Construction of high-performance starch debranching enzyme chimera strain, production method and application thereof

文档序号：1856583 发布日期：2021-11-19 浏览：22次中文

阅读说明：本技术 高性能淀粉脱支酶嵌合体菌种构建及其生产方法和应用 (Construction of high-performance starch debranching enzyme chimera strain, production method and application thereof ) 是由范岩牟洋于 2021-07-05 设计创作，主要内容包括：本发明公开了高性能淀粉脱支酶嵌合体菌种构建及其生产方法和应用。所述高性能淀粉脱支酶嵌合体由两个亲代普鲁兰酶氨基酸序列的有效嵌合得到,且所述高性能淀粉脱支酶嵌合体与亲代普鲁兰酶氨基酸序列同源性不低于95％；所述高性能淀粉脱支酶嵌合体是由Bacillus acidopullulyticus的亲代普鲁兰酶的N-末端1-476氨基酸残基和Bacillus deramificans的亲代普鲁兰酶C-末端465-976氨基酸残基嵌合而获得的。嵌合普鲁兰酶具有较高的糖化速率,这个嵌合普鲁兰酶同时还具有比亲本普鲁兰酶更高的耐热性和热稳定性,适用于糖化反应和淀粉液化。(The invention discloses construction of a high-performance starch debranching enzyme chimera strain, a production method and application thereof. The high-performance starch debranching enzyme chimera is obtained by effectively embedding amino acid sequences of two parent pullulanases, and the homology of the high-performance starch debranching enzyme chimera and the amino acid sequences of the parent pullulanases is not lower than 95%; the high-performance starch debranching enzyme chimera is obtained by chimerizing the N-terminal 1-476 amino acid residues of parent pullulanase of Bacillus acidopululyticus and the C-terminal 465-976 amino acid residues of parent pullulanase of Bacillus deramificans. The chimeric pullulanase has higher saccharification rate, has higher heat resistance and thermal stability than parent pullulanase, and is suitable for saccharification reaction and starch liquefaction.)

1. The high-performance starch debranching enzyme chimera is characterized in that the high-performance starch debranching enzyme chimera is obtained by effectively embedding amino acid sequences of two parent pullulanases, and the homology of the high-performance starch debranching enzyme chimera and the amino acid sequences of the parent pullulanases is not lower than 95%;

preferably, both of said parent pullulanases are Bacillus acidopulullulans pullulanase and/or Bacillus deramificans pullulanase;

preferably, the high-performance starch debranching enzyme chimera is obtained by chimerizing the N-terminal 1-476 amino acid residues of a parent pullulanase of Bacillus acidopululyticus and the C-terminal 465-976 amino acid residues of a parent pullulanase of Bacillus deramificans.

2. An expression vector encoding the high performance starch debranching enzyme chimera of claim 1, characterized by consisting of a synthetic polynucleotide;

preferably, the expression vector comprises an expression component consisting of a promoter sequence, a ribosome binding site, a high-performance starch debranching enzyme chimera coding gene sequence and a terminator sequence;

preferably, the expression vector further comprises a signal sequence for directing secretion of the high performance starch debranching enzyme chimera, the signal sequence being located upstream of the codon.

3. A recombinant microbial host cell comprising the expression vector of claim 2;

preferably, the high performance starch debranching enzyme chimera encoding gene sequence of the expression vector is integrated into the chromosome of the recombinant host cell.

4. A method for producing a high-performance starch debranching enzyme chimera is characterized by comprising the following steps:

s1: culturing the recombinant microbial host cell of claim 3 for expression of a high performance starch debranching enzyme chimera;

s2: obtaining a high performance starch debranching enzyme chimera in the cultured recombinant microbial host cells or supernatant thereof.

5. A composition comprising the high performance starch debranching enzyme chimera of claim 1;

preferably, the composition further comprises an amylase.

6. Use of the composition of claim 5 for catalyzing carbohydrate glycation.

7. Use of a composition according to claim 6 for catalysing the glycation of carbohydrates, characterised in that the carbohydrates contain at least one alpha-1, 6 glycosidic bond.

8. A method of catalyzing a carbohydrate, comprising: reacting a carbohydrate with the composition of claim 5.

9. The method of catalyzing carbohydrates as claimed in claim 7 wherein the carbohydrate is starch, amylopectin, dextran, maltodextrin, pullulan or glycogen.

10. The method of catalyzing carbohydrates as claimed in claim 7, wherein the reaction conditions are: the pH is 4.0-4.5 and the temperature is 60-64 ℃.

Technical Field

The invention relates to the technical field of starch debranching enzyme, in particular to construction of a high-performance starch debranching enzyme chimera strain as well as a production method and application thereof.

Background

For the existing pullulanase, the defects of low saccharification efficiency, low enzyme activity under the acidic high-temperature condition and the like exist. Therefore, in order to meet the requirement of industrial production, the problems of the saccharification efficiency of the pullulanase and the enzyme activity under the acidic high-temperature condition (especially when the temperature reaches 60 ℃ and the pH value is lower than 4.5) need to be solved.

Disclosure of Invention

Based on the technical problems in the background art, the invention provides the construction of the high-performance starch debranching enzyme chimera strain, the production method and the application thereof, compared with the parent pullulanase from bacillus, the saccharification efficiency is improved, the requirements of industrial production are met, and the enzyme activity and other properties of the chimeric pullulanase are improved compared with those of parents.

The high-performance starch debranching enzyme chimera provided by the invention is obtained by effectively embedding amino acid sequences of two parent pullulanases, and the homology of the high-performance starch debranching enzyme chimera and the amino acid sequences of the parent pullulanases is not less than 95%.

Preferably, both of said parent pullulanases are Bacillus acidopulullulans pullulanase and/or Bacillus deramificans pullulanase.

Preferably, the high-performance starch debranching enzyme chimera is obtained by chimerizing the N-terminal 1-476 amino acid residues of a parent pullulanase of Bacillus acidopululyticus and the C-terminal 465-976 amino acid residues of a parent pullulanase of Bacillus deramificans.

The expression vector for coding the high-performance starch debranching enzyme chimera consists of a synthetic polynucleotide.

Preferably, the expression vector comprises an expression component consisting of a promoter sequence, a ribosome binding site, a high-performance starch debranching enzyme chimera coding gene sequence and a terminator sequence.

Preferably, the expression vector further comprises a signal sequence for directing secretion of the high performance starch debranching enzyme chimera, the signal sequence being located upstream of the codon.

The invention provides a recombinant microbial host cell, which comprises the expression vector.

Preferably, the high performance starch debranching enzyme chimera encoding gene sequence of the expression vector is integrated into the chromosome of the recombinant host cell.

The invention provides a method for producing a high-performance starch debranching enzyme chimera, which comprises the following steps:

s1: culturing the recombinant microbial host cell for expressing the high-performance starch debranching enzyme chimera;

s2: obtaining a high performance starch debranching enzyme chimera in the cultured recombinant microbial host cells or supernatant thereof.

The invention provides a composition which comprises the high-performance starch debranching enzyme chimera.

Preferably, the composition further comprises an amylase.

The invention provides application of the composition in catalyzing carbohydrate saccharification.

Preferably, the carbohydrate contains at least one alpha-1, 6 glycosidic bond.

The method for catalyzing carbohydrate provided by the invention adopts the carbohydrate and the composition to react.

Preferably, the carbohydrate is starch, amylopectin, dextran, maltodextrin, pullulan or glycogen.

Preferably, the reaction conditions are: the pH is 4.0-4.5 and the temperature is 60-64 ℃.

Has the advantages that:

the high-performance starch debranching enzyme chimera is obtained by effectively embedding amino acid sequences of two parent pullulanases, and has better pH tolerance, heat resistance and thermal stability. In addition, the chimeric pullulanase and the saccharifying enzyme derived from the aspergillus niger are mixed into the composite saccharifying enzyme, so that the composite saccharifying enzyme has excellent performance, is convenient for production operation, and can meet the requirements of most customers for producing glucose (or derivatives thereof) by using starch raw materials. In saccharification, under the condition of 0.300PU/gDS dosage, the glucose yield of the parent pullulanase for 24 hours is 94.5%, and under the condition of 0.150PU/gDS dosage, the glucose yield of the high-performance starch debranching enzyme chimera prepared by the method for 24 hours can reach 94.6%, so that the application effect of the high-performance starch debranching enzyme chimera prepared by the method for saccharification is remarkably improved.

Drawings

FIG. 1 is a schematic representation of the pYF-tsDE vector proposed by the present invention;

FIG. 2 is a schematic diagram of a pUC57-KS-erm vector as set forth in the present invention;

FIG. 3 is a schematic diagram of pYF-tsINT-pul vector according to the present invention.

Detailed Description

(1) Construction of pYF-tsDE plasmid

pYF-tsDE is shown in FIG. 1, and is prepared from pUC57-KS-erm (Asclepiadaceae Biotechnology Co., Ltd., Nanjing) as raw material by CN 107532155A.

(2) Construction of protease deficient Bacillus subtilis strains

The invention takes bacillus subtilis as host cell, directly used as template of PCR reaction after being processed, and the following primers are synthesized by Genscript and are respectively used for PCR reaction amplification flanking sequences Apr, Npr and SpoIIAC:

the primers for amplifying the upstream sequence of the Apr gene are as follows:

pksb-Apr_czF1：GGTATCGATA AGCTTCCTGC AGATCTCTCA GGAGCATTTAACCT

pksb-Apr_R1：GCACCTACTG CAATAGTAAG GAACAGATTG CGCAT

the primers for amplifying the downstream sequence of the Apr gene are as follows:

pksb-Apr_F2：ATGCGCAATC TGTTCCTTAC TATTGCAGTA GGTGC

pksb-Apr_czR2：AATATGGCGG CCGCGAATTC AGATCTCTAA TGCTGTCTCG CGT

the primers for amplifying the upstream sequence of the Npr gene are as follows:

pksb-Npr_czF1：GGTATCGATA AGCTTCCTGC AGATCTCATC TTCCCCTTGAT

pksb-Npr_R1：CAGTCTTCTG TATCGTTACG CTTTTAATTC GGCT

the primers for amplifying the downstream sequence of the Npr gene are as follows:

pksb-Npr_F2：AGCCGAATTAAAAGCGTAAC GATACAGAAG ACTG

pksb-Npr_czR2：TATGGCGGCC GCGAATTCAG ATCTCCTGGC CAGGAGAATC T

amplification of SpoIIAC: the primers for the gene upstream sequence were:

pksb-Spo_czF1：GGTATCGATA AGCTTCCTGC AGGAACAATC TGAACAGCAG GCACTC

pksb-Spo_R1：TTGTCAAACCATTTTTCTTC GCCCGATGCA GCCGATCTG

the primers for amplifying the downstream sequence of the SpoIIAC gene are as follows:

pksb-Spo_F2：CAGATCGGCT GCATCGGGCG AAGAAAAATG GTTTGACAA

pksb-Spo_czR2：ATATGGCGGC CGCGAATTCA GATCTGTTCA TGATGGCAAGACAC

the reaction conditions were as described in CN107532155A, and the amplification product was detected and purified after the reaction was completed.

To obtain a strain for gene replacement at the designed site, several selected monoclonals were inoculated into 2YT medium and cultured at 30 ℃ for 5-7 days (subculture every 2 days). Strains sensitive to erythromycin were subjected to PCR screening (see SEQ ID NO.7, 8, 9). Protease deficient phenotype validation was performed on 1% skim milk plates.

(3) Construction of pullulanase-producing strains

The pullulanase expression cassette mainly comprises: a promoter sequence (Seq ID: NO 10), a ribosome binding site (Seq ID: NO 11), a chimeric pullulan-encoding gene, and a termination sequence (Seq ID: NO 12). In addition, the invention also inserts a strong natural signal sequence (Seq ID: NO 13) screened from bacillus subtilis into the upstream of the promoter of the pullulanase coding gene, and is used for enhancing the secretion efficiency of the pullulanase. The complete pullulanase expression cassette was inserted into the BglII site in the linearized pYF-tsDE using the assembly master mix kit (New England Biolabs) and the resulting integrated plasmid was named pYF-tsINT-puI (FIG. 3).

(4) Shake flask fermentation for pullulanase production

The fermentation method refers to CN107532155A, and SDS-PAGE analysis is carried out on the fermented product to determine the effective secretion of the chimeric pullulanase.

(5) Pullulanase step-by-step feeding fermentation process

And (4) streaking the gene engineering bacillus strain which is obtained in the step (3) and is frozen and stored at the temperature of-80 ℃ on an agar slant, and recovering and culturing at the temperature of 37 ℃ overnight. The concrete fed-batch fermentation process refers to CN 107532155A.

(6) Pullulanase activity assay

The enzyme activity was measured according to the description of CN 107532155A.

(7) Application of chimeric pullulanase

All the following results are based on a chimeric pullulanase (SEQ ID NO.6) obtained by the N-terminal 1-470 amino acid residues of a parent pullulanase of Bacillus acidopulullulanus and the C-terminal 469-928 amino acid residues of a parent pullulanase of Bacillus deramificans. The homology of the amino acid sequence of the chimeric pullulanase reaches at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100%.

Chimeric pullulanase saccharification test conditions: 31% dry matter (DS), mixed well and pH adjusted to 4.3 with 50% (w/v) NaOH. Pullulanase and glucoamylase (mixed concentration: 0.225GU/gDS) and blank marker water were added at 0.3 and 0.15PU/gDS, respectively. The original unmodified pullulanase (from Bacillus deramificans, 0.300PU/gDS) was added to a shake flask as a control. The results are shown in Table 1.

Table 1: at pH4.3, the saccharification application of truncated pullulanase and parent pullulanase is compared

As can be seen from Table 1, the chimeric pullulanase maintained or even increased the glucose yield (94.6-97.8%) compared to the parent glucose yield (94.5-96.4%) at an enzyme dosage (0.150PU/gDS) at which the addition was reduced by half, which confirms that the chimeric pullulanase did not affect the pullulanase activity. Importantly, the yield of the glucose is 97.8% within 48h, which shows that in practical application, the chimeric pullulanase only needs lower dosage to bring good production performance, and can help customers to reduce the use cost of the enzyme preparation. In addition, within 24h of the initial reaction, the rate of saccharification catalyzed by the chimeric pullulanase was higher than that of the parent pullulanase (results not shown). In conclusion, the chimeric pullulanase resulted in better structural stability and higher enzymatic activity.

Secondly, we determined the tolerance of the chimeric pullulanase to pH by saccharification experiments under low pH conditions. The saccharification reaction conditions were as described above, and the pH was 4.0. The results are shown in Table 2.

Table 2: at pH4.0, the saccharification application of truncated pullulanase and parent pullulanase is compared

As shown in table 2, under the conditions of ph4.0, the glucose yield of the parent pullulanase was only 96.1% at 48h of reaction, which is lower than the minimal percentage of glucose yield (96.5%) necessary in the starch industry. And for the chimeric pullulanase, the catalytic activity is higher under the condition of pH4.0 under acidic condition, and the final glucose yield of the chimeric pullulanase can reach 97.2% at the reaction time of 48 hours (Table 2).

In addition, the heat resistance and heat stability of the chimeric pullulanase and the parental pullulanase were measured, and the results are shown in table 3.

Table 3: comparison of saccharification applications of chimeric pullulanase and parent pullulanase at different temperatures (pH4.3)

From table 3, it can be seen that the chimeric pullulanase can significantly maintain a sustained higher glucose yield at a high temperature of 64 ℃, that is, the heat resistance and the heat stability of the chimeric pullulanase are significantly improved compared with those of the parent pullulanase.

Finally, the saccharification reaction of the chimeric pullulanase was also studied. The pH was adjusted to 5.2 with 50% (w/v) NaOH, and the rest of the reaction conditions were as described in CN 107532155A. The results are shown in Table 4.

Table 4: comparison of applications of maltose yields of chimeric and pro-pullulanases

As shown in table 4, the chimeric pullulanase performed better under the same conditions as compared with the procallopullulanase. The yield of maltose is obviously higher than that of the parent pullulanase, which shows that the catalytic activity of the chimeric pullulanase in the industrial production of maltose is greatly improved.

In summary, according to the experimental results in the present invention, the chimeric pullulanase has better pH tolerance, heat resistance and thermostability than the parent pullulanase. The chimeric pullulanase of the present invention also exhibits certain advantages under certain conditions. Therefore, the chimeric pullulanase of the present invention can be applied to the saccharification process of starch raw materials, especially in the starch processing industry.

Sequence listing

<110> Nanjing Newyoda science and technology Co., Ltd

<120> construction of high-performance starch debranching enzyme chimera strain, production method and application thereof

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<213> Bacillus deramificans (Bacillus debranching)

<400> 3

gatgggaaca cgacaacgat cattgtccac tattttcgcc ctgctggtga ttatcaacct 60

tggagtctat ggatgtggcc aaaagacgga ggtggggctg aatacgattt caatcaaccg 120

gctgactctt ttggagctgt tgcaagtgct gatattccag gaaacccaag tcaggtagga 180

attatcgttc gcactcaaga ttggaccaaa gatgtgagcg ctgaccgcta catagattta 240

agcaaaggaa atgaggtgtg gcttgtagaa ggaaacagcc aaatttttta taatgaaaaa 300

gatgctgagg atgcagctaa acccgctgta agcaacgctt atttagatgc ttcaaaccag 360

gtgctggtta aacttagcca gccgttaact cttggggaag gcgcaagcgg ctttacggtt 420

catgacgaca cagcaaataa ggatattcca gtgacatctg tgaaggatgc aagtcttggt 480

caagatgtaa ccgctgtttt ggcaggtacc ttccaacata tttttggagg ttccgattgg 540

gcacctgata atcacagtac tttattaaaa aaggtgacta acaatctcta tcaattctca 600

ggagatcttc ctgaaggaaa ctaccaatat aaagtggctt taaatgatag ctggaataat 660

ccgagttacc catctgacaa cattaattta acagtccctg ccggcggtgc acacgtcact 720

ttttcgtata ttccgtccac tcatgcagtc tatgacacaa ttaataatcc taatgcggat 780

ttacaagtag aaagcggggt taaaacggat ctcgtgacgg ttactctagg ggaagatcca 840

gatgtgagcc atactctgtc cattcaaaca gatggctatc aggcaaagca ggtgatacct 900

cgtaatgtgc ttaattcatc acagtactac tattcaggag atgatcttgg gaatacctat 960

acacagaaag caacaacctt taaagtctgg gcaccaactt ctactcaagt aaatgttctt 1020

ctttatgaca gtgcaacggg ttctgtaaca aaaatcgtac ctatgacggc atcgggccat 1080

ggtgtgtggg aagcaacggt taatcaaaac cttgaaaatt ggtattacat gtatgaggta 1140

acaggccaag gctctacccg aacggctgtt gatccttatg caactgcgat tgcaccaaat 1200

ggaacgagag gcatgattgt ggacctggct aaaacagatc ctgctggctg gaacagtgat 1260

aaacatatta cgccaaagaa tatagaagat gaggtcatct atgaaatgga tgtccgtgac 1320

ttttccattg accctaattc gggtatgaaa aataaaggga agtatttggc tcttacagaa 1380

aaaggaacaa agggccctga caacgtaaag acggggatag attccttaaa acaacttggg 1440

attactcatg ttcagcttat gcctgttttc gcatctaaca gtgtcgatga aactgatcca 1500

acccaagata attggggtta tgaccctcgc aactatgatg ttcctgaagg gcagtatgct 1560

acaaatgcga atggtaatgc tcgtataaaa gagtttaagg aaatggttct ttcactccat 1620

cgtgaacaca ttggggttaa catggatgtt gtctataatc atacctttgc cacgcaaatc 1680

tctgacttcg ataaaattgt accagaatat tattaccgta cggatgatgc aggtaattat 1740

accaacggat caggtactgg aaatgaaatt gcagccgaaa ggccaatggt tcaaaaattt 1800

attattgatt cccttaagta ttgggtcaat gagtatcata ttgacggctt ccgttttgac 1860

ttaatggcgc tgcttggaaa agacacgatg tccaaagctg cctcggagct tcatgctatt 1920

aatccaggaa ttgcacttta cggtgagcca tggacgggtg gaacctctgc actgccagat 1980

gatcagcttc tgacaaaagg agctcaaaaa ggcatgggag tagcggtgtt taatgacaat 2040

ttacgaaacg cgttggacgg caatgtcttt gattcttccg ctcaaggttt tgcgacaggt 2100

gcaacaggct taactgatgc aattaagaat ggcgttgagg ggagtattaa tgactttacc 2160

tcttcaccag gtgagacaat taactatgtc acaagtcatg ataactacac cctttgggac 2220

aaaatagccc taagcaatcc taatgattcc gaagcggatc ggattaaaat ggatgaactc 2280

gcacaagcag ttgttatgac ctcacaaggc gttccattca tgcaaggcgg ggaagaaatg 2340

cttcgtacaa aaggcggcaa cgacaatagt tataatgcag gcgatgcggt caatgagttt 2400

gattggagca ggaaagctca atatccagat gttttcaact attatagcgg gctaatccac 2460

cttcgtcttg atcacccagc cttccgcatg acgacagcta atgaaatcaa tagccacctc 2520

caattcctaa atagtccaga gaacacagtg gcctatgaat taactgatca tgttaataaa 2580

gacaaatggg gaaatatcat tgttgtttat aacccaaata aaactgtagc aaccatcaat 2640

ttgccgagcg ggaaatgggc aatcaatgct acgagcggta aggtaggaga atccaccctt 2700

ggtcaagcag agggaagtgt ccaagtacca ggtatatcta tgatgatcct tcatcaagag 2760

gtaagcccag accacggtaa aaagtaa 2787

<210> 4

<211> 928

<212> PRT

<213> Bacillus deramificans (Bacillus debranching)

<400> 4

Asp Gly Asn Thr Thr Thr Ile Ile Val His Tyr Phe Arg Pro Ala Gly

1 5 10 15

Asp Tyr Gln Pro Trp Ser Leu Trp Met Trp Pro Lys Asp Gly Gly Gly

20 25 30

Ala Glu Tyr Asp Phe Asn Gln Pro Ala Asp Ser Phe Gly Ala Val Ala

35 40 45

Ser Ala Asp Ile Pro Gly Asn Pro Ser Gln Val Gly Ile Ile Val Arg

50 55 60

Thr Gln Asp Trp Thr Lys Asp Val Ser Ala Asp Arg Tyr Ile Asp Leu

65 70 75 80

Ser Lys Gly Asn Glu Val Trp Leu Val Glu Gly Asn Ser Gln Ile Phe

85 90 95

Tyr Asn Glu Lys Asp Ala Glu Asp Ala Ala Lys Pro Ala Val Ser Asn

100 105 110

Ala Tyr Leu Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln Pro

115 120 125

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

130 135 140

Ala Asn Lys Asp Ile Pro Val Thr Ser Val Lys Asp Ala Ser Leu Gly

145 150 155 160

Gln Asp Val Thr Ala Val Leu Ala Gly Thr Phe Gln His Ile Phe Gly

165 170 175

Gly Ser Asp Trp Ala Pro Asp Asn His Ser Thr Leu Leu Lys Lys Val

180 185 190

Thr Asn Asn Leu Tyr Gln Phe Ser Gly Asp Leu Pro Glu Gly Asn Tyr

195 200 205

Gln Tyr Lys Val Ala Leu Asn Asp Ser Trp Asn Asn Pro Ser Tyr Pro

210 215 220

Ser Asp Asn Ile Asn Leu Thr Val Pro Ala Gly Gly Ala His Val Thr

225 230 235 240

Phe Ser Tyr Ile Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn

245 250 255

Pro Asn Ala Asp Leu Gln Val Glu Ser Gly Val Lys Thr Asp Leu Val

260 265 270

Thr Val Thr Leu Gly Glu Asp Pro Asp Val Ser His Thr Leu Ser Ile

275 280 285

Gln Thr Asp Gly Tyr Gln Ala Lys Gln Val Ile Pro Arg Asn Val Leu

290 295 300

Asn Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr

305 310 315 320

Thr Gln Lys Ala Thr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln

325 330 335

Val Asn Val Leu Leu Tyr Asp Ser Ala Thr Gly Ser Val Thr Lys Ile

340 345 350

Val Pro Met Thr Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn

355 360 365

Gln Asn Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly

370 375 380

Ser Thr Arg Thr Ala Val Asp Pro Tyr Ala Thr Ala Ile Ala Pro Asn

385 390 395 400

Gly Thr Arg Gly Met Ile Val Asp Leu Ala Lys Thr Asp Pro Ala Gly

405 410 415

Trp Asn Ser Asp Lys His Ile Thr Pro Lys Asn Ile Glu Asp Glu Val

420 425 430

Ile Tyr Glu Met Asp Val Arg Asp Phe Ser Ile Asp Pro Asn Ser Gly

435 440 445

Met Lys Asn Lys Gly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys

450 455 460

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

465 470 475 480

Ile Thr His Val Gln Leu Met Pro Val Phe Ala Ser Asn Ser Val Asp

485 490 495

Glu Thr Asp Pro Thr Gln Asp Asn Trp Gly Tyr Asp Pro Arg Asn Tyr

500 505 510

Asp Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Asn Ala Arg

515 520 525

Ile Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Arg Glu His Ile

530 535 540

Gly Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile

545 550 555 560

Ser Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp

565 570 575

Ala Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile Ala Ala

580 585 590

Glu Arg Pro Met Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Tyr Trp

595 600 605

Val Asn Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Leu Met Ala Leu

610 615 620

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

625 630 635 640

Asn Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser

645 650 655

Ala Leu Pro Asp Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys Gly Met

660 665 670

Gly Val Ala Val Phe Asn Asp Asn Leu Arg Asn Ala Leu Asp Gly Asn

675 680 685

Val Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu

690 695 700

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

705 710 715 720

Ser Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr

725 730 735

Thr Leu Trp Asp Lys Ile Ala Leu Ser Asn Pro Asn Asp Ser Glu Ala

740 745 750

Asp Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Val Val Met Thr Ser

755 760 765

Gln Gly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys

770 775 780

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

785 790 795 800

Asp Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser

805 810 815

Gly Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr

820 825 830

Ala Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn

835 840 845

Thr Val Ala Tyr Glu Leu Thr Asp His Val Asn Lys Asp Lys Trp Gly

850 855 860

Asn Ile Ile Val Val Tyr Asn Pro Asn Lys Thr Val Ala Thr Ile Asn

865 870 875 880

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

885 890 895

Glu Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln Val Pro Gly Ile

900 905 910

Ser Met Met Ile Leu His Gln Glu Val Ser Pro Asp His Gly Lys Lys

915 920 925

<210> 5

<211> 2793

<212> DNA

<213> Debranching enzyme chimera

<400> 5