examples:

1. bacterial strain and carrier

Escherichia coli BL21(DE3) and expression vector pET-28a (+) are available from Novagen; plasmid pEASY-E1-Z2-5 was supplied by university of Master Yunan.

2. Enzymes and other biochemical reagents

HRV3C protease and restriction enzyme were obtained from TaKaRa, Nickel-NTA Agarose was obtained from QIAGEN, DNA polymerase, dNTP and

the II kit is purchased from Nanjing Novodka company, the inulin is purchased from Sigma company, and other kits are made in China (all can be purchased from common biochemical reagents company).

3. Culture medium

LB culture medium: peptone 10g, Yeast extract 5g, NaCl 10g, distilled water to 1000mL, natural pH (about 7). On the basis of the solid medium, 2.0% (w/v) agar was added.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Experimental example 1 construction and transformation of wild enzyme InuAGN25DVS expression vector

(1) Designing primers 5'-TGGAAGTTCTGTTCCAGGGGCCCCAGACGGGACAGCATAAACAAG-3' (SEQ ID NO.11) and 5'-GGTGGTGGTGTTATCTCTTAAATGCAGAAATACCGAT-3' (SEQ ID NO.12) according to a low-temperature inulinase nucleotide sequence JQ863108(SEQ ID NO.4) recorded by GenBank, carrying out PCR amplification by taking a plasmid pEASY-E1-Z2-5 as a template to obtain an inulinase mature peptide coding sequence Z2-5, adding an HRV3C protease enzyme cutting site coding sequence to the 5' end of Z2-5, and simultaneously forming recombination regions at the 5' end and the 3' end of Z2-5, wherein the recombination regions are matched with the recombination regions at the two ends of the linearized vector obtained in the step (2). Z2-5 can also be obtained by gene synthesis.

(2) Primers 5'-AGAGATAACACCACCACCACCACCACTG-3' (SEQ ID NO.13) and 5'-CCTGGAACAGAACTTCCAGGAATTCGGATCCGCGACC-3' (SEQ ID NO.14) are designed, PCR amplification is carried out by taking pET-28a (+) plasmid as a template to obtain a linearized pET-28a (+) vector, and recombination regions are formed at the 5 'end and the 3' end of the linearized pET-28a (+) vector and matched with the recombination regions at the two ends of Z2-5.

(3) The PCR reaction parameters are as follows: denaturation at 95 ℃ for 30 sec; then denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 15sec, extension at 72 ℃ for 3min for 30sec, and heat preservation at 72 ℃ for 5min after 30 cycles.

(4) To 50. mu.L of the PCR product of the linearized pET-28a (+) vector, 1. mu.L of DpnI was added and digested at 37 ℃ for 1 hour.

(5) According to

II, the instructions of the kit are that the digested product in the step (4) and Z2-5 are recombined and connected for 30min at 37 ℃, and then the expression vector of the wild enzyme InuAGN25DVS can be obtained.

(6) The wild enzyme expression vector is transformed into escherichia coli BL21(DE3) by a heat shock mode, and a recombinant strain for expressing the wild enzyme InuAGN25DVS is obtained.

Experimental example 2 construction and transformation of expression vectors for mutant enzymes MutQ 23. delta.6 and MutQ 23. delta.3

(1) mutQ23 Δ 6 was synthesized, together with the gene sequence mutQ23 Δ 3(SEQ ID NO.6) encoding the mutant enzyme mutQ23 Δ 3(SEQ ID NO.5), supplemented at the 5 'end of the encoding gene with the HRV3C protease cleavage site coding sequence and the EcoRI restriction site sequence (5'-GAATTCCTGGAAGTTCTGTTCCAG-3'), and at the 3' end of the encoding gene with the XhoI restriction site sequence (5 '-CTCGAG-3').

(2) Carrying out EcoRI and XhoI double enzyme digestion on the sequences synthesized in the step (1) respectively; meanwhile, EcoRI and XhoI double enzyme digestion is carried out on the expression vector pET-28a (+).

(3) And (3) respectively connecting the enzyme digestion products in the step (2) by using DNA ligase to obtain expression vectors containing mutQ23 delta 6 and mutQ23 delta 3.

(4) The ligation products were transformed into E.coli BL21(DE3), respectively, to obtain recombinant strains containing mutQ23 Δ 6 and mutQ23 Δ 3, respectively.

EXAMPLE 3 preparation of the wild enzyme InuAGN25DVS and the mutant enzymes MutQ23 Δ 6 and MutQ23 Δ 3

The recombinant strains containing Z2-5, mutQ 23. DELTA.6 and mutQ 23. DELTA.3 were inoculated in LB (containing 50. mu.g mL of each) at an inoculum size of 0.1% respectively^-1Kanamycin) culture solution, and rapidly shaking at 37 ℃ for 16 h.

The activated bacterial suspension was inoculated into fresh LB (50. mu.g mL) at 1% inoculum size^-1Kanamycin) culture solution, and rapidly shaking and culturing for about 2-3 h (OD)₆₀₀0.6-1.0) was reached, induction was carried out by adding IPTG at a final concentration of 0.25mM, and shaking culture was continued at 20 ℃ for about 20 hours. Centrifugation was carried out at 12000rpm for 5min to collect the cells. After the cells were suspended in an appropriate amount of pH 7.0McIlvaine buffer, the cells were disrupted by ultrasonic waves in a low-temperature water bath.

After the crude enzyme solution concentrated in the above cells was centrifuged at 13,000rpm for 10min, the supernatant was aspirated and the objective protein was respectively subjected to affinity purification using Nickel-NTAAgarose and 0-500 mM imidazole. The recombinant protein expressed and purified by the recombinant strain containing Z2-5 is named as InuAGN25DVSHIs, and the amino acid sequence is shown as SEQ ID NO. 7; the recombinant protein expressed and purified by the recombinant strain containing mutQ23 delta 6 is named as mutQ23 delta 6His, and the amino acid sequence is shown as SEQ ID NO. 8; the recombinant protein expressed and purified by the recombinant strain containing mutQ23 delta 3 is named as mutQ23 delta 3His, and the amino acid sequence is shown as SEQ ID NO. 9.

According to the HRV3C protease specification, InuAGN25DVSHI, MutQ23 delta 6His and MutQ23 delta 3His are respectively enzyme-cut by HRV3C protease, histidine tags and pET-28a (+) vectors positioned at the N ends of the InuAGN25DVSHI, the MutQ23 delta 6His and the MutQ23 delta 3His are removed, and the enzyme-cut products are purified by Nickel-NTAAgarose to obtain InuAGN25DVS (SEQ ID NO.10), MutQ23 delta 6 and MutQ23 delta 3.

Experimental example 4 Properties of the purified wild-type enzyme InuAGN25DVS and mutant enzymes MutQ23 Δ 6 and MutQ23 Δ 3

(1) Enzyme Activity assay

The activity determination method adopts a 3, 5-dinitrosalicylic acid (DNS) method: dissolving the substrate inulin in a buffer solution to make the final concentration of the inulin be 0.5% (w/v); the reaction system contains 50 mu L of proper enzyme solution and 450 mu L of substrate; preheating a substrate at a reaction temperature for 5min, adding an enzyme solution, reacting for 10min, adding 750 mu L DNS to terminate the reaction, boiling in boiling water for 5min, cooling to room temperature, and measuring an OD value at a wavelength of 540 nm; 1 enzyme activity unit (U) is defined as the amount of enzyme required to break down the substrate under the given conditions to produce 1. mu. mol reducing sugars (calculated as fructose) per minute.

(2) Determination of the thermal Activity of an enzyme

Determination of the thermal activity of the enzyme: the enzymatic reaction was carried out at 20-50 ℃ in a buffer at pH 6.0. Using inulin as a substrate, the reaction was carried out for 10min, and the enzymatic properties of the wild enzyme InuAGN25DVS and the mutant enzymes MutQ23 Delta 6 and MutQ23 Delta 3 were determined.

The results show that: the enzyme activity at 45 ℃ is 100%, the enzyme activities of MutQ23 delta 6 at 20 ℃ and 50 ℃ are 41% and 26% respectively, the enzyme activities of a wild enzyme InuAGN25DVS at 20 ℃ and 50 ℃ are 36% and 89% respectively, and the enzyme activities of a mutant enzyme MutQ23 delta 3 at 20 ℃ and 50 ℃ are 46% and 77% respectively, namely the activity of MutQ23 delta 6 at 50 ℃ is lower than that of InuAGN25DVS and MutQ23 delta 3 (see figure 1).

(3) Determination of the thermostability of the enzyme

Determination of the thermostability of the enzymes: after treating the enzyme solution with the same amount of enzyme at 55 deg.C for 5-30min, the enzyme reaction was carried out at pH 6.0 and 37 deg.C, and the untreated enzyme solution was used as a control. Using inulin as a substrate, the reaction was carried out for 10min, and the enzymatic properties of the wild enzyme InuAGN25DVS and the mutant enzymes MutQ23 Delta 6 and MutQ23 Delta 3 were determined.

The results show that: after treatment for 5 min-30 min at 55 ℃, the enzyme activity of MutQ23 delta 6 is reduced from 62% to 8%, the enzyme activity of a wild enzyme InuAGN25DVS is reduced from 71% to 20%, and the enzyme activity of a mutant enzyme MutQ23 delta 3 is reduced from 90% to 22%, namely the thermal stability of MutQ23 delta 6 at 55 ℃ is poorer than that of InuAGN25DVS and MutQ23 delta 3 (see figure 2).

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be limited only by the attached claims.

Sequence listing

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<120> thermolabile exoinulase mutant MutQ23 delta 6 and preparation and application thereof

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Asn Pro Gly Ile Trp Asp Phe Arg Asp Pro Lys Val Met Trp His Val

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Phe Tyr Ser Ser Lys Asp Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe

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Gly Lys Glu Val Gly Ala His Gly Gly Val Trp Glu Cys Pro Asp Leu

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Ile Pro Met Asp Tyr Lys Gly Thr Thr Lys Trp Val Leu Leu Val Ser

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Ile Asn Pro Gly Gly Pro Asn Gly Gly Ser Val Thr Gln Tyr Phe Val

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Gly Asp Phe Asp Gly His Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys

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Trp Leu Asp Trp Gly Pro Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn

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Leu Gly Asp Arg Gln Leu Met Ile Gly Trp Met Ser Asn Trp Gln Tyr

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Ala Asn Val Val Pro Thr Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro

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Arg Val Leu Ser Leu Gly Lys Val Gly Gln Ser Phe Tyr Val Ala Ser

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His Gly Gly Gly Thr Lys Glu Val Ser Phe Glu Gln His Leu Pro Gln

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Ile Leu Ser Asn Glu Ser Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu

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Lys Gly Ala Leu Ser Phe Ser Leu Tyr Val Asp Val Ser Ser Val Glu

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Leu Phe Ala Asp Glu Gly Leu Thr Val Met Thr Ser Leu Phe Phe Pro

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aaagatttga aaacatggca gaaggaaagt gagtttggca aagaggtggg ggcccatggt 600

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ttgttggtga gcatcaatcc aggtggaccc aacggtggct cggtgacaca atattttgtg 720

ggtgactttg atgggcatca gttccgaaca acggatacta aaataaagtg gttggattgg 780

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actgtgccca aagaaattga aaccgcattt cgtccactaa aaaaatacca tggcggtgga 1020

acgaaggaag tttcgttcga gcaacatctc ccacaagcgt accgtttgga cctcaaagat 1080

ttgaagcagc aggcttttaa agtcatctta tcgaatgaat ctggcgacga gttggttatt 1140

ggttatcgtg aagaccaaaa tgcctattat attgatcgtt ctaagtccgg tgaagtgtct 1200

tttaatggag aatttccaaa gatagcgttg gcccaacggc cgctaaataa gggagcactt 1260

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gtaatgacca gtttattttt tccgaacaag ccgataacca agctgcgtat cgtgggagat 1380

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<213> wild enzyme (AGC01503)

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Phe Gly Thr Ser Leu Ala Gln Thr Gly Gln His Lys Gln Gly Asp Pro

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Glu Glu Lys Tyr Arg Pro Leu Tyr His Phe Thr Pro Gln Gln Gly Trp

35 40 45

Met Asn Asp Pro Asn Gly Leu Val Tyr Leu Asp Gly Asn Tyr His Leu

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Phe Phe Gln His Asn Pro Glu Lys Pro Val Trp Gly Pro Met His Trp

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Gly His Ala Ile Ser Lys Asp Leu Ile His Trp Asp Glu Gln Lys Ile

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Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile Phe Ser Gly Ser Ala Val

100 105 110

Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly Lys Gly Ala Met Val Ala

115 120 125

Ile Phe Thr His His Asn His Gln Glu Glu Asp Arg Lys Thr Gly Leu

130 135 140

His Gln Asn Gln Ser Leu Ala Tyr Ser Leu Asp Lys Gly Leu Thr Trp

145 150 155 160

Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro Asn Pro Gly Ile Trp Asp

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Phe Arg Asp Pro Lys Val Met Trp His Val His Ser Gln Arg Trp Ile

180 185 190

Met Thr Leu Ala Thr Lys Asp Cys Ile Thr Phe Tyr Ser Ser Lys Asp

195 200 205

Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe Gly Lys Glu Val Gly Ala

210 215 220

His Gly Gly Val Trp Glu Cys Pro Asp Leu Ile Pro Met Asp Tyr Lys

225 230 235 240

Gly Thr Thr Lys Trp Val Leu Leu Val Ser Ile Asn Pro Gly Gly Pro

245 250 255

Asn Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp Phe Asp Gly His

260 265 270

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275 280 285

Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn Leu Gly Asp Arg Gln Leu

290 295 300

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305 310 315 320

Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro Arg Val Leu Ser Leu Gly

325 330 335

Lys Val Gly Gln Ser Phe Tyr Val Ala Ser Thr Val Pro Lys Glu Ile

340 345 350

Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr His Gly Gly Gly Thr Lys

355 360 365

Glu Val Ser Phe Glu Gln His Leu Pro Gln Ala Tyr Arg Leu Asp Leu

370 375 380

Lys Asp Leu Lys Gln Gln Ala Phe Lys Val Ile Leu Ser Asn Glu Ser

385 390 395 400

Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr

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Ile Asp Arg Ser Lys Ser Gly Glu Val Ser Phe Asn Gly Glu Phe Pro

420 425 430

Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn Lys Gly Ala Leu Ser Phe

435 440 445

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Leu Arg Ile Val Gly Asp Lys Lys Leu Glu Phe Gln Glu Ile Gly Ile

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aattatcatc ttttttttca gcataatccc gaaaaacccg tttgggggcc tatgcattgg 240

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tttggaaaag gtgctatggt cgccattttt acccatcaca atcatcagga agaggatcga 420

aaaacaggat tgcatcaaaa tcaaagtttg gcttatagtt tggacaaggg ccttacctgg 480

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attactttct attcttccaa agatttgaaa acatggcaga aggaaagtga gtttggcaaa 660

gaggtggggg cccatggtgg tgtctgggaa tgccctgatc ttatccccat ggattacaaa 720

ggaactacaa aatgggtttt gttggtgagc atcaatccag gtggacccaa cggtggctcg 780

gtgacacaat attttgtggg tgactttgat gggcatcagt tccgaacaac ggatactaaa 840

ataaagtggt tggattgggg acccgataac tatgccggcg taacctggag taacctaggg 900

gataggcaac tgatgattgg ctggatgagc aactggcaat atgccaatgt ggttcctacc 960

acaaaatggc gttcctcctc aaccatacct cgtgttttat ctttaggaaa agttggacag 1020

tcgttctacg ttgcgagtac tgtgcccaaa gaaattgaaa ccgcatttcg tccactaaaa 1080

aaataccatg gcggtggaac gaaggaagtt tcgttcgagc aacatctccc acaagcgtac 1140

cgtttggacc tcaaagattt gaagcagcag gcttttaaag tcatcttatc gaatgaatct 1200

ggcgacgagt tggttattgg ttatcgtgaa gaccaaaatg cctattatat tgatcgttct 1260

aagtccggtg aagtgtcttt taatggagaa tttccaaaga tagcgttggc ccaacggccg 1320

ctaaataagg gagcactttc tttcagtttg tatgttgatg tgagctctgt ggaacttttt 1380

gcagacgagg ggctaacggt aatgaccagt ttattttttc cgaacaagcc gataaccaag 1440

ctgcgtatcg tgggagataa aaagttggag tttcaggaaa tcggtatttc tgcatttaag 1500

agataa 1506

<210> 5

<211> 478

<212> PRT

<213> mutant enzyme (MutQ 23. DELTA.3)

<400> 5

Gly Pro Gln His Lys Gln Gly Asp Pro Glu Glu Lys Tyr Arg Pro Leu

1 5 10 15

Tyr His Phe Thr Pro Gln Gln Gly Trp Met Asn Asp Pro Asn Gly Leu

20 25 30

Val Tyr Leu Asp Gly Asn Tyr His Leu Phe Phe Gln His Asn Pro Glu

35 40 45

Lys Pro Val Trp Gly Pro Met His Trp Gly His Ala Ile Ser Lys Asp

50 55 60

Leu Ile His Trp Asp Glu Gln Lys Ile Ala Leu Tyr Pro Asp Ser Leu

65 70 75 80

Gly Thr Ile Phe Ser Gly Ser Ala Val Ile Asp Lys Asp Asn Thr Ala

85 90 95

Gly Phe Gly Lys Gly Ala Met Val Ala Ile Phe Thr His His Asn His

100 105 110

Gln Glu Glu Asp Arg Lys Thr Gly Leu His Gln Asn Gln Ser Leu Ala

115 120 125

Tyr Ser Leu Asp Lys Gly Leu Thr Trp Thr Lys Tyr Lys Gly Asn Pro

130 135 140

Val Leu Pro Asn Pro Gly Ile Trp Asp Phe Arg Asp Pro Lys Val Met

145 150 155 160

Trp His Val His Ser Gln Arg Trp Ile Met Thr Leu Ala Thr Lys Asp

165 170 175

Cys Ile Thr Phe Tyr Ser Ser Lys Asp Leu Lys Thr Trp Gln Lys Glu

180 185 190

Ser Glu Phe Gly Lys Glu Val Gly Ala His Gly Gly Val Trp Glu Cys

195 200 205

Pro Asp Leu Ile Pro Met Asp Tyr Lys Gly Thr Thr Lys Trp Val Leu

210 215 220

Leu Val Ser Ile Asn Pro Gly Gly Pro Asn Gly Gly Ser Val Thr Gln

225 230 235 240

Tyr Phe Val Gly Asp Phe Asp Gly His Gln Phe Arg Thr Thr Asp Thr

245 250 255

Lys Ile Lys Trp Leu Asp Trp Gly Pro Asp Asn Tyr Ala Gly Val Thr

260 265 270

Trp Ser Asn Leu Gly Asp Arg Gln Leu Met Ile Gly Trp Met Ser Asn

275 280 285

Trp Gln Tyr Ala Asn Val Val Pro Thr Thr Lys Trp Arg Ser Ser Ser

290 295 300

Thr Ile Pro Arg Val Leu Ser Leu Gly Lys Val Gly Gln Ser Phe Tyr

305 310 315 320

Val Ala Ser Thr Val Pro Lys Glu Ile Glu Thr Ala Phe Arg Pro Leu

325 330 335

Lys Lys Tyr His Gly Gly Gly Thr Lys Glu Val Ser Phe Glu Gln His

340 345 350

Leu Pro Gln Ala Tyr Arg Leu Asp Leu Lys Asp Leu Lys Gln Gln Ala

355 360 365

Phe Lys Val Ile Leu Ser Asn Glu Ser Gly Asp Glu Leu Val Ile Gly

370 375 380

Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr Ile Asp Arg Ser Lys Ser Gly

385 390 395 400

Glu Val Ser Phe Asn Gly Glu Phe Pro Lys Ile Ala Leu Ala Gln Arg

405 410 415

Pro Leu Asn Lys Gly Ala Leu Ser Phe Ser Leu Tyr Val Asp Val Ser

420 425 430

Ser Val Glu Leu Phe Ala Asp Glu Gly Leu Thr Val Met Thr Ser Leu

435 440 445

Phe Phe Pro Asn Lys Pro Ile Thr Lys Leu Arg Ile Val Gly Asp Lys

450 455 460

Lys Leu Glu Phe Gln Glu Ile Gly Ile Ser Ala Phe Lys Arg

465 470 475

<210> 6

<211> 1437

<212> DNA

<213> mutant Gene (mutQ 23. DELTA.3)

<400> 6

gggccccagc ataaacaagg agatcccgaa gagaagtatc gcccacttta ccattttaca 60

ccacagcagg gttggatgaa cgatcccaat ggactggttt atctggacgg taattatcat 120

cttttttttc agcataatcc cgaaaaaccc gtttgggggc ctatgcattg gggccatgcg 180

atcagtaaag atttaatcca ttgggacgag cagaagatcg cattgtaccc cgatagtttg 240

gggactattt tctccggtag tgctgtaatc gataaagata acacggcagg ttttggaaaa 300

ggtgctatgg tcgccatttt tacccatcac aatcatcagg aagaggatcg aaaaacagga 360

ttgcatcaaa atcaaagttt ggcttatagt ttggacaagg gccttacctg gacaaagtat 420

aaaggcaatc cggtattgcc aaaccctggt atatgggatt tcagagatcc aaaggtgatg 480

tggcatgtgc atagccagcg ttggattatg accttggcta caaaagattg tattactttc 540

tattcttcca aagatttgaa aacatggcag aaggaaagtg agtttggcaa agaggtgggg 600

gcccatggtg gtgtctggga atgccctgat cttatcccca tggattacaa aggaactaca 660

aaatgggttt tgttggtgag catcaatcca ggtggaccca acggtggctc ggtgacacaa 720

tattttgtgg gtgactttga tgggcatcag ttccgaacaa cggatactaa aataaagtgg 780

ttggattggg gacccgataa ctatgccggc gtaacctgga gtaacctagg ggataggcaa 840

ctgatgattg gctggatgag caactggcaa tatgccaatg tggttcctac cacaaaatgg 900

cgttcctcct caaccatacc tcgtgtttta tctttaggaa aagttggaca gtcgttctac 960

gttgcgagta ctgtgcccaa agaaattgaa accgcatttc gtccactaaa aaaataccat 1020

ggcggtggaa cgaaggaagt ttcgttcgag caacatctcc cacaagcgta ccgtttggac 1080

ctcaaagatt tgaagcagca ggcttttaaa gtcatcttat cgaatgaatc tggcgacgag 1140

ttggttattg gttatcgtga agaccaaaat gcctattata ttgatcgttc taagtccggt 1200

gaagtgtctt ttaatggaga atttccaaag atagcgttgg cccaacggcc gctaaataag 1260

ggagcacttt ctttcagttt gtatgttgat gtgagctctg tggaactttt tgcagacgag 1320

gggctaacgg taatgaccag tttatttttt ccgaacaagc cgataaccaa gctgcgtatc 1380

gtgggagata aaaagttgga gtttcaggaa atcggtattt ctgcatttaa gagataa 1437

<210> 7

<211> 523

<212> PRT

<213> recombinant wild enzyme (InuAGN25DVSHIs)

<400> 7

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Leu Glu Val Leu Phe Gln Gly Pro Gln Thr Gly Gln

35 40 45

His Lys Gln Gly Asp Pro Glu Glu Lys Tyr Arg Pro Leu Tyr His Phe

50 55 60

Thr Pro Gln Gln Gly Trp Met Asn Asp Pro Asn Gly Leu Val Tyr Leu

65 70 75 80

Asp Gly Asn Tyr His Leu Phe Phe Gln His Asn Pro Glu Lys Pro Val

85 90 95

Trp Gly Pro Met His Trp Gly His Ala Ile Ser Lys Asp Leu Ile His

100 105 110

Trp Asp Glu Gln Lys Ile Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile

115 120 125

Phe Ser Gly Ser Ala Val Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly

130 135 140

Lys Gly Ala Met Val Ala Ile Phe Thr His His Asn His Gln Glu Glu

145 150 155 160

Asp Arg Lys Thr Gly Leu His Gln Asn Gln Ser Leu Ala Tyr Ser Leu

165 170 175

Asp Lys Gly Leu Thr Trp Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro

180 185 190

Asn Pro Gly Ile Trp Asp Phe Arg Asp Pro Lys Val Met Trp His Val

195 200 205

His Ser Gln Arg Trp Ile Met Thr Leu Ala Thr Lys Asp Cys Ile Thr

210 215 220

Phe Tyr Ser Ser Lys Asp Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe

225 230 235 240

Gly Lys Glu Val Gly Ala His Gly Gly Val Trp Glu Cys Pro Asp Leu

245 250 255

Ile Pro Met Asp Tyr Lys Gly Thr Thr Lys Trp Val Leu Leu Val Ser

260 265 270

Ile Asn Pro Gly Gly Pro Asn Gly Gly Ser Val Thr Gln Tyr Phe Val

275 280 285

Gly Asp Phe Asp Gly His Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys

290 295 300

Trp Leu Asp Trp Gly Pro Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn

305 310 315 320

Leu Gly Asp Arg Gln Leu Met Ile Gly Trp Met Ser Asn Trp Gln Tyr

325 330 335

Ala Asn Val Val Pro Thr Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro

340 345 350

Arg Val Leu Ser Leu Gly Lys Val Gly Gln Ser Phe Tyr Val Ala Ser

355 360 365

Thr Val Pro Lys Glu Ile Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr

370 375 380

His Gly Gly Gly Thr Lys Glu Val Ser Phe Glu Gln His Leu Pro Gln

385 390 395 400

Ala Tyr Arg Leu Asp Leu Lys Asp Leu Lys Gln Gln Ala Phe Lys Val

405 410 415

Ile Leu Ser Asn Glu Ser Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu

420 425 430

Asp Gln Asn Ala Tyr Tyr Ile Asp Arg Ser Lys Ser Gly Glu Val Ser

435 440 445

Phe Asn Gly Glu Phe Pro Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn

450 455 460

Lys Gly Ala Leu Ser Phe Ser Leu Tyr Val Asp Val Ser Ser Val Glu

465 470 475 480

Leu Phe Ala Asp Glu Gly Leu Thr Val Met Thr Ser Leu Phe Phe Pro

485 490 495

Asn Lys Pro Ile Thr Lys Leu Arg Ile Val Gly Asp Lys Lys Leu Glu

500 505 510

Phe Gln Glu Ile Gly Ile Ser Ala Phe Lys Arg

515 520

<210> 8

<211> 517

<212> PRT

<213> recombinant mutant enzyme (MutQ 23. DELTA.6 His)

<400> 8

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Leu Glu Val Leu Phe Gln Gly Pro Gln Gly Asp Pro

35 40 45

Glu Glu Lys Tyr Arg Pro Leu Tyr His Phe Thr Pro Gln Gln Gly Trp

50 55 60

Met Asn Asp Pro Asn Gly Leu Val Tyr Leu Asp Gly Asn Tyr His Leu

65 70 75 80

Phe Phe Gln His Asn Pro Glu Lys Pro Val Trp Gly Pro Met His Trp

85 90 95

Gly His Ala Ile Ser Lys Asp Leu Ile His Trp Asp Glu Gln Lys Ile

100 105 110

Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile Phe Ser Gly Ser Ala Val

115 120 125

Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly Lys Gly Ala Met Val Ala

130 135 140

Ile Phe Thr His His Asn His Gln Glu Glu Asp Arg Lys Thr Gly Leu

145 150 155 160

His Gln Asn Gln Ser Leu Ala Tyr Ser Leu Asp Lys Gly Leu Thr Trp

165 170 175

Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro Asn Pro Gly Ile Trp Asp

180 185 190

Phe Arg Asp Pro Lys Val Met Trp His Val His Ser Gln Arg Trp Ile

195 200 205

Met Thr Leu Ala Thr Lys Asp Cys Ile Thr Phe Tyr Ser Ser Lys Asp

210 215 220

Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe Gly Lys Glu Val Gly Ala

225 230 235 240

His Gly Gly Val Trp Glu Cys Pro Asp Leu Ile Pro Met Asp Tyr Lys

245 250 255

Gly Thr Thr Lys Trp Val Leu Leu Val Ser Ile Asn Pro Gly Gly Pro

260 265 270

Asn Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp Phe Asp Gly His

275 280 285

Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys Trp Leu Asp Trp Gly Pro

290 295 300

Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn Leu Gly Asp Arg Gln Leu

305 310 315 320

Met Ile Gly Trp Met Ser Asn Trp Gln Tyr Ala Asn Val Val Pro Thr

325 330 335

Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro Arg Val Leu Ser Leu Gly

340 345 350

Lys Val Gly Gln Ser Phe Tyr Val Ala Ser Thr Val Pro Lys Glu Ile

355 360 365

Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr His Gly Gly Gly Thr Lys

370 375 380

Glu Val Ser Phe Glu Gln His Leu Pro Gln Ala Tyr Arg Leu Asp Leu

385 390 395 400

Lys Asp Leu Lys Gln Gln Ala Phe Lys Val Ile Leu Ser Asn Glu Ser

405 410 415

Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr

420 425 430

Ile Asp Arg Ser Lys Ser Gly Glu Val Ser Phe Asn Gly Glu Phe Pro

435 440 445

Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn Lys Gly Ala Leu Ser Phe

450 455 460

Ser Leu Tyr Val Asp Val Ser Ser Val Glu Leu Phe Ala Asp Glu Gly

465 470 475 480

Leu Thr Val Met Thr Ser Leu Phe Phe Pro Asn Lys Pro Ile Thr Lys

485 490 495

Leu Arg Ile Val Gly Asp Lys Lys Leu Glu Phe Gln Glu Ile Gly Ile

500 505 510

Ser Ala Phe Lys Arg

515

<210> 9

<211> 520

<212> PRT

<213> recombinant mutant enzyme (MutQ 23. DELTA.3 His)

<400> 9

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Leu Glu Val Leu Phe Gln Gly Pro Gln His Lys Gln

35 40 45

Gly Asp Pro Glu Glu Lys Tyr Arg Pro Leu Tyr His Phe Thr Pro Gln

50 55 60

Gln Gly Trp Met Asn Asp Pro Asn Gly Leu Val Tyr Leu Asp Gly Asn

65 70 75 80

Tyr His Leu Phe Phe Gln His Asn Pro Glu Lys Pro Val Trp Gly Pro

85 90 95

Met His Trp Gly His Ala Ile Ser Lys Asp Leu Ile His Trp Asp Glu

100 105 110

Gln Lys Ile Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile Phe Ser Gly

115 120 125

Ser Ala Val Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly Lys Gly Ala

130 135 140

Met Val Ala Ile Phe Thr His His Asn His Gln Glu Glu Asp Arg Lys

145 150 155 160

Thr Gly Leu His Gln Asn Gln Ser Leu Ala Tyr Ser Leu Asp Lys Gly

165 170 175

Leu Thr Trp Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro Asn Pro Gly

180 185 190

Ile Trp Asp Phe Arg Asp Pro Lys Val Met Trp His Val His Ser Gln

195 200 205

Arg Trp Ile Met Thr Leu Ala Thr Lys Asp Cys Ile Thr Phe Tyr Ser

210 215 220

Ser Lys Asp Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe Gly Lys Glu

225 230 235 240

Val Gly Ala His Gly Gly Val Trp Glu Cys Pro Asp Leu Ile Pro Met

245 250 255

Asp Tyr Lys Gly Thr Thr Lys Trp Val Leu Leu Val Ser Ile Asn Pro

260 265 270

Gly Gly Pro Asn Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp Phe

275 280 285

Asp Gly His Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys Trp Leu Asp

290 295 300

Trp Gly Pro Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn Leu Gly Asp

305 310 315 320

Arg Gln Leu Met Ile Gly Trp Met Ser Asn Trp Gln Tyr Ala Asn Val

325 330 335

Val Pro Thr Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro Arg Val Leu

340 345 350

Ser Leu Gly Lys Val Gly Gln Ser Phe Tyr Val Ala Ser Thr Val Pro

355 360 365

Lys Glu Ile Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr His Gly Gly

370 375 380

Gly Thr Lys Glu Val Ser Phe Glu Gln His Leu Pro Gln Ala Tyr Arg

385 390 395 400

Leu Asp Leu Lys Asp Leu Lys Gln Gln Ala Phe Lys Val Ile Leu Ser

405 410 415

Asn Glu Ser Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu Asp Gln Asn

420 425 430

Ala Tyr Tyr Ile Asp Arg Ser Lys Ser Gly Glu Val Ser Phe Asn Gly

435 440 445

Glu Phe Pro Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn Lys Gly Ala

450 455 460

Leu Ser Phe Ser Leu Tyr Val Asp Val Ser Ser Val Glu Leu Phe Ala

465 470 475 480

Asp Glu Gly Leu Thr Val Met Thr Ser Leu Phe Phe Pro Asn Lys Pro

485 490 495

Ile Thr Lys Leu Arg Ile Val Gly Asp Lys Lys Leu Glu Phe Gln Glu

500 505 510

Ile Gly Ile Ser Ala Phe Lys Arg

515 520

<210> 10

<211> 481

<212> PRT

<213> HRV3C protease cleavage recombinant wild enzyme product (InuAGN25DVS)

<400> 10

Gly Pro Gln Thr Gly Gln His Lys Gln Gly Asp Pro Glu Glu Lys Tyr

1 5 10 15

Arg Pro Leu Tyr His Phe Thr Pro Gln Gln Gly Trp Met Asn Asp Pro

20 25 30

Asn Gly Leu Val Tyr Leu Asp Gly Asn Tyr His Leu Phe Phe Gln His

35 40 45

Asn Pro Glu Lys Pro Val Trp Gly Pro Met His Trp Gly His Ala Ile

50 55 60

Ser Lys Asp Leu Ile His Trp Asp Glu Gln Lys Ile Ala Leu Tyr Pro

65 70 75 80

Asp Ser Leu Gly Thr Ile Phe Ser Gly Ser Ala Val Ile Asp Lys Asp

85 90 95

Asn Thr Ala Gly Phe Gly Lys Gly Ala Met Val Ala Ile Phe Thr His

100 105 110

His Asn His Gln Glu Glu Asp Arg Lys Thr Gly Leu His Gln Asn Gln

115 120 125

Ser Leu Ala Tyr Ser Leu Asp Lys Gly Leu Thr Trp Thr Lys Tyr Lys

130 135 140

Gly Asn Pro Val Leu Pro Asn Pro Gly Ile Trp Asp Phe Arg Asp Pro

145 150 155 160

Lys Val Met Trp His Val His Ser Gln Arg Trp Ile Met Thr Leu Ala

165 170 175

Thr Lys Asp Cys Ile Thr Phe Tyr Ser Ser Lys Asp Leu Lys Thr Trp

180 185 190

Gln Lys Glu Ser Glu Phe Gly Lys Glu Val Gly Ala His Gly Gly Val

195 200 205

Trp Glu Cys Pro Asp Leu Ile Pro Met Asp Tyr Lys Gly Thr Thr Lys

210 215 220

Trp Val Leu Leu Val Ser Ile Asn Pro Gly Gly Pro Asn Gly Gly Ser

225 230 235 240

Val Thr Gln Tyr Phe Val Gly Asp Phe Asp Gly His Gln Phe Arg Thr

245 250 255

Thr Asp Thr Lys Ile Lys Trp Leu Asp Trp Gly Pro Asp Asn Tyr Ala

260 265 270

Gly Val Thr Trp Ser Asn Leu Gly Asp Arg Gln Leu Met Ile Gly Trp

275 280 285

Met Ser Asn Trp Gln Tyr Ala Asn Val Val Pro Thr Thr Lys Trp Arg

290 295 300

Ser Ser Ser Thr Ile Pro Arg Val Leu Ser Leu Gly Lys Val Gly Gln

305 310 315 320

Ser Phe Tyr Val Ala Ser Thr Val Pro Lys Glu Ile Glu Thr Ala Phe

325 330 335

Arg Pro Leu Lys Lys Tyr His Gly Gly Gly Thr Lys Glu Val Ser Phe

340 345 350

Glu Gln His Leu Pro Gln Ala Tyr Arg Leu Asp Leu Lys Asp Leu Lys

355 360 365

Gln Gln Ala Phe Lys Val Ile Leu Ser Asn Glu Ser Gly Asp Glu Leu

370 375 380

Val Ile Gly Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr Ile Asp Arg Ser

385 390 395 400

Lys Ser Gly Glu Val Ser Phe Asn Gly Glu Phe Pro Lys Ile Ala Leu

405 410 415

Ala Gln Arg Pro Leu Asn Lys Gly Ala Leu Ser Phe Ser Leu Tyr Val

420 425 430

Asp Val Ser Ser Val Glu Leu Phe Ala Asp Glu Gly Leu Thr Val Met

435 440 445

Thr Ser Leu Phe Phe Pro Asn Lys Pro Ile Thr Lys Leu Arg Ile Val

450 455 460

Gly Asp Lys Lys Leu Glu Phe Gln Glu Ile Gly Ile Ser Ala Phe Lys

465 470 475 480

Arg

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 11

tggaagttct gttccagggg ccccagacgg gacagcataa acaag 45

<210> 12

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 12

ggtggtggtg ttatctctta aatgcagaaa taccgat 37

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 13

agagataaca ccaccaccac caccactg 28

<210> 14

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 14

cctggaacag aacttccagg aattcggatc cgcgacc 37

Claims

1. A heat-labile exoinulase mutant MutQ23 delta 6, characterized in that the amino acid sequence of the mutant MutQ23 delta 6 is shown as SEQ ID number 1.

2. A mutant MutQ23 Δ 6 as claimed in claim 1, wherein the gene mutQ23 Δ 6 encodes a mutant MutQ23 Δ 6 having the nucleotide sequence shown in SEQ ID number 2.

3. A recombinant vector comprising the coding gene mutQ23 Δ 6 of claim 2.

4. A recombinant bacterium comprising the encoding gene mutQ23 Δ 6 according to claim 2.

5. A method for preparing the mutant MutQ23 Δ 6 according to claim 1, comprising:

the addition of HRV3C protease cleavage site coding sequence at the 5' end of mutQ23 Δ 6 as defined in claim 2;

culturing the recombinant strain, inducing the expression of the recombinant exoinulase mutant MutQ23 delta 6, and carrying out enzyme digestion on the obtained recombinant exoinulase mutant MutQ23 delta 6 by using HRV3C protease to obtain the exoinulase mutant MutQ23 delta 6.

6. The method according to claim 5, wherein the recombinant strain is cultured in a culture medium containing 50 μ g-mL⁻¹LB broth of kanamycin.

7. The method according to claim 6, wherein the recombinant strain is cultured at 50 μ g-mL at 37 ℃⁻¹The culture was performed with shaking in LB medium containing kanamycin.

8. The method of claim 5, wherein the inducing is performed when OD is greater than OD₆₀₀When the concentration reaches 0.6-1.0, adding IPTG for induction.

9. Use of the mutant MutQ23 Δ 6 according to claim 1 in food products.

10. Use according to claim 9, wherein the food product comprises: and (5) brewing wine.