CN102899339A - Alkalescence xylanase xynHBs nucleotide optimization sequence and high-efficiency expression method - Google Patents

Abstract

The invention provides an alkalescence xylanase gene xynHBs nucleotide optimization sequence and a high-efficiency expression method, which is characterized in that a xynHB gene (Bacillussp.HBP8, the GenBank registration No. is AY954639) through site directed mutagenesis is obtained by using DNAworks software according to codon bias of pichia pastoris through optimization of a xynHB gene sequence. and the gene optimization sequence of the alkalescence xylanase xynHBs can be obtained through the processes of PCR amplification, connection conversion and sequencing verification. The sequence is used for constructing a recombinant plasmid xynHBs-905A, and the recombinant plasmid xynHBs-905A is conversed to the pichia pastoris GS115 to obtain a masculine recombinant bacterial strain GS115/xynHBs-905A. The masculine recombinant bacterial strain is performed fermentation culture, compared with an original recombinant bacterial strain containing a gene without sequence optimization, thereby the xylanase protein with higher protein activity than that of the original heatproof alkalescence xylanase can be prepared. The alkalescence xylanase xynHBs nucleotide optimization sequence is used for producing the alkalescence xylanase by the pichia pastoris, target protein expression amount is high, the purifying process is simple, the production cost of the alkalescence xylanase is greatly reduced, and the utilization rate by the enterprises is increased.

Description

A kind of alkalescent xylanase xynHBs Nucleotide majorizing sequence and high-efficiency expression method thereof

Technical field

The present invention relates to biological technical field, be specifically related to alkalescent xylanase xynHBs Nucleotide majorizing sequence and high-efficiency expression method thereof.

Background technology

The alkalescence high-temperature xylanase has impayable effect in papermaking and Pulp industry.Along with the development of society, country requires more and more higher to enterprise, and energy-saving and emission-reduction become the hot issue of enterprise.The importance of zytase in papermaking and Pulp industry is that it has replaced poisonous chemical substance, can reclaim by product useful in the sector by the enzyme process pre-treatment simultaneously, compare method of chemical treatment, the pre-treatment of xylanase can reduce pharmaceutical chemicals usage quantity in the bleaching process, and improves the advantages such as bleaching effect.Thereby zytase has splendid application prospect in the bleaching of paper pulp, also is the main development direction of following paper industry.

But, needed high-temperature alkaline zytase generally derives from genus bacillus and intestinal bacteria in the process of association with pulp bleaching, and they can great expression in prokaryotic cell prokaryocyte, if can expect a large amount of purer zytases, just must carry out the purge process of series of complex.For the ease of purifying, people begin to adopt take pichia spp as expression strain, pichia spp is only secreted oneself protein seldom, add and only have a small amount of albumen in the pichia spp growth medium, the foreign protein that this means secretion is the major ingredient of albumen in the substratum, yet because the problem of source gene codon preference makes the output of alkalescent xylanase on the low side.

In the early-stage Study, the people such as Zhang Guimin are with xylanase gene xynHB (the Bacillus sp.HBP8 of bacillus pumilus, GenBank accession number: the site-directed mutagenesis that AY954630) mediates by Dpn I, make the 639th and 640 Nucleotide AA be mutated into GC, so that 214 amino acids N become A (patent publication No. CN:1924002A), this xylan Thermostability is significantly improved, brought up to 65 minutes 60 ℃ transformation period by 12 minutes.On this basis, the present invention's xynHB gene that site-directed mutagenesis is crossed is done further optimization.

Therefore, select suitable expressive host, make the albumen of expression be easier to purifying and have higher activity, reduce greatly the production cost of alkalescent xylanase, increase enterprise and its utilization ratio is become our research theme.

Summary of the invention

The objective of the invention is:

1, the invention provides and disclose a kind of restriction enzyme sites such as Sal I, Cpo I that do not contain, be adapted at optimized gene sequence and the gene preparation optimization method thereof of the alkalescent xylanase gene xynHBs of Pichia anomala expression.2, the invention allows for the high expression method of above-mentioned xynHBs Nucleotide majorizing sequence.

The step of one of the object of the invention is:

(1) optimizes.Adopt the conventional overlapping primer extension of PCR, xynHB gene order (the Bacillus sp.HBP8 that site-directed mutagenesis is crossed, GenBank accession number: AY954630) input DNAworks software, (Codon Frequency Table) selects P.pastoris, series of optimum gene order and primer sequence that the xynHB that obtains being correlated with expresses in pichia spp in the codon frequency table.

(2) transform primer.For the ease of protein expression, at 5 of primer 1 ' added base sequence GTCA, 5 of primer 22 ' end has added base sequence GGCCA.

(3) pcr amplification.With above-mentioned primer sequence template each other, adopt the method for successively decreasing from the two ends primer to middle primer concentration to carry out the amplification of total length, namely primer 1, primer 22 concentration are 40nmol/L, successively decrease successively, but middle primer 11, primer 12 concentration are minimum is 0.625nmol/L.Concrete reaction conditions is 94 ℃ of denaturation 5min, 94 ℃ of 30s then, and 60 ℃ of 30s, 72 ℃ of 90s, 30 circulations, 72 ℃ are extended 10min again.

(4) detect.The PCR product detects output and specificity with 0.7% agarose gel electrophoresis, and is connected conversion with DNA purification kit purifying with the T carrier, selects transformant and obtains the xynHBs majorizing sequence consistent with design result through after the sequence verification.

(5) relatively.XynHBs sequence after the optimization is compared with original series xynHB, optimised and the pichia spp low frequency codon wherein of the base of 15%-25% in the sequence is all replaced by high frequency or inferior high frequency codon, the impact of the secondary structure that forms in the dna sequence dna on expressing, the xynHBs gene order has been removed 26 amino acid whose signal peptide sequences of coded product self.XynHBs sequence after wherein optimizing is compared with original series xynHB, and effect is best is that the optimised and pichia spp low frequency codon wherein of 139 bases in the sequence is all replaced by high frequency or inferior high frequency codon.

Two the step of the object of the invention is:

(1) with the xynHBs majorizing sequence that obtains in the object of the invention one through T ₄After archaeal dna polymerase and dTTP process, be connected construction recombination plasmid xynHBs-905A with the expression vector pHBM905A that is connected with Not I through Cpo I.

(2) recombinant plasmid xynHBs-905A is changed in the Pichia pastoris GS115 after Sal I linearizing, obtain positive recombinant bacterial strain GS115/xynHBs-905A.

(3) positive recombinant bacterial strain is inoculated in the BMGY substratum of 25ml, 28 ℃, 200rpm cultivates 48h, to OD ₅₄₀Be 10-20;

(4) with centrifugal 5 minutes of cultured bacterium liquid 5000rpm, remove supernatant, transfer in the BMMY of 25ml substratum, 25 ℃, 200rpm cultivates 84h, during add methyl alcohol to final concentration 1% every 12h and induce.

(5) temperature is 4 ℃, and the centrifugal 10min of 5000rpm collects supernatant, contains a large amount of active alkali body acidic xylanases in the supernatant liquor.

(6) the same method makes up the not recombinant plasmid xynHB-905A of majorizing sequence, changes in the Pichia pastoris GS115 after the Sal I linearizing, screens positive recombinant bacterial strain GS115/xynHB-905A, and carries out abduction delivering under identical condition.

(7) step (3) and step (6) expression supernatant liquor neutral and alkali zytase carries out active comparison.

The carrier that sets out described in the above-mentioned steps (2) is pHBM905A, or carrier pPIC9k or carrier pPICZ α;

The present invention has adopted take Pichia pastoris GS115 as expression strain, uses full gene synthesis method to synthesize new gene (xynHBs).According to the preferences of pichia spp codon, utilize the site-directed mutagenesis sequence of the xynHB gene that the DNAworks software optimization retrieves from GenBank, all select Preference greater than 10% codon, the xynHBs gene of optimizing has been synthesized in design.For the ease of protein expression, synthetic sequence is processed with T4 archaeal dna polymerase and dTTP, and do not contained the restriction enzyme sites such as Sal I, Cpo I in the xynHBs sequence of optimizing.Express for the ease of protein excretion simultaneously, in the selection of expression vector, adopted the pHBM905A expression vector that contains yeast saccharomyces cerevisiae alpha factor signal peptide sequence, pilot protein carries out exocytosis expresses, the content of target protein reaches more than 95% in the supernatant liquor, makes the albumen of expression be easier to purifying and have higher activity.Greatly reduce the production cost of alkalescent xylanase, increased the utilization ratio of enterprise to it.

The alkalescent xylanase of xynHBs nucleotide sequence after the optimization through transforming Pichia yeast fermentation generation reaches 907U/ml in the fermentation supernatant, the enzymic activity 650U/ml of the alkalescent xylanase of the xynHB nucleotide sequence before ratio is optimized through transforming the generation of Pichia yeast fermentation supernatant has improved 39.5%.

Description of drawings

Fig. 1 detects picture for 0.7% agarose gel electrophoresis of synthetic xynHBs gene product.

Fig. 2 is the front xynHB gene of optimization and optimizes rear xynHBs gene institute inulinase-producing activity relatively.

Embodiment

The present invention is further illustrated below in conjunction with specific embodiment:

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Synthesizing of enforcement one, xynHBs optimized gene sequence:

(1) the overlapping primer extension of PCR of employing laboratory routine.Xylanase gene xynHB (the Bacillus sp.HBP8 of bacillus pumilus, GenBank accession number: the site-directed mutagenesis that AY954630) mediates by DpnI, make the 639th and 640 Nucleotide AA be mutated into GC, brought up to 65 minutes so that 214 amino acids N become the transformation period of 60 ℃ of its thermostabilitys of A (patent publication No. CN:1924002A) by 12 minutes.Gene order input DNAworks software with this mutagenesis, (Codon Frequency Table) selects P.pastoris in the codon frequency table, the series of optimum gene order that the xynHBs that obtains being correlated with expresses in pichia spp, wherein effect is best is that 139 bases in the sequence are optimised, and has removed xynHBs optimized gene sequence (as shown in table 1) and the primer sequence (as shown in table 2 below) of 26 amino acid signal peptide-coding sequences in the sequence.

XynHBs optimized gene sequence table 1

(wherein vertical line represents that corresponding up and down base is identical, and the space represents that corresponding base is different)

Primer sequence table table 2

(2) for the ease of protein expression, added base sequence GTCA at 5 of primer 1 ' end, 5 of primer 22 ' end has added base sequence GGCCA (grey sign as shown in table 2).

(3) with above-mentioned primer sequence template each other, the method that employing is successively decreased from the two ends primer to middle primer concentration is carried out the amplification of total length, be that primer 1, primer 22 concentration are 40nmol/L, successively decrease successively to middle primer concentration from the two ends primer again, but middle primer 11, primer 12 concentration are minimum is 0.625nmol/L.Concrete reaction conditions is 94 ℃ of denaturation 5min, 94 ℃ of 30s then, and 60 ℃ of 30s, 72 ℃ of 90s, 30 circulations, 72 ℃ are extended 10min again.

(4) the PCR product detects output and specificity with 0.7% agarose gel electrophoresis, and with DNA purification kit purifying, the PCR product is connected conversion with the T carrier, selects to obtain the xynHBs Nucleotide majorizing sequence consistent with design result after transformant checks order.

Efficiently expressing of the zytase xynHBs gene of enforcement two, optimization

1, the structure of expression vector xynHBs-905A

(1) will implement the plasmid that contains xynHBs that order-checking is correct in and process with T4DNA polysaccharase and dTTP, obtain the sticky end of NotI and Cpo I, agarose electrophoresis recycling product.

(2) expression vector pHBM905A is processed with Not I and Cpo I double digestion, agarose electrophoresis reclaims enzyme and cuts product.The xynHBs gene fragment of handling well is connected 4 hours with carrier pHBM905A with dna ligase Soulution I (Takara company).

Linked system (4 μ l): 1.5 μ l (1.5 μ g) T ₄The xynHBs dna fragmentation that archaeal dna polymerase and dTTP process;

0.5 the pHBM905A dna fragmentation that μ l (0.5 μ g) Not I and Cpo I process;

2 μ l Soulution I ligase enzymes.

(3) connect product and be converted into competent escherichia coli cell DH5 α, it is dull and stereotyped to coat afterwards the LB that contains 50 μ g/ml penbritins, 37 ℃ of incubated overnight are screened positive transformant and are carried out sequence verification with the PCR method, the recombinant plasmid called after xynHBs-905A that order-checking is correct.

(4) by above-mentioned same method construction recombination plasmid xynHB-905A.

2, the preparation of engineering bacteria

(1) recombinant plasmid xynHBs-905A is shocked by electricity after Sal I linearizing changes Pichia yeast GS115 over to, coat on MD (minimum medium that does not the contain Histidine) flat board, 28 ℃, 48h cultivates, obtain containing the transformant of xynHBs-905A gene, the transformant dibbling is being contained on the substrate flat board of crosslinked xylan, selecting the minimum bacterial strain of hydrolysis circle, this positive recombinant bacterium is being denoted as GS115/xynHBs-905A.

(2) replace xynHBs-905A with xynHB-905A, transform Pichia yeast GS115, step is the same, obtain containing the xynHB-905A transformant, the transformant dibbling is being contained on the substrate flat board of crosslinked xylan, selecting the minimum bacterial strain of hydrolysis circle, in contrast bacterium.The positive recombinant bacterium of this xynHB-905A of containing gene is denoted as GS115/xynHB-905A.

3, the fermentation culture recombinant bacterial strain prepares alkalescent xylanase

In the BMGY substratum that positive recombinant bacterial strain GS115/xynHBs-905A and the GS115/xynHB-905A of preparation in the step 2 is incubated at respectively 25ml, 28 ℃, shaking table 200rpm cultivates 48h; When OD600=15, with centrifugal 5 minutes of cultured bacterium liquid 5000rpm, remove supernatant, go in the BMMY substratum, 25 ℃ of 200rpm continue to cultivate 84h, during add methyl alcohol to final concentration 1% every 12h and induce.

The BMGY substratum is 1% yeast powder, 2% peptone, 0.34%YNB, 1% (NH ₄) ₂SO ₄100mmol/L potassium phosphate buffer pH6.0,1% glycerine.

The BMMY substratum is 1% yeast powder, 2% peptone, 0.34% YNB, 1% (NH ₄) ₂SO ₄, 100mmol/L potassium phosphate buffer pH6.0.

After cultivation is finished, 4 ℃ of temperature, centrifugal collection supernatant under 5000rpm, the 10min condition, and carry out the comparison of SDS-PAGE electrophoresis detection and enzyme activity, contain a large amount of active alkali body acidic xylanases in the supernatant liquor with recombinant bacterial strain GS115/xynHBs-905A cultivation.

4, the mensuration of enzyme activity

Comprise in the reaction system that thick enzyme diluent gets 1ml, add 1% xylan substrate 1ml; Reaction conditions is 50 ℃ of reaction 30min, and then boiling water bath 5min termination reaction adds 2.5ml DNS, and boiling water bath 5min adds water 8ml at last, puts upside down mixing, measures its absorbance at the 540nm place.Blank is the enzyme liquid reaction (that is: add first the thick enzyme diluent of 1ml, add substrate reactions 30min again) of non-activity.

The result:

One, the synthetic result of full gene primer

Shown in (table 2), 1-22 is designed primer, wherein 5 of primer 1 ' end adds base sequence GTCA (grey sign), and 5 of primer 22 ' end has added base sequence GGCCA (grey sign), and 15 complementary sequences about bp are arranged between every pair of adjacent primer.

Two, the xynHBs gene order contrast after the xynHB gene order before the optimization and the optimization

Sequence after the optimization is compared with original series, and the base of total 15%-25% is optimised, and pichia spp low frequency codons all in the sequence are all replaced by high frequency or inferior high frequency codon, shown in (table 1).

Three, composition sequence PCR result

The xynHBs sequence size of removing after the signal peptide optimization is 606bp, and other 9bp is for ease of construction expression base sequence that plasmid adds.The PCR product detects through 0.7% agarose gel electrophoresis, and a specific band appears in the position between 400-900bp, conforms to the theoretical value size.(Fig. 1)

Four, the comparison of enzyme activity

Shown in (Fig. 2), alkalescent xylanase that GS115/xynHBs-905A produces activity is 907U/ml after optimizing, the active 650U/ml of being of alkalescent xylanase that GS115/xynHB-905A produces before optimizing, and enzyme work has improved 39.5%.

Sequence table

＜110〉Hubei University

＜120〉a kind of alkalescent xylanase XynHBsNucleotide majorizing sequence and high-efficiency expression method thereof

<140>

<141>

<160> 1

<210> 1

<211> 606

<212> DNA

＜213〉artificial sequence

<220>

<222> （1）…（606）

<400> 1

1 gcggaaacga tttatgataa tagaataggc acacacagcg gatacgattt tgaattatgg

61 aaggattacg gaaatacctc gatgacactc aataacggcg gggcatttag tgcaagctgg

121 aacaatatcg gaaatgcctt atttcgaaaa ggaaagaagt ttgattccac taaaactcat

181 catcaacttg gcaacatctc catcaactac aacgcagcct ttaacccggg cgggaattcc

241 tatttatgtg tctatggctg gacacaatct ccattagctg aatactacat tgttgagtca

301 tggggcacat atcgtccaac agggacgtat aaaggatcat tttatgccga tggaggcaca

361 tatgacatat atgaaacgct ccgtgtcaat cagccttcta tcattggaga cgctaccttc

421 aaacaatatt ggagtgtacg tcaaacaaaa cgcacaagcg gaactgtctc tgtcagtgag

481 cattttaaaa aatgggaaag cttaggcatg ccaatgggaa aaatgtatga aacagcatta

541 actgtagaag gctaccgaag caacggaagt gcgaatgtca tgacgaatca gctgatgatt

601 cgataa

Signal peptide sequence:

<210>

<211> 26

<212>

＜213〉bacillus pumilus ( Bacillus sp.HBP8)

<400>

MNLKRLRLLFVMCIGFVLTLTAVPAH

Claims (4)

1. alkalescent xylanase XynHBsThe Nucleotide majorizing sequence, after it is characterized in that optimizing XynHBsSequence and original series XynHBCompare, the optimised and pichia spp low frequency password wherein of 139 bases is all replaced by high frequency or inferior high frequency password in the sequence, has removed 26 amino acid whose signal coding sequences in the sequence, through T ₄After archaeal dna polymerase and dTTP process, the 5' of sequence end formed with CpoThe sticky end that the I restriction enzyme site is identical, 3' end formed with NotThe sticky end that the I restriction enzyme site is identical.

2. alkalescent xylanase XynHBsNucleotide majorizing sequence method is characterized in that optimization step is:

(1) optimizes, adopt the overlapping primer extension of PCR of laboratory routine, site-directed mutagenesis is crossed XynHBGene order input DNAworks software, (Codon Frequency Table) selects in the codon frequency table P. pastoris, obtain being correlated with XynHBsThe optimized gene sequence of in pichia spp, expressing XynHBsAnd primer sequence;

(2) transform primer, for the ease of protein expression, added base sequence GTCA at the 5' of primer 1, the 5' end of primer 22 has added base sequence GGCCA;

(3) pcr amplification, with above-mentioned primer sequence template each other, adopt the method for successively decreasing from the two ends primer to middle primer concentration to carry out the amplification of total length, namely primer 1, primer 22 concentration are 40nmol/L, successively decrease successively, but middle primer 11, primer 12 concentration are minimum is 0.625nmol/L; Concrete reaction conditions is 94 ℃ of denaturation 5min, 94 ℃ of 30s then, and 60 ℃ of 30s, 72 ℃ of 90s, 30 circulations, 72 ℃ are extended 10min again;

(4) detect, the PCR product detects output and specificity with 0.7% agarose gel electrophoresis, and is connected conversion with DNA purification kit purified pcr product with the T carrier, select obtain after transformant checks order consistent with design result XynHBsMajorizing sequence.

3. according to claim 1,2 or 3 described a kind of alkalescent xylanases XynHBsThe Nucleotide majorizing sequence is characterized in that described recombinant vectors is with alkalescent xylanase XynHBsThe Nucleotide majorizing sequence is inserted the carrier pHBM905A that sets out, or the recombinant expression plasmid that obtains of the cloning site of carrier pPIC9k or carrier pPICZa; Described recombinant bacterial strain is that above-mentioned recombinant expression plasmid conversion Pichia pastoris GS115 obtains.

4. alkalescent xylanase XynHBsThe high-efficiency expression method of Nucleotide majorizing sequence is characterized in that step is:

(1) with claim 1 or 2 described alkalescent xylanase XynHBsThe Nucleotide majorizing sequence is synthetic through PCR, passes through T again ₄After archaeal dna polymerase and dTTP process, with process CpoI and NotThe expression vector pHBM905A that I processes connects, construction recombination plasmid xynHBs-905A;

(2) with recombinant plasmid xynHBs-905A warp SalChange in the Pichia pastoris GS115 after the I linearizing, obtain positive recombinant bacterial strain GS115/xynHBs-905A;

(3) positive recombinant bacterial strain is inoculated in the BMGY substratum of 25ml, 28 ℃, 200rpm cultivates 48h, to OD ₅₄₀Be 10-20;

(4) with centrifugal 5 minutes of cultured bacterium liquid 5000rpm, remove supernatant, transfer in the BMMY of 25ml substratum, 25 ℃, 200rpm cultivates 84h, during add methyl alcohol to final concentration 1% every 12 h and induce;

(5) temperature is 4 ℃, and the centrifugal 10min of 5000rpm collects supernatant, contains a large amount of active alkali body acidic xylanases in the supernatant liquor.

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