[2]The invention belongs to the technical field of, genetic engineering, and particularly relates to a pichia pastoris strain and. application thereof in xylanase production.
[4]The xylanase which is the most representative in the natural world of xylan, is the most representative of the, hemicellulose-based 1/3-1/2, xylanase which is the, most representative of the xylanase to. improve the fermentation, degree, and the xylanase is used, in the industry for increasing the fermentation level of the. xylanase to improve the fermentation level, β-1, 4.
[5]A great deal of, fund and technical effort are added to the beer production raw material. in China, so that β- the production cost of the beer is, greatly affected by the, fact that, the processing technology is complicated. and the production cost of the beer is too high . β.
[6]Content of the invention
[7]The pichia pastoris mutant strain is obtained from, constructed Pichia pastoris engineering bacteria through ultraviolet mutagenesis . the expression amount of xylanase can be greatly improved, and the original enzymatic property of, xylanase is not influenced.
[8]In one aspect, the invention provides a pichia pastoris engineering strain, and the strain is carried with a recombinant. plasmid for recombinant expression of a xylanase gene.
[9]The amino acid sequence of the xylanase is. SEQ ID NO:1The, coding nucleotide sequences thereof are as follows. SEQ ID NO:2.
[10]In one aspect, the present invention provides a mutant strain Pichia pastoris XL2(. Pichia pastorisThe XL2), collection number of the 2018 China Center 6 for Type Culture 15 (China Center for Type Culture) of China is preserved in China Cushu CCTCC NO:M2018375. Sushu Juyu, and the preservation number of China Center for, Type Culture (China Center for Type Culture).
[11]The invention also provides application. of the Pichia pastoris mutant strain in production of xylanase.
[12]Compared with the starting bacteria, the xylanase enzyme activity of XL2 the mutant strain pichia pastoris, can be widely applied to the production of xylanase 146 U/ml, is beneficial to remarkably 67.8%;20L reducing the production cost of the enzyme, 813 U/mL, and further promotes the wide 62%, application of the enzyme in the field. of, beer production . XL2, pH 3-7, 60 °C. XL2.
[13]Description of drawings
[14]The relative enzyme activity curve of 1: the fermentation supernatants of pH- the bacteria and the mutant XL2 pichia pastoris from the bacteria and the mutant bacteria Pichia pastoris is; shown in FIGS.
[15]The temperature of 2: the supernatant of the fermentation supernatant of the bacteria - and the mutant; XL2 pichia pastoris from the bacteria and the mutant bacteria is relatively close to the enzyme activity curve.
[16]The fermentation process chart of 3: the bacteria and the mutant Pichia pastoris (XL2 pichia pastoris). is shown in FIGS.
[18]The present invention will now be better understood and appreciated by. those skilled in the art in conjunction with the following, examples in which the, present J. invention (Sambrook) will be better understood and appreciated by those skilled in the art in, the following Examples.
[19]Construction of 1: recombinant plasmids
[20]The has first codon-optimized for the xylanase gene sequence according to the pichia pastoris, codons, 1 with an increase in the first base of the first amino acid codon 6 up to GAATTC( a number of bases. EcoRAfter I its stop), codon was terminated, the cleavage site of SEQ TAA GCGGCCGC( ID NO was increased. NotThe I nucleotide sequence), which is optimized by the synthesis of the strapeukiragin from, the Sagile Co. Ltd. is as follows. SEQ ID NO:2.
[21]Restriction enzymeEcoROryza® I. NotEnzyme I(Fermentas)-cleavage of a xylanase gene with a restriction enzyme of SEQ; ID NO :DNA at the same, time as restriction endonucleaseEcoROryza® I. NotThe I enzyme-pPIC9K cut product. was purified by means of a gel-purification, kit T4 DNA by (Fermentas) using a gel-purification kit and. the two endonuclease products were Trans5α subjected to (Transgen), a selection by ligation. of the two, enzyme-cut product by (Invitrogen). ligation of the two enzyme-product ligation-product-product ligation-product.
[22]Using the plasmid small-amount preparation method, the plasmid DNA was (Axygen) purified from the coli clone whose sequencing 1 result was correct, and the resulting sequence. DNA was shown as a result of sequencing by the sequencing results of the three, recombinant plasmids were obtained from the sequencing result. SEQ ID NO:2The, amino acid sequences encoded by them are as follows. SEQ ID NO:1The. naming of the recombinant plasmid construct was thus described in henceforth in, the pPIC9KXL. range of from.
[23]Construction of 2: Pichia pastoris engineering strain
[24]The recombinant plasmid was used as pPIC9K-XL a plasmid. SalThe I linearized gene, expression plasmid linearization fragment is transformed into the pichia pastoris by GS115, an MD electroporation method to obtain the pichia pastoris recombinant GS115/pPIC9K-XL, strain and then screened for multi-copy YPD transformants on the same plate containing different concentrations. of genetic streptomycin.
[25]The expression amount of the xylanase is determined to be as, 30 °C 250rpm BMGY that 1 d the, expression amount BMM of the xylanase 30 °C 250rpm according to, the size of 0.5% the xylanase,can be 4 d determined, to be as, high as the expression level of SDS-PAGE the xylanase, to obtain the expression level 40 kDa of, the Pichia pastoris engineering, strain, and the expression amount of the xylanase 87U/ml, is as shown in the specification . XL(Pichia pastorisXL).
[26]Definition of (1) Enzyme activity units
[27]The amount 37 °C, pH of enzyme 5.5 required to liberate, and reduce the sugar from a solution of xylan at a concentration in a range 5 mg/ml of from about pH at a value of from about 1 μ mol pH of from about pH of from about pH of from about pH of U. from about pH of about one is an enzyme activity unit.
[29]After the 2 ml reaction was 1% ended, the (pH5.5 reaction was - terminated after the), reaction was terminated by, 37 °C equilibration 10 min, of 2 ml the pH5.5 acidic - xylanase enzyme solution with an 37 °C acidic xylanase enzyme solution having a, concentration 37 °C in the range 30 min. of from pH, of 5 ml DNS the, solution in a standard 540 nm. blank and then 5 min, cooled to room temperature and, then cooled to room temperature 25 ml, and, then cooled to room temperature, AE.
[30]Enzyme activity calculation formula:
[32]Method for improving :XD activity of xylanase in diluted enzyme solution, U/ml; in formula (I)AE Light absorbance; of enzyme reaction liquidAB The absorbance of the enzyme blank is determined ;K as the ;C slope of the standard curve.0 For the standard curve, the molar mass of ;M the xylose is equal to the molar mass, 150.2 g/mol;t of the xylose ;1000, and the enzymatic hydrolysis reaction time is, min;N determined as the enzyme, 1 mmol=1000 μ mol. solution dilution multiple of the conversion factor.
[33]Mutagenesis screening of 3: Pichia pastoris XL (Pichia pastoris)
[34]The effect that the mutation caused by the ultraviolet, mutagenesis is very strong due to the mutation, randomness is. also, random and it is difficult, for the person skilled in the art to perform, the multi-wheel UV,directed mutagenesis at a large amount of time. and there is a possibility that effective positive, mutation can, not be obtained.
[35]The has further improved the yield of XL xylanase, by carrying out the genetic modification of the strain, from the pichia pastoris by an. ultraviolet mutagenesis method through the ultraviolet mutagenesis method.
[36]A suspension of pichia pastoris from Pichia XL pastoris, 30 °C, 2-3d, for YPD example, inoculated with sterile water-washed thalists in an, amount, of at least twice 1 × 10 as diluted with sterile water-washed thalists.6 The irradiation of the UV/mL, lamp 2-10min, with an ultraviolet lamp at a distance, of from (40W) about half of the ultraviolet lamp is at a 22cm, rate of about equal to 90% or greater than about two times to reach 48h. a coating flat-plate culture of .30 °C more than or equal to about two times.
[37]The activity of first the zymotic enzyme is increased 360 by centrifugation to remove, the thallus from the supernatant containing the xylanase by 200ul BMGY centrifugation to remove 96 the, 30 °C 250rpm thallus from 1 d the, culture supernatant containing the, xylanase 200ul BMM, 30 °C 250rpm and the 2 d, activity of the 0.5% zymotic enzyme. is remarkably 2 d improved, by fermenting the, zymophytic acid from the zymophyte, to produce the xylanase,containing XL supernatant .
[38]The result shows, first that the enzyme activity of the xylanase in, the mutant strain obtained by the ultraviolet mutagenesis screening is higher than the enzyme 3. activity of the xylanase in the mutant strain 15 fermentation supernatant . XL1,XL2,XL3.
[39]The strains obtained by the screening 3 are respectively XL1,XL2,XL3 switched into the BMGY culture supernatants of, 30 °C, 250rpm the fermentation 1 d supernatant, of the BMM Pichia pastoris, 30 °C, 250rpm and the, zymophyase activity detection 0.5% result shows; that the 4 d enzyme, activity of the, fermentation supernatant of; the pichia pastoris in the mutant strain is. greater than, or equal to and higher than XL2 that of the pichia pastoris, 146U/ml, XL 67.8%.
[40]Have found that mutant 2018 strain 6 is 15 Pichia pastoris XL2( (Pichia pastoris), for example, over a period of from day-to-day for the last year. Pichia pastorisThe collection number of XL2) the China Center for Type Culture (China Center for Type Culture) of CCTCC NO:M2018375. China in, the Chinese Wushu Sushu college college
[41]Enzymatic property analysis of 4: xylanase of example
[42]1, Optimal action value pH
[43]The xylanase pH activity is determined 2.0, 2.5, 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0 by respectively, diluting the fermentation supernatant of the XL pichia pastoris and the XL2 mutant Pichia pastoris from the fermentation supernatant of, the Pichia pastoris and the mutant Pichia pH pastoris, respectively, at 37 °C, the highest enzyme activity of, the enzyme, activity of the enzyme activity 100%, pH- 1, XL XL2 -pH pH 3.5-6.5.
[44]2, Optimum action temperature
[45]The xylanase activity of 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, pH5.5 the, fermentation supernatant of the pichia pastoris XL and the mutant pichia XL2 pastoris is calculated at the highest enzyme, activity, and the result 100%, of the relative, enzyme activity - of the xylanase. relative to 2 the enzyme, activity is substantially the XL same as the temperature XL2 change curve of the xylanase shown - in FIGS, 70 °C.
[46]In summary, according to the present invention, the xylanase XL2 having the recombinant expression, of the mutant strain of the present invention obtained by pH ultraviolet screening 3.5-6.5, has an enzymatic property and 70 °C. the same optimal action temperature as the most optimal action (s) at the, same time as before the mutation, and the most optimal acting temperature of the xylanase is in the range of from zero.
[47]Fermentable amplification 5:
[48]The formula of 20L the culture medium used for fermenting XL and fermenting the pichia XL2 pastoris and, the mutant pichia pastoris from 5.5 g/L, the fermentation: broth 20.3 g/L, 1.1 g/L, of the pichia pastoris and the 55 g/L, mutant pichia 16.4 g/L, pastoris 1.65 g/L, 0.05%.
[49]The fermentation production process :pH is 5.0, controlled 25 °C, by a 300rpm, fermentation 1.0-1.5(v/v), production process, 20% namely . a stirring rate of the air quantity of the air in the air volume of not more than the air quantity of the air in the air flow rate of not more than the air volume.
[50]The whole fermentation process is divided into three: first phases, namely, the, fermentation 7% process is divided, 30 °C into 24-26 h, three phases, namely, the; second incubation period of the, cell culture is performed according, to the cell culture, stage at a rate 80% of from about + ml. 30-60 min; third 20%, 160 h.
[51]The final fermentation enzyme activity of the mutant pichia pastoris is substantially higher, than the final fermentation. enzyme activity 3 of the, mutant 50h pichia, pastoris, and the final fermentation XL2 enzyme activity of the mutant pichia pastoris is; higher 62.3%, than that, which is higher than XL that of the starting bacteria 501 U/ml, XL2 813 U/ml.
[53]<110> Bluish-island-current blue biological cluster limited company
[54]<120> Pichia pastoris strain and application thereof in xylanase production
[56]<170>SIPOSequenceListing 1.0
[60]<213> Aspergillus aculeatus (Aspergillus aculeatus)
[62]Val Gly Leu Asp Gln Ala Ala Val Ala Lys Gly Leu Gln Tyr Phe Gly
[64]Thr Ala Thr Asp Asn Pro Glu Leu Thr Asp Ile Pro Tyr Val Thr Gln
[66]Leu Asn Asn Thr Ala Asp Phe Gly Gln Ile Thr Pro Gly Asn Ser Met
[68]Lys Trp Asp Ala Thr Glu Pro Ser Gln Gly Thr Phe Thr Phe Thr Asn
[70]Gly Asp Val Ile Ala Asp Leu Ala Glu Gly Asn Gly Gln Tyr Leu Arg
[72]Cys His Thr Leu Val Trp Tyr Asn Gln Leu Pro Ser Trp Val Thr Ser
[74]Gly Thr Trp Thr Asn Ala Thr Leu Thr Ala Ala Leu Lys Asn His Ile
[76]Thr Asn Val Val Ser His Tyr Lys Gly Lys Cys Leu His Trp Asp Val
[78]Val Asn Glu Ala Leu Asn Asp Asp Gly Thr Tyr Arg Thr Asn Ile Phe
[80]Tyr Thr Thr Ile Gly Glu Ala Tyr Ile Pro Ile Ala Phe Ala Ala Ala
[82]Ala Ala Ala Asp Pro Asp Ala Lys Leu Phe Tyr Asn Asp Tyr Asn Leu
[84]Glu Tyr Gly Gly Ala Lys Ala Ala Ser Ala Arg Ala Ile Val Gln Leu
[86]Val Lys Asn Ala Gly Ala Lys Ile Asp Gly Val Gly Leu Gln Ala His
[88]Phe Ser Val Gly Thr Val Pro Ser Thr Ser Ser Leu Ile Ser Val Leu
[90]Gln Ser Phe Thr Ala Leu Gly Val Glu Val Ala Tyr Thr Glu Ala Asp
[92]Val Arg Ile Leu Leu Pro Thr Thr Ala Thr Thr Leu Ala Gln Gln Ser
[94]Ser Asp Phe Gln Ala Leu Val Gln Ser Cys Val Gln Thr Thr Gly Cys
[96]Val Gly Phe Thr Ile Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro
[98]Ser Thr Phe Ser Gly Tyr Gly Ala Ala Leu Pro Trp Asp Glu Asn Leu
[100]Val Lys Lys Pro Ala Tyr Asn Gly Leu Leu Ala Gly Met Gly Val Thr
[102]Val Thr Thr Thr Thr Thr Thr Thr Thr Ala Thr Ala Thr Gly Lys Thr
[104]Thr Thr Thr Thr Ala Gly Ala Ala Ser Thr Gly Thr Thr Ala Ala His
[106]Trp Gly Gln Cys Gly Gly Leu Asn Trp Ser Gly Pro Thr Val Cys Ala
[108]Ser Gly Tyr Thr Cys Thr Tyr Val Asn Asp Tyr Tyr Ser Gln Cys Leu
[113]<213> Aspergillus aculeatus (Aspergillus aculeatus)
[115]gttggtttgg accaagctgc tgttgctaag ggtctgcaat acttcggtac tgctactgac 60
[116]aacccagagt tgactgacat cccatacgtt acccagttga acaacactgc tgacttcggt 120
[117]cagatcactc caggtaactc tatgaagtgg gacgctactg aaccatctca gggtactttc 180
[118]actttcacca acggtgacgt tatcgctgac ttggctgaag gtaacggtca gtacttgaga 240
[119]tgtcacaccc tggtttggta caaccagttg ccttcttggg ttacttccgg tacttggact 300
[120]aacgctactt tgactgctgc cctgaagaac cacatcacca acgttgtttc tcactacaag 360
[121]ggaaagtgct tgcactggga cgttgttaac gaagctttga acgacgacgg tacttacagg 420
[122]accaacatct tctacactac tatcggtgag gcttacatcc caattgcttt tgctgctgct 480
[123]gcagctgctg atccagatgc taagttgttc tacaacgact acaacttgga gtacggtggt 540
[124]gctaaggctg cttctgctag agctatcgtt cagttggtta agaacgctgg tgccaagatt 600
[125]gacggtgttg gattgcaagc tcacttctcc gttggtactg ttccttctac ttcctccttg 660
[126]atctctgtct tgcagtcctt cactgctttg ggtgttgagg ttgcttacac tgaggctgac 720
[127]gttagaatct tgttgccaac tactgctacc actttggctc aacaatcctc cgacttccaa 780
[128]gctttggttc agtcctgtgt tcagaccact ggttgtgttg gtttcactat ctgggactgg 840
[129]actgacaagt actcttgggt tccatccact ttctctggtt acggtgctgc tttgccatgg 900
[130]gacgaaaact tggtcaagaa gccagcttac aacggtttgt tggctggtat gggtgttact 960
[131]gttactacca ccactactac aactaccgct actgccactg gtaagactac taccacaact 1020
[132]gctggtgctg catccactgg tactactgct gctcattggg gtcaatgtgg tggtttgaat 1080
[133]tggtccggtc caactgtttg tgcttccggt tacacttgta cctacgtgaa cgactactac 1140
[134]tcccagtgtt tgtaa 1155
