CN106434586B - Trehalose synthetase mutant and gene thereof - Google Patents

Trehalose synthetase mutant and gene thereof Download PDF

Info

Publication number
CN106434586B
CN106434586B CN201610876031.2A CN201610876031A CN106434586B CN 106434586 B CN106434586 B CN 106434586B CN 201610876031 A CN201610876031 A CN 201610876031A CN 106434586 B CN106434586 B CN 106434586B
Authority
CN
China
Prior art keywords
leu
asp
arg
pro
ala
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.)
Active
Application number
CN201610876031.2A
Other languages
Chinese (zh)
Other versions
CN106434586A (en
Inventor
黄和
王东生
江凌
周家海
徐晴
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Institute of Organic Chemistry of CAS
Nanjing Tech University
Original Assignee
Shanghai Institute of Organic Chemistry of CAS
Nanjing Tech University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shanghai Institute of Organic Chemistry of CAS, Nanjing Tech University filed Critical Shanghai Institute of Organic Chemistry of CAS
Priority to CN201610876031.2A priority Critical patent/CN106434586B/en
Publication of CN106434586A publication Critical patent/CN106434586A/en
Application granted granted Critical
Publication of CN106434586B publication Critical patent/CN106434586B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01245—Alpha,alpha-trehalose synthase (2.4.1.245)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a trehalose synthetase mutant and a gene thereof, the trehalose synthetase mutant is obtained by mutating trehalose synthetase of mycobacterium smegmatis (mycobacterium smegmatis), and particularly relates to a method for mutating amino acids at 167 th, 169 th, 174 th, 175 th and 176 th positions of the trehalose synthetase into alanine, wherein the amino acid at 169 th position is optionally mutated into alanine, and the amino acids at 167 th, 174 th, 175 th and 176 th positions are mutated into alanine to be combined.

Description

Trehalose synthetase mutant and gene thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to the field of enzyme engineering, namely a mutant, a gene, a recombinant vector, a transgenic cell of trehalose synthase and application of a composition in catalytic synthesis of trehalose.
Background
Trehalose (Trehalose) with the molecular formula of C 12 H 22 O 11. 2H 2 O is disaccharide formed by combining two glucose molecules by alpha, alpha-1, 1 glycosidic bonds, has no hemiacetal hydroxyl group, is non-reducing sugar, and has stable chemical properties, and is known as 'vital sugar' because the Trehalose can form a unique protective film on the cell surface under the severe environmental conditions of high temperature, high cold, high osmotic pressure, drying and dehydration and the like, and can effectively protect protein molecules from being inactivated, so that the Trehalose can be used as an activity protective agent of biological products and can maintain the activity of cells, and has great use in cosmetics and foods, thereby having wide market prospect and application value.
Currently, among all methods for producing trehalose, the simplest process is: trehalose synthase (Trehalose synthase) takes maltose as a substrate, and Trehalose is directly generated through one-step catalytic reaction of intramolecular transglycosylation. The reaction does not need intermediate steps, the raw material cost is low, the process is simple, and the industrial application value is high; however, trehalose synthase also has some disadvantages, mainly including low conversion rate, high by-product yield, poor temperature and pH tolerance, etc.
The method utilizes genetic engineering and enzyme engineering means to improve the activity and tolerance characteristic of trehalose synthase, and the improvement of the trehalose efficiency in catalytic synthesis reaction is a necessary way to realize the high-efficiency production of trehalose.
In the present intensive research on trehalose synthase, it was proved that trehalose synthase is one member of glycosyl hydrolase 13 Family (also called α -amylase Family), which has a typical (β/α) 8 barrel catalytic domain, it has been found that there are three different sources of trehalose synthase structures that have been resolved, and they are respectively from Mycobacterium tuberculosis, Deinococcusradiodurans,Mycobacterium smegmatis three different bacteria, and their PDB numbers are 4LXF, 4TVU and 3 ZO., and based on the comparison of different structures and the hydrophobicity and hydrophilicity of amino acids, after trying site mutation in different regions, the mutated enzyme is found to be significantly improved compared with the original wild type, thus completing the scheme of the present invention.
disclosure of Invention
The technical problem to be solved by the invention is that the activity of the existing trehalose synthase from mycobacterium smegmatis Mycobacterium smegmatis is too low, and in order to improve the activity of the enzyme, the invention provides a trehalose synthase mutant from mycobacterium smegmatis Mycobacterium smegmatis, wherein the amino acid sequence of the enzyme is shown as SEQ ID NO 2, 4, 6, 8 or 10.
In the present invention, the inventors tried site mutation of the enzyme in different regions, and found that amino acids at positions 167, 169, 174, 175 and 176 were mutated to alanine; compared with the wild type, the mutant enzyme is obviously improved, and the scheme of the invention is completed based on the improvement.
Specifically, the scheme of the invention is as follows:
The 167 th, 169 th, 174 th, 175 th and 176 th amino acids derived from the wild-type trehalose synthetase of mycobacterium smegmatis Mycobacterium smegmatis are mutated into alanine, wherein the 169 th amino acid is optionally mutated into alanine, and the 167 th, 174 th, 175 th and 176 th amino acids are mutated into alanine, which are combined to obtain five mutants of R169A, R169A-D174A, R169A-D174A-D167A, R169A-D174A-D737167 3-T38175 42, R169A-D174 167-D82167-T175A-E176A.
Preferably, the amino acid 169 is mutated to alanine.
Correspondingly, the nucleotide sequence of the mutant gene of the enzyme is shown as SEQ ID NO: 1. 3, 5, 7 or 9.
The recombinant vector of the present invention can be realized by introducing the gene sequence of the present invention into a conventional vector or plasmid, which comprises the above gene sequence.
Transgenic cells containing the above gene include, for example, Escherichia coli, yeast cells, Bacillus subtilis cells, etc. Preferably, the gene is expressed in Escherichia coli or Bacillus subtilis.
A composition, wherein any one of the above enzyme mutants is used as an active ingredient in the composition, preferably for catalyzing the synthesis of trehalose.
The enzyme mutant, the gene, the recombinant vector, the transgenic cell and the composition are applied to the catalytic synthesis of trehalose.
The mutant has the advantages that the mutant has five mutants of R169A, R169A-D174A, R169A-D174A-D167A, R169A-D174A-D167A-T175A and R169A-D174A-D167A-T175A-E176A, and the enzyme activity after mutation is respectively improved by 1.07 times, 1.05 times, 2.56 times, 2.41 times and 1.94 times compared with the enzyme activity of a wild type.
Detailed Description
the present invention will be further described with reference to the following examples. The present embodiments are to be considered as illustrative and not restrictive, and the spirit and scope of the invention is not to be limited to the details and modifications thereof.
EXAMPLE 1 preparation of trehalose synthase mutants
Primers for introducing five mutants of R169A, R169A-D174A, R169A-D174A-D167A, R169A-D174A-D167A-T175A and R169A-D174A-D167A-T175A-E176A are respectively designed and sequentially synthesized according to gene sequences of the trehalose synthetase derived from mycobacterium smegmatis, and the DNA coding sequence is determined by sequentially carrying out site-directed mutagenesis on the trehalose synthetase in five steps.
The Arg codon at position 169 was identified as the Ala codon.
In the last step, the Asp codon 174 was changed to Ala codon.
In the last step, the 167 th Asp codon was changed to Ala codon.
In the last step, Thr codon 175 was changed to Ala codon.
In the last step, the codon Glu 176 was changed to Ala.
The mutant gene is connected with a proper expression vector and is introduced into escherichia coli for expression, and the trehalose synthetase with five different mutations is obtained.
In particular, the method comprises the following steps of,
The first step is as follows: the original plasmid TreS-pET22b is taken as a template, and the site-directed mutation primer for introducing R169A mutation is as follows:
A forward primer: 5-aggtatcccgacgcggccatcatcttcgtcgac-3
Reverse primer: 5-gtcgacgaagatgatggccgcgtcgggatacct-3
The second step is that: the site-directed mutagenesis primer for introducing the D174A mutation by taking the mutant R169A as a template is as follows:
A forward primer: 5-gccatcatcttcgtcgccaccgaggagtcgaac-3
Reverse primer: 5-gttcgactcctcggtggcgacgaagatgatggc-3
The third step: the site-directed mutagenesis primer for introducing the D167A mutation by taking the mutant R169A-D174A as a template is as follows:
A forward primer: 5-agcgacaggtatcccgccgcggccatcatcttc-3
Reverse primer: 5-gaagatgatggccgcggcgggatacctgtcgct-3
The fourth step: the site-directed mutagenesis primer for introducing the T175A mutation by taking the mutant R169A-D174A-D167A as a template is as follows:
A forward primer: 5-atcatcttcgtcgccgccgaggagtcgaactgg-3
reverse primer: 5-ccagttcgactcctcggcggcgacgaagatgat-3
The fifth step: the site-directed mutagenesis primer for introducing the E176A mutation is prepared by taking the mutant R169A-D174A-D167A-T175A as a template:
A forward primer: 5-atcttcgtcgccgccgcggagtcgaactggacg-3
Reverse primer: 5-cgtccagttcgactccgcggcggcgacgaagat-3.
The PCR reaction systems are as follows: 5 XPS buffer10 uL, dNTPsMix 4 uL, forward primer (10 uM) 1 uL, reverse primer (10 uM) 1 uL, template DNA1 uL, Primerstar HS (5U/. mu.L) 0.5 uL, and double distilled water was added to 50 uL.
The PCR amplification conditions were all: pre-denaturation at 98 ℃ for 5min followed by 28 cycles (98 ℃ for 15s, 58 ℃ for 30s, 68 ℃ for 6 min), extension at 68 ℃ for 10 min.
Digesting the PCR product by Dpn I, transforming escherichia coli DH5 alpha competence, culturing competent cells in LB solid culture medium overnight (ampicillin resistance), selecting and culturing in LB liquid culture medium, extracting plasmids, correctly sequencing mutant plasmids, and transforming into expression host escherichia coli BL21(DE3) competent cells.
Example 2 expression and purification of trehalose synthase
The constructed plasmid pET22b-TreS is transferred into E.coli BL21(DE3) host for expression. A single clone was inoculated into 5 mL of LB liquid medium containing 100 mg/L of ampicillin, and cultured with shaking at 37 ℃ for 4 hours in a shaker at 200 rpm. Transferring the cultured strain to 1L LB liquid culture medium with 100 mg/L ampicillin concentration, culturing at 37 deg.C until OD600=0.6, cooling to 30 deg.C, adding IPTG (final concentration of 0.2 mM) to induce expression for 14 h, centrifuging at 6000 rpm for 10min, collecting thallus, and freezing at-80 deg.C in refrigerator.
10 g of the frozen cells were taken and resuspended in BufferA (25 mM Tris, 300mM NaCl, pH 8.0). Crushing the cells by using a high-pressure cell crusher, centrifuging for 30min at 16000 rpm after crushing, and collecting the supernatant; secondly, Ni-NTA affinity chromatography: the Ni-NTA column was equilibrated with BufferA, the disrupted supernatant after centrifugation was slowly applied to the column, and unbound protein was washed away with BufferB (300 mM Tris, 300mM NaCl, pH 8.0). Elution was then performed with 5%, 10%, 20%, 100% BufferB and the permeate was collected. The eluted samples were purified of protein using SDS-PAGE, the high purity protein eluates were pooled and concentrated to 2 mL using 30kDa Millipore ultrafiltration tubes at 4 ℃ at 2750 rpm; finally, separation was performed by Superdex 200 gel chromatography: the gel column was equilibrated first with one column volume of BufferC (25 mM Tris, 300mM NaCl, 2mM DTT, pH 8.0). Loading and Collection Using AKTA Purifier FPLC, 2 mL of the concentrated protein sample was loaded at 1 mL/min, eluted using BufferC, and collected in a fraction collector at 1 mL/min. The collected samples were subjected to protein detection using SDS-PAGE, pooled high-purity proteins, dispensed at a volume of 50. mu.L/tube, snap frozen using liquid nitrogen and then stored in a freezer at-80 ℃.
Example 3 expression of trehalose synthase mutants in Bacillus subtilis
(1) Construction of Bacillus subtilis expression vector
Five mutants of trehalose synthase are respectively cloned to a pMA5 vector by molecular biological operations such as double enzyme digestion, gel recovery, T4 ligase ligation and the like to obtain five different pMA5-TreS for expression in bacillus subtilis.
(2) Expression of recombinant bacteria WB600 (pMA 5-tres)
Introducing the constructed recombinant vector into bacillus subtilis WB600 competence, activating recombinant bacteria WB600, diluting and coating, then selecting a ring of recombinant bacteria with a single colony and a moderate size, further activating in LB, and culturing at 37 ℃ at 250r/min overnight. And (3) microscopic examination is carried out on the next day to determine whether the strain is infected, then the strain is inoculated into an LB culture medium according to the inoculation amount of 3% in an aseptic environment, and the culture is carried out for 24 hours at 37 ℃. 1mL of the bacterial solution was centrifuged at 12000r/min for 2min, washed repeatedly with distilled water for 2 times, and finally resuspended in 100. mu.L of distilled water. The same procedure was used for the wild-type trehalose synthase WB600 (pMA 5-tres) control and the blank WB600 control. Taking 10 mu L of the recombinant bacteria and blank bacteria suspension respectively, mixing with 10 mu L of loading buffer solution, carrying out boiling water bath for 10min, and carrying out SDS-PAGE gel electrophoresis. The expression of each mutant in B.glumae was observed. Electrophoresis results show that the expression effect of the mutant and the wild trehalose synthetase in the bacillus subtilis is consistent.
EXAMPLE 4 determination of trehalose synthetase enzymatic Activity
(1) definition of enzyme activity unit: the amount of trehalose synthase required to produce 1. mu. moL of trehalose at 25 ℃ in less than 1min is one unit of enzyme activity.
(2) Enzyme activity determination: 0.54g of maltose reagent was dissolved in 10ml of phosphate buffer solution with pH =7.4 to give a final concentration of 150 mmol/L. Mu.l maltose solution and 200. mu.l crude enzyme solution were mixed well (three in each group in parallel), 1ml reaction system at 25 deg.C for 30min, boiling for 10min to inactivate enzyme. After cooling, centrifuging at 5000r/min for 10min, taking supernatant, and measuring the content of trehalose.
(3) And (3) determining a product and a substrate by using high performance liquid chromatography:
The measurement conditions were as follows: the mobile phase is acetonitrile: water = 75: 25; flow rate: 0.6 ml/min; selecting an amino column (the column temperature is 35 ℃) as a chromatographic column; shodex RI101 parallax refraction detector was chosen.
(4) Shaking culture of wild type trehalose synthetase and mutant enzyme for 16h, detecting by high performance liquid chromatography and calculating to obtain enzyme activity, which is listed in Table 1.
TABLE 1 enzymatic Activity of wild-type trehalose synthase and mutant enzymes
Note that enzyme activity/U.ml -1 enzyme activity/U.ml -1
The experimental results show that compared with the wild type, the enzyme activity of the mutant enzyme is improved by 1.07 times, 1.05 times, 2.56 times, 2.41 times and 1.94 times when R169A with the number of 1 is expressed in escherichia coli, R169A-D174A with the number of 2 is expressed in escherichia coli, R169A-D174A-D167A with the number of 3 is expressed in bacillus subtilis, R169A-D174A-D167A-T175A with the number of 4 is expressed in bacillus subtilis and R169A-D174A-D167A-T175A-E176A with the number of 5 is expressed in escherichia coli.
EXAMPLE 5 analysis of the enzymatic Properties of the mutants
All reactions were carried out according to standard reaction systems: wherein, the mutants of the mutant enzymes 1, 2, 3, 4 and 5 are R169A, R169A-D174A, R169A-D174A-D167A, R169A-D174A-D167A-T175A and R169A-D174A-D167A-T175A-E176A respectively.
(1) Determination of the optimum pH of the enzyme: the effect on the activity of the recombinant enzyme was determined in a series of PBS buffers at different pH values (5.5, 6.0, 6.5, 7.0, 7.5, 8.0). The pH value at which the content of the reaction product is the highest is the optimum pH value of the enzyme.
(2) Determination of optimum reaction temperature: the enzyme activities of the recombinant enzymes at a series of temperatures (10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃) are respectively measured to determine the optimal reaction temperature of the enzymes.
(3) Under the optimal temperature and pH value, the influence of various metal ions such as Fe 3+, Cu 2+, Zn 2+, Fe 2+, Ni 2+, Mn 2+ and the like on the enzyme activity is measured, the final concentration of the metal ions in a reaction system is 5mM, and PBS buffer solution without other metal ions is used as a control (100 percent relative enzyme activity).
(4) Under the optimal reaction condition, the product composition of the recombinase catalytic substrate with different action time is determined.
(5) Under the optimum reaction conditions, the substrate specificity of the recombinase is determined (the substrate includes trehalose, sucrose, cellobiose, starch, isomaltulose, trehalulose, maltotriose, etc.). After the samples had reacted for 5 h under optimal conditions, the product was checked by HPLC.
The experimental result shows that the optimum pH values of the wild enzyme and the enzymes of each mutant are respectively 7.2, 7.3, 7.2, 7.0 and 6.9, the optimum reaction temperatures are respectively 28 ℃, 27.5 ℃, 28 ℃, 29 ℃ and 29 ℃, the effects of metal ions on the mutant enzymes are shown, the experiment shows that 8 metal ions such as Ni 2+, Fe 3+, Mn 2+ and the like have no activation effect on the enzyme activities of the wild trehalose synthase and the mutants thereof, and the inhibition effect of most metal ions on the enzyme activities is not obvious, wherein the Cu 2+ ions have strong inhibition effects on the enzyme activities of the wild trehalose and the mutants, and in the aspect of substrate specificity, according to the detection data of HPLC, the optimum substrates of the wild trehalose and the mutant enzymes are maltose.
The results of the qualitative enzymology experiments are combined to show that the enzymology property of each mutant is similar to that of the wild enzyme.
Example 6 optimization of fermentation conditions of trehalose synthase mutants in E.coli
by adopting a single-factor test and an orthogonal test, the influence of 4 factors of the bacteria concentration OD600 nm, the induction temperature, the induction time and the trehalose conversion time of the trehalose synthase on the fermentation yield and the conversion rate when an inducer is added in the fermentation process of the trehalose synthase mutant is researched, and the optimal fermentation condition and the optimal conversion condition of the trehalose synthase mutant in escherichia coli are determined. The result shows that the transformation time and the induction temperature have larger influence on the transformation rate, the inducer is added when the Escherichia coli grows to the OD600 nm value of 0.8, the induction temperature is kept at 37 ℃ for 14 h, and the transformation rate is the highest when the temperature is 37 ℃, the pH value is 7.2 and the time is 3h in the transformation process. Under the optimal condition, the conversion rate of converting maltose into trehalose by different trehalose synthetase mutants can reach 69.29 percent at most.
Sequence listing
<110> Nanjing university of industry
Shanghai organic chemistry institute of Chinese academy of sciences
<120> trehalose synthase mutant and gene thereof
<130> xb16100802
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 1782
<212> DNA
<213> Artificial
<220>
<223> mutant enzyme 1
<220>
<221> CDS
<222> (1)..(1782)
<400> 1
atg gag gag cac acg cag ggc agc cat gtc gag gcg ggg atc gtc gag 48
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
cat ccg aac gcc gag gac ttc ggt cat gcg cgc acg ctt ccc acc gac 96
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
acc aac tgg ttc aag cac gcg gtg ttc tat gag gtg ctg gtg cgg gcg 144
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
ttc tac gac tcg aac gcc gac ggt atc ggt gat ctg cgc ggc ctc acc 192
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
gaa aaa ctc gac tac atc aag tgg ctc ggg gtc gac tgc ctg tgg ctg 240
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
ccg ccg ttc tac gat tcg ccg ctg cgc gac ggc ggt tac gac atc cgc 288
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
gac ttc tac aag gtg ctg ccc gag ttc ggc acg gtc gac gac ttc gtc 336
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
acc ctg ctg gac gcg gcc cac cgc agg ggc atc cgc atc atc acc gac 384
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
ctg gtg atg aac cac acc tcc gat cag cac gag tgg ttc cag gaa tcc 432
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
cgg cac aat ccc gac ggc ccc tac ggc gac ttc tac gtc tgg agc gac 480
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
acc agc gac agg tat ccc gac gcg gcc atc atc ttc gtc gac acc gag 528
Thr Ser Asp Arg Tyr Pro Asp Ala Ala Ile Ile Phe Val Asp Thr Glu
165 170 175
gag tcg aac tgg acg ttc gac ccc gtc cgc agg cag ttc tac tgg cac 576
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
cgc ttc ttc agc cac cag ccc gac ctc aac tac gac aac ccg gct gtg 624
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
cag gag gcg atg ctc gac gtg ctg agg ttc tgg ctg gac ctc ggt atc 672
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
gac ggt ttc cgt ctg gac gcg gtg ccg tac ctg ttc gag cgc gag ggc 720
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
acc aac tgc gag aac ctg ccc gag acc cac gcg ttc ctc aag cgg tgc 768
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
cgc aag gcg atc gac gac gag tac ccg ggg cgc gtg ctg ctg gcc gag 816
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
gcc aat cag tgg ccc gcc gac gtc gtc gcg tac ttc ggc gac ccg gac 864
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
acc ggc ggc gac gaa tgc cac atg gcg ttc cac ttc ccg ctg atg cca 912
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
cgg atc ttc atg gcc gtg cgg cgc gag tcc cgc ttc ccg atc tcg gag 960
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
atc ctg gcg cag acc ccg ccg atc ccc gac acc gcg cag tgg ggc atc 1008
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
ttc ctg cgc aac cac gac gag ctc acg ctc gag atg gtc acc gac gaa 1056
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
gaa cgt gac tac atg tac gcc gag tac gcc aag gac ccg cgg atg aag 1104
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
gcc aac gtc ggc atc cgg cgc agg ctc gca ccg ctg ttg gag aac gac 1152
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
cgc aac cag atc gag ttg ttc acc gcg ctg ctg ctc tcg ctg ccg ggg 1200
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
tcg ccg gtg ctg tac tac ggt gac gag ata ggt atg ggg gac atc atc 1248
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
tgg ctc ggc gac cgc gac agc gtg cgt acg ccc atg cag tgg acc ccc 1296
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
gac cgc aat gcg ggc ttc tcc aag gcg act ccg ggc cgg ctc tac ctg 1344
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
ccg ccc aac cag gac gcg gtg tac ggc tac cac tct gtg aac gtc gag 1392
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
gcg caa ctc gac agc tcc agc tcg ctg ttg aac tgg acg cgc aac atg 1440
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
ctg gcc gtg cgc agc cgc cac gat gcc ttc gca gtc ggc acg ttc cgc 1488
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
gaa ctg ggc ggc tcc aat ccg tcg gtg ctg gcg tac atc cgg gag gtc 1536
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
acg cgc caa cag ggc gac ggc ggc gcg aag acc gat gcc gtc ctg tgt 1584
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
gtc aac aac ctg tcc cgc ttc ccc cag ccc atc gag ttg aat ctc cag 1632
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
cag tgg gcc ggt tac ata ccc gtc gag atg acc ggc tac gtc gag ttc 1680
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
ccg agt atc ggc cag ttg ccg tac ctg ctc acc ctg ccc ggt cac ggg 1728
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
ttc tac tgg ttc cag ctt cga gaa ccc gat cca gaa ccg gga gcg cag 1776
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
caa tga 1782
Gln
<210> 2
<211> 593
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 2
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
Thr Ser Asp Arg Tyr Pro Asp Ala Ala Ile Ile Phe Val Asp Thr Glu
165 170 175
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
Gln
<210> 3
<211> 1782
<212> DNA
<213> Artificial
<220>
<223> mutant enzyme 2
<220>
<221> CDS
<222> (1)..(1782)
<400> 3
atg gag gag cac acg cag ggc agc cat gtc gag gcg ggg atc gtc gag 48
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
cat ccg aac gcc gag gac ttc ggt cat gcg cgc acg ctt ccc acc gac 96
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
acc aac tgg ttc aag cac gcg gtg ttc tat gag gtg ctg gtg cgg gcg 144
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
ttc tac gac tcg aac gcc gac ggt atc ggt gat ctg cgc ggc ctc acc 192
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
gaa aaa ctc gac tac atc aag tgg ctc ggg gtc gac tgc ctg tgg ctg 240
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
ccg ccg ttc tac gat tcg ccg ctg cgc gac ggc ggt tac gac atc cgc 288
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
gac ttc tac aag gtg ctg ccc gag ttc ggc acg gtc gac gac ttc gtc 336
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
acc ctg ctg gac gcg gcc cac cgc agg ggc atc cgc atc atc acc gac 384
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
ctg gtg atg aac cac acc tcc gat cag cac gag tgg ttc cag gaa tcc 432
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
cgg cac aat ccc gac ggc ccc tac ggc gac ttc tac gtc tgg agc gac 480
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
acc agc gac agg tat ccc gac gcg gcc atc atc ttc gtc gcc acc gag 528
Thr Ser Asp Arg Tyr Pro Asp Ala Ala Ile Ile Phe Val Ala Thr Glu
165 170 175
gag tcg aac tgg acg ttc gac ccc gtc cgc agg cag ttc tac tgg cac 576
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
cgc ttc ttc agc cac cag ccc gac ctc aac tac gac aac ccg gct gtg 624
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
cag gag gcg atg ctc gac gtg ctg agg ttc tgg ctg gac ctc ggt atc 672
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
gac ggt ttc cgt ctg gac gcg gtg ccg tac ctg ttc gag cgc gag ggc 720
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
acc aac tgc gag aac ctg ccc gag acc cac gcg ttc ctc aag cgg tgc 768
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
cgc aag gcg atc gac gac gag tac ccg ggg cgc gtg ctg ctg gcc gag 816
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
gcc aat cag tgg ccc gcc gac gtc gtc gcg tac ttc ggc gac ccg gac 864
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
acc ggc ggc gac gaa tgc cac atg gcg ttc cac ttc ccg ctg atg cca 912
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
cgg atc ttc atg gcc gtg cgg cgc gag tcc cgc ttc ccg atc tcg gag 960
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
atc ctg gcg cag acc ccg ccg atc ccc gac acc gcg cag tgg ggc atc 1008
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
ttc ctg cgc aac cac gac gag ctc acg ctc gag atg gtc acc gac gaa 1056
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
gaa cgt gac tac atg tac gcc gag tac gcc aag gac ccg cgg atg aag 1104
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
gcc aac gtc ggc atc cgg cgc agg ctc gca ccg ctg ttg gag aac gac 1152
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
cgc aac cag atc gag ttg ttc acc gcg ctg ctg ctc tcg ctg ccg ggg 1200
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
tcg ccg gtg ctg tac tac ggt gac gag ata ggt atg ggg gac atc atc 1248
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
tgg ctc ggc gac cgc gac agc gtg cgt acg ccc atg cag tgg acc ccc 1296
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
gac cgc aat gcg ggc ttc tcc aag gcg act ccg ggc cgg ctc tac ctg 1344
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
ccg ccc aac cag gac gcg gtg tac ggc tac cac tct gtg aac gtc gag 1392
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
gcg caa ctc gac agc tcc agc tcg ctg ttg aac tgg acg cgc aac atg 1440
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
ctg gcc gtg cgc agc cgc cac gat gcc ttc gca gtc ggc acg ttc cgc 1488
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
gaa ctg ggc ggc tcc aat ccg tcg gtg ctg gcg tac atc cgg gag gtc 1536
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
acg cgc caa cag ggc gac ggc ggc gcg aag acc gat gcc gtc ctg tgt 1584
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
gtc aac aac ctg tcc cgc ttc ccc cag ccc atc gag ttg aat ctc cag 1632
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
cag tgg gcc ggt tac ata ccc gtc gag atg acc ggc tac gtc gag ttc 1680
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
ccg agt atc ggc cag ttg ccg tac ctg ctc acc ctg ccc ggt cac ggg 1728
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
ttc tac tgg ttc cag ctt cga gaa ccc gat cca gaa ccg gga gcg cag 1776
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
caa tga 1782
Gln
<210> 4
<211> 593
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 4
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
Thr Ser Asp Arg Tyr Pro Asp Ala Ala Ile Ile Phe Val Ala Thr Glu
165 170 175
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
Gln
<210> 5
<211> 1782
<212> DNA
<213> Artificial
<220>
<223> mutant enzyme 3
<220>
<221> CDS
<222> (1)..(1782)
<400> 5
atg gag gag cac acg cag ggc agc cat gtc gag gcg ggg atc gtc gag 48
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
cat ccg aac gcc gag gac ttc ggt cat gcg cgc acg ctt ccc acc gac 96
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
acc aac tgg ttc aag cac gcg gtg ttc tat gag gtg ctg gtg cgg gcg 144
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
ttc tac gac tcg aac gcc gac ggt atc ggt gat ctg cgc ggc ctc acc 192
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
gaa aaa ctc gac tac atc aag tgg ctc ggg gtc gac tgc ctg tgg ctg 240
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
ccg ccg ttc tac gat tcg ccg ctg cgc gac ggc ggt tac gac atc cgc 288
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
gac ttc tac aag gtg ctg ccc gag ttc ggc acg gtc gac gac ttc gtc 336
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
acc ctg ctg gac gcg gcc cac cgc agg ggc atc cgc atc atc acc gac 384
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
ctg gtg atg aac cac acc tcc gat cag cac gag tgg ttc cag gaa tcc 432
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
cgg cac aat ccc gac ggc ccc tac ggc gac ttc tac gtc tgg agc gac 480
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
acc agc gac agg tat ccc gcc gcg gcc atc atc ttc gtc gcc acc gag 528
Thr Ser Asp Arg Tyr Pro Ala Ala Ala Ile Ile Phe Val Ala Thr Glu
165 170 175
gag tcg aac tgg acg ttc gac ccc gtc cgc agg cag ttc tac tgg cac 576
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
cgc ttc ttc agc cac cag ccc gac ctc aac tac gac aac ccg gct gtg 624
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
cag gag gcg atg ctc gac gtg ctg agg ttc tgg ctg gac ctc ggt atc 672
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
gac ggt ttc cgt ctg gac gcg gtg ccg tac ctg ttc gag cgc gag ggc 720
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
acc aac tgc gag aac ctg ccc gag acc cac gcg ttc ctc aag cgg tgc 768
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
cgc aag gcg atc gac gac gag tac ccg ggg cgc gtg ctg ctg gcc gag 816
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
gcc aat cag tgg ccc gcc gac gtc gtc gcg tac ttc ggc gac ccg gac 864
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
acc ggc ggc gac gaa tgc cac atg gcg ttc cac ttc ccg ctg atg cca 912
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
cgg atc ttc atg gcc gtg cgg cgc gag tcc cgc ttc ccg atc tcg gag 960
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
atc ctg gcg cag acc ccg ccg atc ccc gac acc gcg cag tgg ggc atc 1008
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
ttc ctg cgc aac cac gac gag ctc acg ctc gag atg gtc acc gac gaa 1056
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
gaa cgt gac tac atg tac gcc gag tac gcc aag gac ccg cgg atg aag 1104
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
gcc aac gtc ggc atc cgg cgc agg ctc gca ccg ctg ttg gag aac gac 1152
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
cgc aac cag atc gag ttg ttc acc gcg ctg ctg ctc tcg ctg ccg ggg 1200
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
tcg ccg gtg ctg tac tac ggt gac gag ata ggt atg ggg gac atc atc 1248
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
tgg ctc ggc gac cgc gac agc gtg cgt acg ccc atg cag tgg acc ccc 1296
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
gac cgc aat gcg ggc ttc tcc aag gcg act ccg ggc cgg ctc tac ctg 1344
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
ccg ccc aac cag gac gcg gtg tac ggc tac cac tct gtg aac gtc gag 1392
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
gcg caa ctc gac agc tcc agc tcg ctg ttg aac tgg acg cgc aac atg 1440
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
ctg gcc gtg cgc agc cgc cac gat gcc ttc gca gtc ggc acg ttc cgc 1488
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
gaa ctg ggc ggc tcc aat ccg tcg gtg ctg gcg tac atc cgg gag gtc 1536
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
acg cgc caa cag ggc gac ggc ggc gcg aag acc gat gcc gtc ctg tgt 1584
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
gtc aac aac ctg tcc cgc ttc ccc cag ccc atc gag ttg aat ctc cag 1632
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
cag tgg gcc ggt tac ata ccc gtc gag atg acc ggc tac gtc gag ttc 1680
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
ccg agt atc ggc cag ttg ccg tac ctg ctc acc ctg ccc ggt cac ggg 1728
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
ttc tac tgg ttc cag ctt cga gaa ccc gat cca gaa ccg gga gcg cag 1776
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
caa tga 1782
Gln
<210> 6
<211> 593
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 6
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
Thr Ser Asp Arg Tyr Pro Ala Ala Ala Ile Ile Phe Val Ala Thr Glu
165 170 175
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
Gln
<210> 7
<211> 1782
<212> DNA
<213> Artificial
<220>
<223> mutant enzyme 4
<220>
<221> CDS
<222> (1)..(1782)
<400> 7
atg gag gag cac acg cag ggc agc cat gtc gag gcg ggg atc gtc gag 48
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
cat ccg aac gcc gag gac ttc ggt cat gcg cgc acg ctt ccc acc gac 96
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
acc aac tgg ttc aag cac gcg gtg ttc tat gag gtg ctg gtg cgg gcg 144
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
ttc tac gac tcg aac gcc gac ggt atc ggt gat ctg cgc ggc ctc acc 192
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
gaa aaa ctc gac tac atc aag tgg ctc ggg gtc gac tgc ctg tgg ctg 240
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
ccg ccg ttc tac gat tcg ccg ctg cgc gac ggc ggt tac gac atc cgc 288
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
gac ttc tac aag gtg ctg ccc gag ttc ggc acg gtc gac gac ttc gtc 336
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
acc ctg ctg gac gcg gcc cac cgc agg ggc atc cgc atc atc acc gac 384
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
ctg gtg atg aac cac acc tcc gat cag cac gag tgg ttc cag gaa tcc 432
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
cgg cac aat ccc gac ggc ccc tac ggc gac ttc tac gtc tgg agc gac 480
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
acc agc gac agg tat ccc gcc gcg gcc atc atc ttc gtc gcc gcc gag 528
Thr Ser Asp Arg Tyr Pro Ala Ala Ala Ile Ile Phe Val Ala Ala Glu
165 170 175
gag tcg aac tgg acg ttc gac ccc gtc cgc agg cag ttc tac tgg cac 576
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
cgc ttc ttc agc cac cag ccc gac ctc aac tac gac aac ccg gct gtg 624
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
cag gag gcg atg ctc gac gtg ctg agg ttc tgg ctg gac ctc ggt atc 672
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
gac ggt ttc cgt ctg gac gcg gtg ccg tac ctg ttc gag cgc gag ggc 720
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
acc aac tgc gag aac ctg ccc gag acc cac gcg ttc ctc aag cgg tgc 768
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
cgc aag gcg atc gac gac gag tac ccg ggg cgc gtg ctg ctg gcc gag 816
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
gcc aat cag tgg ccc gcc gac gtc gtc gcg tac ttc ggc gac ccg gac 864
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
acc ggc ggc gac gaa tgc cac atg gcg ttc cac ttc ccg ctg atg cca 912
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
cgg atc ttc atg gcc gtg cgg cgc gag tcc cgc ttc ccg atc tcg gag 960
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
atc ctg gcg cag acc ccg ccg atc ccc gac acc gcg cag tgg ggc atc 1008
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
ttc ctg cgc aac cac gac gag ctc acg ctc gag atg gtc acc gac gaa 1056
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
gaa cgt gac tac atg tac gcc gag tac gcc aag gac ccg cgg atg aag 1104
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
gcc aac gtc ggc atc cgg cgc agg ctc gca ccg ctg ttg gag aac gac 1152
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
cgc aac cag atc gag ttg ttc acc gcg ctg ctg ctc tcg ctg ccg ggg 1200
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
tcg ccg gtg ctg tac tac ggt gac gag ata ggt atg ggg gac atc atc 1248
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
tgg ctc ggc gac cgc gac agc gtg cgt acg ccc atg cag tgg acc ccc 1296
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
gac cgc aat gcg ggc ttc tcc aag gcg act ccg ggc cgg ctc tac ctg 1344
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
ccg ccc aac cag gac gcg gtg tac ggc tac cac tct gtg aac gtc gag 1392
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
gcg caa ctc gac agc tcc agc tcg ctg ttg aac tgg acg cgc aac atg 1440
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
ctg gcc gtg cgc agc cgc cac gat gcc ttc gca gtc ggc acg ttc cgc 1488
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
gaa ctg ggc ggc tcc aat ccg tcg gtg ctg gcg tac atc cgg gag gtc 1536
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
acg cgc caa cag ggc gac ggc ggc gcg aag acc gat gcc gtc ctg tgt 1584
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
gtc aac aac ctg tcc cgc ttc ccc cag ccc atc gag ttg aat ctc cag 1632
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
cag tgg gcc ggt tac ata ccc gtc gag atg acc ggc tac gtc gag ttc 1680
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
ccg agt atc ggc cag ttg ccg tac ctg ctc acc ctg ccc ggt cac ggg 1728
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
ttc tac tgg ttc cag ctt cga gaa ccc gat cca gaa ccg gga gcg cag 1776
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
caa tga 1782
Gln
<210> 8
<211> 593
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 8
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
Thr Ser Asp Arg Tyr Pro Ala Ala Ala Ile Ile Phe Val Ala Ala Glu
165 170 175
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
Gln
<210> 9
<211> 1782
<212> DNA
<213> Artificial
<220>
<223> mutant enzyme 5
<220>
<221> CDS
<222> (1)..(1782)
<400> 9
atg gag gag cac acg cag ggc agc cat gtc gag gcg ggg atc gtc gag 48
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
cat ccg aac gcc gag gac ttc ggt cat gcg cgc acg ctt ccc acc gac 96
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
acc aac tgg ttc aag cac gcg gtg ttc tat gag gtg ctg gtg cgg gcg 144
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
ttc tac gac tcg aac gcc gac ggt atc ggt gat ctg cgc ggc ctc acc 192
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
gaa aaa ctc gac tac atc aag tgg ctc ggg gtc gac tgc ctg tgg ctg 240
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
ccg ccg ttc tac gat tcg ccg ctg cgc gac ggc ggt tac gac atc cgc 288
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
gac ttc tac aag gtg ctg ccc gag ttc ggc acg gtc gac gac ttc gtc 336
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
acc ctg ctg gac gcg gcc cac cgc agg ggc atc cgc atc atc acc gac 384
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
ctg gtg atg aac cac acc tcc gat cag cac gag tgg ttc cag gaa tcc 432
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
cgg cac aat ccc gac ggc ccc tac ggc gac ttc tac gtc tgg agc gac 480
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
acc agc gac agg tat ccc gcc gcg gcc atc atc ttc gtc gcc gcc gcg 528
Thr Ser Asp Arg Tyr Pro Ala Ala Ala Ile Ile Phe Val Ala Ala Ala
165 170 175
gag tcg aac tgg acg ttc gac ccc gtc cgc agg cag ttc tac tgg cac 576
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
cgc ttc ttc agc cac cag ccc gac ctc aac tac gac aac ccg gct gtg 624
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
cag gag gcg atg ctc gac gtg ctg agg ttc tgg ctg gac ctc ggt atc 672
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
gac ggt ttc cgt ctg gac gcg gtg ccg tac ctg ttc gag cgc gag ggc 720
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
acc aac tgc gag aac ctg ccc gag acc cac gcg ttc ctc aag cgg tgc 768
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
cgc aag gcg atc gac gac gag tac ccg ggg cgc gtg ctg ctg gcc gag 816
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
gcc aat cag tgg ccc gcc gac gtc gtc gcg tac ttc ggc gac ccg gac 864
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
acc ggc ggc gac gaa tgc cac atg gcg ttc cac ttc ccg ctg atg cca 912
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
cgg atc ttc atg gcc gtg cgg cgc gag tcc cgc ttc ccg atc tcg gag 960
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
atc ctg gcg cag acc ccg ccg atc ccc gac acc gcg cag tgg ggc atc 1008
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
ttc ctg cgc aac cac gac gag ctc acg ctc gag atg gtc acc gac gaa 1056
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
gaa cgt gac tac atg tac gcc gag tac gcc aag gac ccg cgg atg aag 1104
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
gcc aac gtc ggc atc cgg cgc agg ctc gca ccg ctg ttg gag aac gac 1152
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
cgc aac cag atc gag ttg ttc acc gcg ctg ctg ctc tcg ctg ccg ggg 1200
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
tcg ccg gtg ctg tac tac ggt gac gag ata ggt atg ggg gac atc atc 1248
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
tgg ctc ggc gac cgc gac agc gtg cgt acg ccc atg cag tgg acc ccc 1296
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
gac cgc aat gcg ggc ttc tcc aag gcg act ccg ggc cgg ctc tac ctg 1344
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
ccg ccc aac cag gac gcg gtg tac ggc tac cac tct gtg aac gtc gag 1392
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
gcg caa ctc gac agc tcc agc tcg ctg ttg aac tgg acg cgc aac atg 1440
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
ctg gcc gtg cgc agc cgc cac gat gcc ttc gca gtc ggc acg ttc cgc 1488
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
gaa ctg ggc ggc tcc aat ccg tcg gtg ctg gcg tac atc cgg gag gtc 1536
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
acg cgc caa cag ggc gac ggc ggc gcg aag acc gat gcc gtc ctg tgt 1584
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
gtc aac aac ctg tcc cgc ttc ccc cag ccc atc gag ttg aat ctc cag 1632
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
cag tgg gcc ggt tac ata ccc gtc gag atg acc ggc tac gtc gag ttc 1680
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
ccg agt atc ggc cag ttg ccg tac ctg ctc acc ctg ccc ggt cac ggg 1728
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
ttc tac tgg ttc cag ctt cga gaa ccc gat cca gaa ccg gga gcg cag 1776
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
caa tga 1782
Gln
<210> 10
<211> 593
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 10
Met Glu Glu His Thr Gln Gly Ser His Val Glu Ala Gly Ile Val Glu
1 5 10 15
His Pro Asn Ala Glu Asp Phe Gly His Ala Arg Thr Leu Pro Thr Asp
20 25 30
Thr Asn Trp Phe Lys His Ala Val Phe Tyr Glu Val Leu Val Arg Ala
35 40 45
Phe Tyr Asp Ser Asn Ala Asp Gly Ile Gly Asp Leu Arg Gly Leu Thr
50 55 60
Glu Lys Leu Asp Tyr Ile Lys Trp Leu Gly Val Asp Cys Leu Trp Leu
65 70 75 80
Pro Pro Phe Tyr Asp Ser Pro Leu Arg Asp Gly Gly Tyr Asp Ile Arg
85 90 95
Asp Phe Tyr Lys Val Leu Pro Glu Phe Gly Thr Val Asp Asp Phe Val
100 105 110
Thr Leu Leu Asp Ala Ala His Arg Arg Gly Ile Arg Ile Ile Thr Asp
115 120 125
Leu Val Met Asn His Thr Ser Asp Gln His Glu Trp Phe Gln Glu Ser
130 135 140
Arg His Asn Pro Asp Gly Pro Tyr Gly Asp Phe Tyr Val Trp Ser Asp
145 150 155 160
Thr Ser Asp Arg Tyr Pro Ala Ala Ala Ile Ile Phe Val Ala Ala Ala
165 170 175
Glu Ser Asn Trp Thr Phe Asp Pro Val Arg Arg Gln Phe Tyr Trp His
180 185 190
Arg Phe Phe Ser His Gln Pro Asp Leu Asn Tyr Asp Asn Pro Ala Val
195 200 205
Gln Glu Ala Met Leu Asp Val Leu Arg Phe Trp Leu Asp Leu Gly Ile
210 215 220
Asp Gly Phe Arg Leu Asp Ala Val Pro Tyr Leu Phe Glu Arg Glu Gly
225 230 235 240
Thr Asn Cys Glu Asn Leu Pro Glu Thr His Ala Phe Leu Lys Arg Cys
245 250 255
Arg Lys Ala Ile Asp Asp Glu Tyr Pro Gly Arg Val Leu Leu Ala Glu
260 265 270
Ala Asn Gln Trp Pro Ala Asp Val Val Ala Tyr Phe Gly Asp Pro Asp
275 280 285
Thr Gly Gly Asp Glu Cys His Met Ala Phe His Phe Pro Leu Met Pro
290 295 300
Arg Ile Phe Met Ala Val Arg Arg Glu Ser Arg Phe Pro Ile Ser Glu
305 310 315 320
Ile Leu Ala Gln Thr Pro Pro Ile Pro Asp Thr Ala Gln Trp Gly Ile
325 330 335
Phe Leu Arg Asn His Asp Glu Leu Thr Leu Glu Met Val Thr Asp Glu
340 345 350
Glu Arg Asp Tyr Met Tyr Ala Glu Tyr Ala Lys Asp Pro Arg Met Lys
355 360 365
Ala Asn Val Gly Ile Arg Arg Arg Leu Ala Pro Leu Leu Glu Asn Asp
370 375 380
Arg Asn Gln Ile Glu Leu Phe Thr Ala Leu Leu Leu Ser Leu Pro Gly
385 390 395 400
Ser Pro Val Leu Tyr Tyr Gly Asp Glu Ile Gly Met Gly Asp Ile Ile
405 410 415
Trp Leu Gly Asp Arg Asp Ser Val Arg Thr Pro Met Gln Trp Thr Pro
420 425 430
Asp Arg Asn Ala Gly Phe Ser Lys Ala Thr Pro Gly Arg Leu Tyr Leu
435 440 445
Pro Pro Asn Gln Asp Ala Val Tyr Gly Tyr His Ser Val Asn Val Glu
450 455 460
Ala Gln Leu Asp Ser Ser Ser Ser Leu Leu Asn Trp Thr Arg Asn Met
465 470 475 480
Leu Ala Val Arg Ser Arg His Asp Ala Phe Ala Val Gly Thr Phe Arg
485 490 495
Glu Leu Gly Gly Ser Asn Pro Ser Val Leu Ala Tyr Ile Arg Glu Val
500 505 510
Thr Arg Gln Gln Gly Asp Gly Gly Ala Lys Thr Asp Ala Val Leu Cys
515 520 525
Val Asn Asn Leu Ser Arg Phe Pro Gln Pro Ile Glu Leu Asn Leu Gln
530 535 540
Gln Trp Ala Gly Tyr Ile Pro Val Glu Met Thr Gly Tyr Val Glu Phe
545 550 555 560
Pro Ser Ile Gly Gln Leu Pro Tyr Leu Leu Thr Leu Pro Gly His Gly
565 570 575
Phe Tyr Trp Phe Gln Leu Arg Glu Pro Asp Pro Glu Pro Gly Ala Gln
580 585 590
Gln

Claims (6)

1. A mutant trehalose synthase enzyme derived from mycobacterium smegmatis, wherein the amino acid sequence of the enzyme is as set forth in SEQ ID NO: 6. 8 or 10.
2. The trehalose synthase mutant gene is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO: 5. 7 or 9.
3. A recombinant vector comprising the gene according to claim 2.
4. A transgenic cell comprising the gene of claim 2.
5. A composition comprising the enzyme of claim 1 as an active ingredient.
6. The enzyme mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, the transgenic cell of claim 4 and the composition of claim 5 for the catalytic synthesis of trehalose.
CN201610876031.2A 2016-10-08 2016-10-08 Trehalose synthetase mutant and gene thereof Active CN106434586B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610876031.2A CN106434586B (en) 2016-10-08 2016-10-08 Trehalose synthetase mutant and gene thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610876031.2A CN106434586B (en) 2016-10-08 2016-10-08 Trehalose synthetase mutant and gene thereof

Publications (2)

Publication Number Publication Date
CN106434586A CN106434586A (en) 2017-02-22
CN106434586B true CN106434586B (en) 2019-12-10

Family

ID=58171835

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610876031.2A Active CN106434586B (en) 2016-10-08 2016-10-08 Trehalose synthetase mutant and gene thereof

Country Status (1)

Country Link
CN (1) CN106434586B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107384888B (en) * 2017-08-04 2020-06-30 齐鲁工业大学 Mutation-modified high-temperature-resistant trehalose synthase and application thereof
CN108048439B (en) * 2017-11-20 2021-02-26 齐鲁工业大学 Preparation method and application of mutant trehalose synthase

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization;Yung-Lin Wang;《Acta Crystallographica Section D Biological Crystallography》;20141231;第D70卷;3152-3154跨页段，参见图3d，图8 bcd *
海藻糖合成酶结构和功能的研究进展;王东生;《化学与生物工程》;20170731;第34卷(第7期);全文 *
耻垢分枝杆菌海藻糖合成酶基因TreS的克隆及其在大肠杆菌中的表达;李镭;《食品与生物技术学报》;20071130;第26卷(第6期);全文 *

Also Published As

Publication number Publication date
CN106434586A (en) 2017-02-22

Similar Documents

Publication Publication Date Title
CN112080480B (en) Glycosyltransferase mutants and uses thereof
CN111094563A (en) Composition for preparing tagatose and method for preparing tagatose using the same
TWI700370B (en) A composition for producing tagatose and methods for producing tagatose using the same
CN109609530B (en) Trehalose synthetase and application thereof in trehalose production
CN110462036A (en) A kind of novel D-Psicose 3- epimerase and the method for preparing D-Psicose using the enzyme
CN112301012B (en) Cyclodextrin glucosyltransferase mutant and construction method thereof
CN111133103A (en) Composition for preparing tagatose and method for preparing tagatose using the same
CN114015708B (en) Deep sea bacteria-derived alpha-glucosidase QsGH13 and encoding gene and application thereof
CN106434586B (en) Trehalose synthetase mutant and gene thereof
KR100922085B1 (en) Processes for producing cmp-n-acetylneuraminic acid
CN112852774A (en) Phosphofructosyl aminotransferase encoding gene and preparation and application of enzyme
CN113637652B (en) Adenylyltransferase mutant and application thereof
CN114196696B (en) Recombinant enzyme fused with specific short peptide tag and capable of efficiently catalyzing generation of Reb M
CN113512544B (en) Mannose isomerase mutant with improved heat stability
CN109988778A (en) A kind of saccharose phosphorylation enzyme gene and its application
CN115058398B (en) Arginine mutated nucleic acid ligase
CN105969751B (en) Beta-glucosidase gene and application thereof
CN109576234B (en) Leucine-5-hydroxylase mutant and application thereof
CA2392463C (en) Novel use of uridine diphosphate glucose 4-epimerase
CN113302299A (en) Psicose epimerase variants, methods of producing the same, and methods of producing psicose using the same
CN111534498A (en) Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield
US6841368B1 (en) Enzymatic production of difructose dianhydride IV from sucrose and relevant enzymes and genes coding for them
CN114381446B (en) Heat-resistant acid-resistant arabinosidase gene, and expression protein, recombinant vector, recombinant bacterium and application thereof
CN114591938B (en) Carboxylase mutant and preparation method and application thereof
JP2023554112A (en) Allulose epimerase mutant with excellent thermostability, method for producing the same, and method for producing allulose using the same

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant