CN115717135A - Heat-resistant xylosidase mutant and preparation thereof - Google Patents
Heat-resistant xylosidase mutant and preparation thereof Download PDFInfo
- Publication number
- CN115717135A CN115717135A CN202210992314.9A CN202210992314A CN115717135A CN 115717135 A CN115717135 A CN 115717135A CN 202210992314 A CN202210992314 A CN 202210992314A CN 115717135 A CN115717135 A CN 115717135A
- Authority
- CN
- China
- Prior art keywords
- mutant
- xyl
- xylosidase
- beta
- gene
- 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.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 53
- 108010038658 exo-1,4-beta-D-xylosidase Proteins 0.000 claims abstract description 33
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 18
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 18
- 241000588724 Escherichia coli Species 0.000 claims abstract description 17
- 102200079006 c.152C>T Human genes 0.000 claims abstract description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 15
- 239000013598 vector Substances 0.000 claims description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 9
- 229920001221 xylan Polymers 0.000 claims description 8
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 7
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 150000004823 xylans Chemical class 0.000 claims description 7
- 241001506991 Komagataella phaffii GS115 Species 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 235000013305 food Nutrition 0.000 claims description 5
- 239000010902 straw Substances 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 241000609240 Ambelania acida Species 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims description 2
- 235000007164 Oryza sativa Nutrition 0.000 claims description 2
- 241000209140 Triticum Species 0.000 claims description 2
- 235000021307 Triticum Nutrition 0.000 claims description 2
- 239000010905 bagasse Substances 0.000 claims description 2
- 235000009566 rice Nutrition 0.000 claims description 2
- 240000000111 Saccharum officinarum Species 0.000 claims 1
- 235000007201 Saccharum officinarum Nutrition 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 claims 1
- 238000006731 degradation reaction Methods 0.000 claims 1
- 229920001542 oligosaccharide Polymers 0.000 claims 1
- 239000010907 stover Substances 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 88
- 108090000790 Enzymes Proteins 0.000 abstract description 88
- 230000000694 effects Effects 0.000 abstract description 71
- 239000013612 plasmid Substances 0.000 abstract description 38
- 230000014509 gene expression Effects 0.000 abstract description 33
- 241000235058 Komagataella pastoris Species 0.000 abstract description 18
- 238000000855 fermentation Methods 0.000 abstract description 11
- 230000004151 fermentation Effects 0.000 abstract description 11
- 241001328122 Bacillus clausii Species 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 6
- 238000000605 extraction Methods 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 85
- 108010065282 UDP xylose-protein xylosyltransferase Proteins 0.000 description 83
- 102000010199 Xylosyltransferases Human genes 0.000 description 74
- 239000000243 solution Substances 0.000 description 51
- 238000006243 chemical reaction Methods 0.000 description 28
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 18
- 238000012258 culturing Methods 0.000 description 17
- 239000006228 supernatant Substances 0.000 description 17
- 239000001963 growth medium Substances 0.000 description 15
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 13
- 238000001976 enzyme digestion Methods 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 12
- 238000012216 screening Methods 0.000 description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 11
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 239000011347 resin Substances 0.000 description 11
- 229920005989 resin Polymers 0.000 description 11
- 239000000872 buffer Substances 0.000 description 10
- MLJYKRYCCUGBBV-YTWAJWBKSA-N 4-nitrophenyl beta-D-xyloside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 MLJYKRYCCUGBBV-YTWAJWBKSA-N 0.000 description 9
- 241000193830 Bacillus <bacterium> Species 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 239000013613 expression plasmid Substances 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 238000012795 verification Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- 239000004472 Lysine Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 230000005484 gravity Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000012064 sodium phosphate buffer Substances 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000000593 degrading effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 239000012982 microporous membrane Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 2
- 244000153158 Ammi visnaga Species 0.000 description 2
- 235000010585 Ammi visnaga Nutrition 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 2
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 2
- 108010038447 Chromogranin A Proteins 0.000 description 2
- 102100031186 Chromogranin-A Human genes 0.000 description 2
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- -1 Tris saturated phenol Chemical class 0.000 description 2
- UGXQOOQUZRUVSS-ZZXKWVIFSA-N [5-[3,5-dihydroxy-2-(1,3,4-trihydroxy-5-oxopentan-2-yl)oxyoxan-4-yl]oxy-3,4-dihydroxyoxolan-2-yl]methyl (e)-3-(4-hydroxyphenyl)prop-2-enoate Chemical compound OC1C(OC(CO)C(O)C(O)C=O)OCC(O)C1OC1C(O)C(O)C(COC(=O)\C=C\C=2C=CC(O)=CC=2)O1 UGXQOOQUZRUVSS-ZZXKWVIFSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 229920000617 arabinoxylan Polymers 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 108010079058 casein hydrolysate Proteins 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- CPTIBDHUFVHUJK-NZYDNVMFSA-N mitopodozide Chemical compound C1([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H](CO)[C@@H]2C(=O)NNCC)=CC(OC)=C(OC)C(OC)=C1 CPTIBDHUFVHUJK-NZYDNVMFSA-N 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000012521 purified sample Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 239000007222 ypd medium Substances 0.000 description 2
- LGQKSQQRKHFMLI-SJYYZXOBSA-N (2s,3r,4s,5r)-2-[(3r,4r,5r,6r)-4,5,6-trihydroxyoxan-3-yl]oxyoxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)OC1 LGQKSQQRKHFMLI-SJYYZXOBSA-N 0.000 description 1
- JCSJTDYCNQHPRJ-UHFFFAOYSA-N 20-hydroxyecdysone 2,3-acetonide Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(OC2C(C(O)C(O)OC2)O)OC1 JCSJTDYCNQHPRJ-UHFFFAOYSA-N 0.000 description 1
- LGQKSQQRKHFMLI-UHFFFAOYSA-N 4-O-beta-D-xylopyranosyl-beta-D-xylopyranose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(O)OC1 LGQKSQQRKHFMLI-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 101710101545 Alpha-xylosidase Proteins 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 102100031746 Bone sialoprotein 2 Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 241000193464 Clostridium sp. Species 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- SQNRKWHRVIAKLP-UHFFFAOYSA-N D-xylobiose Natural products O=CC(O)C(O)C(CO)OC1OCC(O)C(O)C1O SQNRKWHRVIAKLP-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 101000707248 Homo sapiens Bone sialoprotein 2 Proteins 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 239000006004 Quartz sand Substances 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229960001931 ampicillin sodium Drugs 0.000 description 1
- KLOHDWPABZXLGI-YWUHCJSESA-M ampicillin sodium Chemical compound [Na+].C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C([O-])=O)(C)C)=CC=CC=C1 KLOHDWPABZXLGI-YWUHCJSESA-M 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- JCSJTDYCNQHPRJ-FDVJSPBESA-N beta-D-Xylp-(1->4)-beta-D-Xylp-(1->4)-D-Xylp Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)C(O)OC2)O)OC1 JCSJTDYCNQHPRJ-FDVJSPBESA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 229960002064 kanamycin sulfate Drugs 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000006098 transglycosylation Effects 0.000 description 1
- 238000005918 transglycosylation reaction Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
- ABKNGTPZXRUSOI-UHFFFAOYSA-N xylotriose Natural products OCC(OC1OCC(OC2OCC(O)C(O)C2O)C(O)C1O)C(O)C(O)C=O ABKNGTPZXRUSOI-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the field of genetic engineering technology of enzymes, and particularly relates to a beta-xylosidase mutant with improved enzyme activity and thermal stability and preparation and application thereof. The invention obtains the wild beta-xylosidase gene of Bacillus clausii (Bacillus clausii) by molecular biotechnology means, randomly mutates the beta-xylosidase gene by using error-prone PCR technology to obtain a beta-xylosidase mutant S51L and a coded gene xylm thereof, reconstructs recombinant plasmids, realizes the high-efficiency expression of the beta-xylosidase in Escherichia coli, pichia pastoris and Bacillus subtilis, and obtains the beta-xylosidase with improved enzyme activity and thermal stability by technologies such as fermentation and extraction.
Description
The technical field is as follows:
the invention belongs to the technical field of genetic engineering of enzymes, and particularly relates to a xylosidase mutant with improved activity and thermal stability, and preparation and application thereof.
The background art comprises the following steps:
beta-Xylosidase (beta-Xylosidase, XYL, EC 3.2.1.37), an exonuclease, is a enzyme that primarily catalyzes the hydrolysis of xyloside and hydrolyzes lower degree of polymerization xylooligosaccharides (xylobiose or xylotriose) from the non-reducing end in an exo-manner to xylose. Meanwhile, the beta-xylosidase can also act on a glycosidic bond formed by aglycone such as terpenes and steroids and xylose to release the aglycone. At present, xylanase systems including XYL (xylosyltransferase) are widely applied to a plurality of fields such as food, medicine, paper making, feed, energy and the like for degrading xylan. In the food industry, beta-xylosidase is used in baking, where flour containing 2-3% arabinoxylan is used, and the addition of a xylan degrading enzyme mixture containing beta-xylosidase to flour releases water retained in arabinoxylan, thereby improving dough machinability, increasing the volume of the final product, improving its crumb structure, and significantly increasing the shelf life of bread in the consumer market; in the pharmaceutical industry, the beta-xylosidase with the transglycosylation activity can promote xylan to form xylo-oligosaccharide (XOS), and the XOS has positive influence on the physiological functions of a human body, such as reducing cholesterol level, increasing the bioavailability of calcium, reducing the risk of colon cancer and the like; in the papermaking sector, xylan hydrolase complexes are also used in the pulp and paper industry, primarily for processing cellulose pulp before whitening.
XYL is widely found in nature and has been isolated from microorganisms such as bacteria, actinomycetes and fungi (including molds, yeasts and mushrooms) and parts of higher plants. Most bacteria or fungi produce only one XYL, and alpha-xylosidase is reported to be derived from Escherichia coli (Escherichia coli) and Aspergillus niger (Aspergillus niger) only; the source of the beta-xylosidase is many, and the beta-xylosidase is mainly derived from fungi and bacteria, such as Bacillus (Bacillus sp.) and clostridium (clostridium sp.). Currently, β -xylosidase enzymes are found in 11 different GH families, GH family 1, 3, 5, 30, 39, 43, 51, 52, 54, 116 and 120, respectively, which are compatible with xylan heterogeneity, whereas β -xylosidase enzymes have been reported to be mostly present in GH3, GH39 and GH 43.
Directed evolution is one of the important means to improve protein function and activity, especially in terms of improving protein thermostability. It belongs to the irrational design of protein, does not need to obtain the high-grade structure of protein and catalytic site, only needs to make random mutation on protein amino acid sequence. It can simulate the evolution mechanism of natural selection in the laboratory, quickly establish a mutant library containing a large number of target protein coding genes in vitro by means of molecular biology, and quickly obtain a protein mutant which accords with the application value of human beings by a high-flux directional screening method. The core steps of directed evolution mainly include the construction of a diverse library of mutants and high throughput screening methods. Commonly used include: error-prone PCR, saturation mutagenesis, DNA shuffling, staggered extension PCR, and the like. Site-directed mutagenesis, i.e., rational design, is purposefully modified on the basis of the spatial structure, active site, catalytic mechanism and the like of protein, and only a few amino acids in natural enzyme protein can be replaced, deleted or inserted, so that the high-level structure of the enzyme protein is not changed, and the modification of the enzyme function is limited. Therefore, for enzymes with unknown structure and function, directed evolution can make up for the deficiency of rational design to some extent.
The colibacillus expression system is the most widely used protein expression system at present, and not only has the advantages of clear genetic background, simple culture operation, high transformation and transduction efficiency, fast growth and reproduction, low cost, and capability of producing target protein rapidly in large scale, but also has the level of expressing foreign gene products far higher than that of other gene expression systems, and the expressed target protein amount even exceeds 30% of the total amount of bacteria, so the colibacillus expression system is widely applied to gene engineering.
The yeast expression system is an economic and efficient eukaryotic protein expression system, can successfully realize intracellular expression or secretory expression, has a relatively cheap amplification culture medium and low requirements on culture conditions, and is suitable for industrial amplification. The yeast expression system comprises a saccharomyces cerevisiae expression system, a methylotrophic yeast expression system and a fission yeast expression system, and has the processes of translation, processing and modification of protein because the expression system is a eukaryotic cell. With the development of biotechnology, yeast expression systems will be used for the expression of more foreign proteins and more widely applied to a plurality of fields.
The bacillus expression system is widely applied to the fields of industry, agriculture, medicine, sanitation, food, animal husbandry, aquatic products and scientific research as a safe, efficient, multifunctional and microorganism strain with great development potential. It can secrete the product expressed by the target gene to the outside of cells, thereby reducing the cost and the workload of further collecting, separating and purifying the gene expression product. Bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus megaterium and the like in the bacillus can be used as expression host bacteria. In the field of microbial genetics, background research of bacillus is also quite clear, and the bacillus has the advantages of unobvious codon preference, simple fermentation, rapid growth, no production of pathogenic toxin, no special requirement on a culture medium and the like. With the development of molecular biology techniques and the intensive research of Bacillus, a large number of genes have been cloned and expressed using Bacillus expression systems, and some have been industrially produced on a large scale, and various enzymes and clinically required chemicals or industrial products are produced by expression using Bacillus.
The invention content is as follows:
based on the problems in the prior art, in order to further promote the application of the beta-xylosidase in the industrial field, the prior property of the beta-xylosidase needs to be further improved, and the invention aims to provide the mutant of the beta-xylosidase with improved enzyme activity and thermal stability.
The technical route for achieving the purpose of the invention is summarized as follows:
the method comprises the steps of obtaining a wild type XYL coding gene of Bacillus clausii (Bacillus clausii) by a basic molecular biology technical means, constructing a recombinant vector by enzyme digestion, connection and the like, obtaining a wild type XYL sequence (shown as SEQ ID NO. 2) by sequencing, carrying out random mutation on the wild type XYL coding gene by an error-prone PCR technology, screening by an Escherichia coli expression system to obtain an XYL mutant S51L and a coding gene xylm thereof, reconstructing the recombinant vector, realizing high-efficiency expression of the recombinant vector in Escherichia coli, pichia pastoris and Bacillus subtilis, and obtaining the XYL mutant with improved enzyme activity and thermal stability by technologies such as fermentation and extraction.
One of the technical schemes provided by the invention is that a beta-Xylosidase (XYL) mutant S51L is obtained by carrying out mutation on the 51 st S51L on the basis of a wild type XYL amino acid sequence shown in SEQ ID NO. 1;
further, the S51L mutant has an amino acid sequence shown in SEQ ID NO. 3.
The second technical scheme provided by the invention is a coding gene of an S51L mutant;
furthermore, the coding gene of the S51L mutant has a nucleotide sequence shown in SEQ ID NO. 4.
The third technical scheme provided by the invention is a recombinant vector or a recombinant strain for expressing the S51L mutant or the coding gene thereof;
further, the expression plasmid adopted by the recombinant vector is pET-22b, pPIC9K or pLY-3 plasmid;
further, the recombinant strains employ host cells including, but not limited to: escherichia coli BL21 (DE 3), pichia pastoris GS115, or Bacillus subtilis WB600.
The fourth technical scheme provided by the invention is the application of the recombinant vector or the recombinant strain, in particular to the application in the production of the beta-xylosidase S51L mutant.
The fifth technical scheme provided by the invention is the application of the S51L mutant, in particular to the application in the preparation of xylose by degrading xylo-oligosaccharide, for example, the S51L mutant can be used alone or synergistically acted with xylanase to hydrolyze xylan to generate xylose, or can be used for preparing xylose from alkali-extracted xylan in wheat straw, rice straw, corn cob and bagasse, and can be applied to multiple fields of food industry, pharmaceutical industry, paper making industry, energy preparation and the like.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in single or three letter code form. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
Identification of XYL mutants
The "amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in the XYL mutant. E.g. Ser51Leu or S51L, indicating a substitution of the amino acid at position 51 from Ser for Leu of the wild type XYL, the numbering of the positions corresponding to the numbering of the amino acid sequence of the wild type XYL in SEQ ID NO. 1.
In the present invention, lower italic XYL represents the gene encoding wild-type XYL, and lower italic xylm represents the gene encoding mutant S51L, the information being as shown in the table below.
| XYL | Amino acid mutation site | Site of gene mutation | Amino acid SEQ ID NO. | Nucleotide SEQ ID NO. |
| Wild type | — | — | 1 | 2 |
| S51L | Ser51Leu | AGT→CTT | 3 | 4 |
The experimental scheme of the invention is as follows:
1. the method for obtaining the XYL mutant coding gene with improved enzyme activity and thermal stability comprises the following steps:
(1) Carrying out error-prone PCR random mutation on a wild type XYL coding gene by taking the wild type XYL coding gene XYL (SEQ ID NO. 2) of the Bacillus clausii as a template;
(2) Constructing a recombinant plasmid by enzyme digestion, connection and the like of the randomly mutated XYL coding gene, transferring the recombinant plasmid into escherichia coli BL21 (DE 3), screening to obtain an XYL mutant with improved enzyme activity and thermal stability, sequencing to obtain an XYL mutant coding gene xylm, and storing a plasmid pET-22b-xylm containing the XYL mutant coding gene with improved enzyme activity and thermal stability.
2. An escherichia coli recombinant strain containing XYL coding genes with improved enzyme activity and thermal stability and a process for preparing XYL mutant with improved activity and thermal stability, comprising the following steps:
(1) Connecting the XYL mutant coding gene xylm with an escherichia coli plasmid pET-22b to obtain a new recombinant plasmid pET-22b-xylm;
(2) And transferring the recombinant plasmid into escherichia coli BL21 (DE 3), screening ampicillin sodium (Amp) resistance, performing enzyme digestion verification to obtain a recombinant strain, and then performing culture fermentation on the recombinant strain to obtain the XYL mutant with improved enzyme activity and heat stability.
3. A pichia pastoris strain containing XYL coding genes with improved enzyme activity and thermal stability and a process for preparing XYL mutants with improved enzyme activity and thermal stability by using the pichia pastoris strain comprise the following steps:
(1) Carrying out homologous recombination on a XYL mutant encoding gene xylm and a plasmid pPIC9K to obtain a new recombinant plasmid, granulating the recombinant plasmid and transferring into JM109, screening kanamycin sulfate (Kan) resistance, selecting a positive clone, extracting the plasmid, carrying out single enzyme digestion by SalI, linearizing the recombinant plasmid, then electrically transferring into a Pichia pastoris competent cell GS115, and carrying out genetic mycin (G418) resistance screening and XYL enzyme activity determination on the obtained recombinant strain to obtain the recombinant strain with improved enzyme activity and thermal stability;
(2) After the recombinant strain is fermented, the XYL mutant with improved enzyme activity and heat stability is prepared.
4. A bacillus subtilis strain containing XYL coding genes with improved enzyme activity and heat stability and a process for preparing XYL mutants with improved enzyme activity and heat stability by using the bacillus subtilis strain comprise the following steps:
(1) Connecting a XYL mutant coding gene xylm with a shuttle plasmid pLY-3 to obtain a new recombinant plasmid pLY-3-xylm, transferring the new recombinant plasmid pLY-3-xylm into escherichia coli JM109, screening chloramphenicol resistance of the obtained recombinant strain, transferring a positive clone into bacillus subtilis WB600, screening Kan resistance of the obtained recombinant strain and measuring the enzyme activity of XYL to obtain the recombinant strain with improved enzyme activity and thermal stability;
(2) Fermenting the recombinant strain to prepare the XYL mutant with improved enzyme activity and thermal stability.
The enzymatic properties of the XYL mutant S51L are as follows:
(1) Specific activity: the specific activity of the XYL mutant S51L was 62U/mg.
(2) Optimum reaction temperature: at 50 deg.C.
(3) Temperature stability: after 30min of water bath heat preservation at 60 ℃ under the condition of pH 8.0, the residual enzyme activity of the mutant S51L is about 86 percent, compared with the wild type, the mutant S51L is completely inactivated.
Has the beneficial effects that:
1. the invention utilizes error-prone PCR technology to carry out random mutation on wild type XYL to obtain mutant S51L with improved enzyme activity. The highest values of the specific enzyme activities of the high-activity beta-xylosidase S51L in fermentation liquids of escherichia coli, pichia pastoris and bacillus subtilis expression systems are respectively 62U/mg, 26U/mg and 32U/mg, and the specific enzyme activities are respectively improved by about 150% compared with wild type.
2. According to the invention, wild type XYL is subjected to random mutation by using an error-prone PCR technology to obtain a mutant S51L with improved thermal stability, and the mutant S51L is subjected to heat preservation at 60 ℃ in a phosphate buffer solution with the pH of 8.0, and the stability is improved along with the increase of the heat preservation time compared with the wild type XYL, after the heat preservation is carried out for 30min, the residual enzyme activity of the mutant XYL is about 86%, and the wild type XYL is inactivated.
3. The invention respectively uses an escherichia coli expression system, a pichia pastoris expression system and a bacillus subtilis expression system to realize the efficient expression of the S51L mutant with improved enzyme activity and thermal stability in different modes.
Description of the drawings:
FIG. 1 is a PCR amplification electrophoresis picture of wild type XYL gene of the present invention
Wherein: m is DNA Marker, and 1 is xyl gene.
FIG. 2 is a restriction enzyme digestion verification diagram of recombinant plasmids pET-22b-xylm and pLY-3-xylm of the invention, wherein: m is DNA Marker,1 is BamHI and HindIII double-enzyme cutting electrophoresis picture of escherichia coli recombinant plasmid, and 2 is BamH I and Sam I double-enzyme cutting electrophoresis picture of recombinant plasmid pLY-3-xylm in bacillus subtilis.
FIG. 3 is the PCR verification chart of the Pichia pastoris positive transformant of the invention
Wherein: m is DNA Marker,1 is pichia pastoris xylm gene electrophoretogram.
FIG. 4 is an SDS-PAGE pattern of a purified sample of mutant S51L of the present invention
Wherein: m is DNA Marker, and 1 is S51L purified sample.
FIG. 5 is a temperature optimum curve of wild type XYL versus mutant S51L with reference to relative viability according to the present invention wherein: WT is the wild type XYL of the invention, and S51L is the XYL mutant of the invention.
FIG. 6 is a temperature optimum curve of wild type XYL and mutant S51L with reference to specific enzyme activity according to the present invention, wherein: WT is the wild type XYL of the invention, and S51L is the XYL mutant of the invention.
FIG. 7 is a graph of a p-nitrophenol standard.
FIG. 8 is a thermal stability curve
Wherein: WT is the wild type XYL of the invention, and S51L is the XYL mutant of the invention.
The specific implementation mode is as follows:
the technical content of the present invention is further illustrated by the following examples, but the present invention is not limited to these examples, and the following examples should not be construed as limiting the scope of the present invention.
1. The culture medium used in the examples of the present invention was as follows:
LB medium (g/L): 5.0 yeast extract, 10.0 tryptone and 10.0 NaCl.
10 XSP salt solution (g/L): k is 2 HPO 4 91.7，KH 2 PO 4 30，(NH4) 2 SO 4 10, sodium citrate 5, mgSO 4 ·7H 2 O 10。
SP I medium: 97.6mL of 1 XSP salt solution, 400. Mu.L of 5% casein hydrolysate, 1mL of 10% yeast juice, and 1mL of 50% glucose. (5% Casein hydrolysate: 0.5g acid hydrolyzed Casein in 10mL ddH 2 O;10% yeast juice: 1g Yeast extract dissolved in 10mL ddH 2 O;50% glucose: 5g glucose dissolved in 10mL ddH 2 O)。
SP II medium: 99mL of SP I medium, 100mM CaCl 2 500μL，500mM MgCl 2 500μL。
YPD medium (g/L): yeast powder 10.0, peptone 20.0, glucose 20.0.
MD Medium (g/L): glucose 20.0, agar 15.0, 10%10 × YNB,0.2% biotin.
The solid culture medium of the above culture medium was supplemented with 2% agar.
2. The relevant solutions used in the examples of the invention are as follows:
lysine Buffer: 20mL of Tris-HCl (1.0 mol/L) and 120mL of NaCl (2.5 mol/L) are respectively measured and dissolved in a proper amount of water, the pH value is adjusted to 8.0 by hydrochloric acid, and then ddH is used 2 And (4) metering the volume of O to 1000mL, filtering through a 0.22 mu m microporous membrane, removing impurities by suction filtration, and storing at 4 ℃.
Wash Buffer: 20mL of Tris-HCl (1.0 mol/L), 120mL of NaCl (2.5 mol/L) and 20mL of imidazole (1.0 mol/L) are respectively measured and dissolved in a proper amount of water, the pH value is adjusted to 8.0 by hydrochloric acid, and then ddH is used 2 And (4) metering the volume of O to 1000mL, filtering through a 0.22 mu m microporous membrane, removing impurities by suction filtration, and storing at 4 ℃ in a dark place.
Elution Buffer: 20mL of Tris-HCl (1.0 mol/L), 120mL of NaCl (2.5 mol/L) and 300mL of imidazole (1.0 mol/L) are respectively measured and dissolved in a proper amount of water, the pH value is adjusted to 8.0 by hydrochloric acid, and then ddH is used 2 And (4) metering the volume of O to 1000mL, filtering the mixture by a 0.22-micron microporous membrane to remove impurities by suction, and storing the mixture at 4 ℃ in a dark place.
The wild type XYL amino acid sequence is shown in SEQ ID NO. 1:
MIRNPVLKGFNPDPSICRVGDDYYMAVSTFEWFPGVQIHHSRDLVNWRLISRPLNRISQLNMIGNPDSGGVWAPCLSYSNGKFWLVYSDVKVVEGNTWKDGHNYLVTCETIDGEWSEPIYLNSSGFDPSLFHDEDGRKYVVNMVWDQRVYNHRFYGICIQEYSVLEKRLVGKPQMIFKGTELGLTEAPHLYQANGYYYLLTAEGGTKYEHAATIARSKEIHGPYEVHPQNPILSSWADPRHPLQKAGHASLVETQHGDWYMAHLLGRPIRRRGKKLLEERGFCPLGRETAIQKIEWKDDWPYVVNGPLPSVEVAGPKLPEVQWPQDYPKCDQFDHPVLNHHYQTLRIPFNQEIGMIDHEAGILRLFGRESLHSKHTQALVARRWQSFHFDAATEVSFYPETFQQAAGLICYYDTENWVSLQVTWHEQKGRILDLVQCDHFHVSQPLQGSEIVVPEQAATVHLKVSVRYDTFSFAYSFDGSHFEDIGVSFDTYKLSDDYIAHGGFFTGAFVGMHCQDTSGVRKHADFHSFSYHELESNSLNQAEGGTSARKSVIHG*
the amino acid sequence of the XYL mutant S51L is shown as SEQ ID NO. 3:
MIRNPVLKGFNPDPSICRVGDDYYMAVSTFEWFPGVQIHHSRDLVNWRLILRPLNRISQLNMIGNPDSGGVWAPCLSYSNGKFWLVYSDVKVVEGNTWKDGHNYLVTCETIDGEWSEPIYLNSSGFDPSLFHDEDGRKYVVNMVWDQRVYNHRFYGICIQEYSVLEKRLVGKPQMIFKGTELGLTEAPHLYQANGYYYLLTAEGGTKYEHAATIARSKEIHGPYEVHPQNPILSSWADPRHPLQKAGHASLVETQHGDWYMAHLLGRPIRRRGKKLLEERGFCPLGRETAIQKIEWKDDWPYVVNGPLPSVEVAGPKLPEVQWPQDYPKCDQFDHPVLNHHYQTLRIPFNQEIGMIDHEAGILRLFGRESLHSKHTQALVARRWQSFHFDAATEVSFYPETFQQAAGLICYYDTENWVSLQVTWHEQKGRILDLVQCDHFHVSQPLQGSEIVVPEQAATVHLKVSVRYDTFSFAYSFDGSHFEDIGVSFDTYKLSDDYIAHGGFFTGAFVGMHCQDTSGVRKHADFHSFSYHELESNSLNQAEGGTSARKSVIHG*
the invention will be further illustrated by the following specific examples.
Example 1: obtaining the wild-type XYL Gene XYL
1. The wild type XYL coding gene is derived from a strain of Bacillus clausii (Bacillus clausii) TCCC11004 stored in a laboratory, and the genome is extracted by using a Bacterial DNA Kit of the American OMEGA company.
(1) Strain activation: dipping a Claus bacillus liquid from a glycerin tube by using an inoculating loop, inoculating the Sporus clausii liquid to an LB flat plate, scribing a three region, and culturing at a constant temperature of 37 ℃ for 12 hours;
(2) Transferring: picking single colonies with neat edges and smooth surfaces from the flat plate, inoculating the single colonies into 5mL of liquid LB culture medium, and culturing for 12h at the temperature of 37 ℃ at 220 r/min;
(3) And (3) collecting thalli: collecting the above cultured bacterial liquid with 1.5mL EP tube, centrifuging at 12000r/min for 1min, discarding supernatant, and collecting thallus;
(4) Add 100. Mu.L of ddH 2 O resuspending the thallus, adding 50 mu L of 50mg/mL lysozyme, and carrying out water bath at 37 ℃ for 10min;
(5) Adding 100 mu L of BTL Buffer and 20 mu L of protease K, and carrying out vortex oscillation;
(6) Water bath at 55 deg.C for 40-50min, shaking every 20-30min, and mixing;
(7) Adding 5 μ L RNase, reversing, mixing for several times, and standing at room temperature for 5min;
(8) Centrifuging at 12000rpm for 2min, removing the undigested part, and transferring the supernatant part to a new 1.5mL EP tube;
(9) Adding 220 mu L BDL Buffer, shaking and mixing evenly, and carrying out water bath at 65 ℃ for 10min;
(10) Adding 220 mu L of absolute ethyl alcohol, blowing, sucking and uniformly mixing;
(11) Transferring to an adsorption column, standing for 1min, centrifuging at 12000rpm for 1min, and removing the filtrate;
(12) Adding 500 μ L HBC Buffer at 12000rpm, centrifuging for 1min, and removing the filtrate;
(13) Adding 700 mu L of DNA Wash Buffer at 12000rpm, centrifuging for 1min, and removing the filtrate;
(14) Adding 500 mu L of DNA Wash Buffer at 12000rpm, centrifuging for 1min, and removing the filtrate;
(15) 12000rpm, idle 2min, metal bath at 55 ℃ for 10min, and air drying;
(16) Add 40. Mu.L of ddH 2 O eluting the genome.
2. Amplification of wild-type XYL encoding Gene XYL
Designing an amplification primer of a wild type XYL coding gene, wherein the sequence is as follows:
upstream P1 (SEQ ID NO. 5):
CGCGGATCCGATACGAAAACCTGTTTAAAGG (BamHI cleavage site underlined)
Downstream P2 (SEQ ID NO. 6):
CCCAAGCTTCCCATGAATCACTTTCCTA (underlined hindlll cleavage site)
The reaction system for PCR amplification is 50 μ L, and the composition thereof is as follows:
| PrimeSTAR Max | 25μL |
| upstream primer P1 (20. Mu. Mol/L) | 2μL |
| Downstream primer P2 (20. Mu. Mol/L) | 2μL |
| Genome | 2μL |
| ddH 2 O | 19μL |
| Total volume | 50μL |
Note: the above-mentioned required reagents are from Takara, a precious bioengineering Co., ltd.
The setting of the amplification program is as follows:
a. pre-denaturation at 98 deg.C for 7min;
b. denaturation: 10s at 98 ℃;
c. annealing: 15s at 57 ℃;
d. extension: 20s at 72 ℃;
b-d reaction for 30 cycles;
f. extension: 10min at 72 ℃.
And (3) carrying out agarose gel electrophoresis on the PCR product to see a band of the wild-type XYL coding gene XYL of the bacillus clausii, wherein the band is about 1600bp (shown in figure 1), recovering the PCR product by using a DNA gel cutting recovery kit, constructing a recombinant plasmid pET-22b-XYL by carrying out enzyme cutting and connection on the PCR product, and sending the recombinant plasmid pET-22b-XYL to a sequencing company for sequencing to obtain a wild-type XYL gene sequence (shown in SEQ ID NO. 2).
Example 2: acquisition of XYL mutant S51L
1. Error-prone PCR: error-prone PCR is carried out by taking wild type XYL coding gene XYL as a template, and the reaction system is as follows:
| ddH 2 O | 21μL |
| recombinant plasmid pET-22b-xyl (5 ng/. Mu.L) | 1μL |
| Upstream primer P1 (10. Mu. Mol/L) | 2μL |
| Downstream primer P2 (10. Mu. Mol/L) | 2μL |
| Taq DNA polymerase | 0.5 |
| 10×Taq buffer | 5μL |
| dATP(10mmol/L) | 1μL |
| dGTP(10mmol/L) | 1μL |
| dTTP(10mmol/L) | 5μL |
| dCTP(10mmol/L) | 5μL |
| MgCl 2 (25mmol/L) | 10μL |
| MnCl 2 (10mmol/L) | 1.25μL |
Note: the above-mentioned required reagents are from Takara, bao bioengineering Co., ltd.
After the system is completed, an error-prone PCR reaction is performed, and the program is set as follows:
a. pre-denaturation at 94 deg.C for 5min;
b. denaturation: 30s at 94 ℃;
c. annealing: 30s at 56 ℃;
d. extension: 120s at 72 ℃;
b-d reaction for 30 cycles;
f. extension at 72 ℃ for 5min.
After the PCR reaction is finished, carrying out double enzyme digestion on the PCR product and the vector plasmid by BamH I and Hind III, purifying and recovering, connecting the error-prone PCR product with the vector plasmid pET-22b which is also subjected to double enzyme digestion, transforming escherichia coli BL21 (DE 3), coating the escherichia coli BL21 and the error-prone PCR product on an LB solid culture medium containing Amp (100 mu g/mL), and carrying out static culture in a 37 ℃ incubator for 12 hours to obtain a transformant.
3. The screening method comprises the following steps: the enzymatic activity of the beta-xylosidase was determined by a p-nitrophenol colorimetric method (p-NPX method). Under a certain condition, beta-xylosidase can hydrolyze a glycosidic bond in p-nitrophenyl-beta-D-xyloside (p-NPX) to generate p-nitrophenol (p-NP), wherein the p-nitrophenol is yellow under an alkaline condition, absorbance at 405nm of the p-nitrophenol is measured by using an enzyme-labeling instrument, the content of the corresponding p-nitrophenol is calculated, and the enzyme activity of the beta-xylosidase is further calculated.
4. Screening of mutant libraries: 720. Mu.L of LB liquid medium containing Amp (100. Mu.g/mL) was added to each well of a 48-well plate, and then, a single clone of each transformant was picked up into the 48-well plate using a sterilized small gun head as much as possible so that just a small amount of the bacteria were attached each time. The 48-well plate was transferred to a shaker culture at 440rpm and 37 ℃ for 6 hours. Then transferred to each well and added with 5mL of LB liquid medium containing Amp (100. Mu.g/mL), shake-cultured at 400rpm for 6h at 37 ℃, and finally added with 3. Mu.L of 1mol/L isopropyl-beta-D-thiogalactoside (IPTG) for induction at 180rpm for 16h at 16 ℃. Centrifuging at 5000rpm for 10min by using a low-temperature centrifuge (4), discarding the supernatant, resuspending the bacterial solution by using a sodium phosphate buffer solution (210 mu L) with pH =7.0, adding 30 mu L of lysozyme (50 mg/mL) to break the bacterial cells, centrifuging at 5000rpm for 10min by using a pore plate centrifuge to obtain a supernatant enzyme solution, adding 20 mu L of the enzyme solution into a 96 pore plate 1 of 100 mu L of reaction solution, reacting the solution for 10min by using 60 solution, adding 100 mu L of the reaction solution into 100 mu L of reaction stop solution, and detecting the light absorption value at 405 nm. The remaining supernatant enzyme solution was incubated at 60 for 10min, followed by centrifugation at 5000rpm for 1min using a well plate centrifuge, and the enzyme activity of the 96 well plate 2 was measured at 60 hours using the same method.
Note: reaction solution: 60 μ L of p-nitrophenyl- β -D-xylopyranoside (1 mg/mL) and 40 μ L of sodium phosphate buffer ph = 8.0; reaction termination solution: 100 μ L of 0.4mol/L Na 2 CO 3 And (3) solution.
5. Selecting the mutant with improved enzyme activity and thermal stability. Calculating the enzyme activity of each mutant according to the conditions of the plate 1 and the plate 2, selecting mutants with improved enzyme activity and thermal stability compared with wild type, performing repeated experiments on the enzyme activity and the thermal stability of the selected mutants to obtain mutants with less enzyme activity reduction than the wild type after 10min of heat preservation at 60 ℃, inoculating the mutant recombinant bacteria into a flat plate, selecting transformants, extracting plasmids, performing enzyme digestion verification (shown as a lane 1 in a figure 2), and sequencing.
Through the error-prone PCR of the steps, the Escherichia coli recombinant strain BL21 (DE 3)/pET-22 b-xylm of the mutant with improved activity and thermal stability is selected, sequencing to obtain an amino acid mutation containing a site, namely S51L: (AGT→CTT), thereby obtaining the XYL mutant S51L (SEQ ID NO. 3) and the gene xylm (SEQ ID NO. 4) encoding it.
Example 3: construction of XYL expression recombinant strain with improved enzyme activity and thermal stability of Pichia pastoris
1. Construction of an enzyme Activity and thermostability enhanced XYL expression plasmid pPIC9K-xylm
The method comprises the steps of carrying out EcoRI and NotI double enzyme digestion on a pichia pastoris expression vector pPIC9K, then connecting the digested product with an S51L mutant coding gene xylm, constructing to obtain a recombinant plasmid pPIC9K-xylm, transforming the recombinant plasmid pPIC9K-xylm to escherichia coli JM109 competent cells, selecting positive transformants, extracting plasmids, carrying out PCR verification and sequencing, and determining that the construction is successful, thus obtaining the recombinant expression plasmid pPIC9K-xylm.
2. Expression plasmid pPIC9K-xylm for transforming Pichia pastoris GS115
Before the constructed recombinant plasmid is electrically transformed into pichia pastoris, the plasmid pPIC9K-xylm needs to be subjected to linearization treatment to improve the integration efficiency of the recombinant plasmid on pichia pastoris chromosomes. Carrying out single enzyme digestion on pPIC9K-xylm by SalI, purifying and recovering to obtain linearized plasmid DNA.
(1) Preparation of Pichia pastoris GS115 competence
(1) Activating strains, inoculating pichia pastoris GS115 to a YPD solid culture medium by using an inoculating loop, and culturing for 48h at 30 ℃;
(2) selecting a single colony, inoculating the single colony into a YPD test tube, and culturing at 30 ℃ and 200rpm for 12h;
(3) inoculating the seed solution into 50mL of fresh YPD medium at an inoculation amount of 2%, and culturing at 30 deg.C for 4-5h to OD 600 Reaching about 1.4;
(4) centrifuging at 5000rpm for 8min at 4 deg.C with a low temperature centrifuge, and discarding the supernatant;
(5) washing thallus with 30mL of precooled sterile distilled water, placing on ice for 5-10min, centrifuging for 8min at 5000rpm by using a low-temperature centrifuge (4 ℃), and discarding the supernatant;
(6) repeating the step (5);
(7) resuspending and mixing the mycelia with 15mL1 mol/L precooled sorbitol, standing on ice for 5min, centrifuging at 5000rpm for 8min by using a low-temperature centrifuge (4 ℃), and discarding the supernatant;
(8) resuspend the cells in 800. Mu.L buffer (1 mol/L sorbitol, 15% glycerol);
(9) subpackaging in 80-100 μ L tube, and storing at-80 deg.C.
(2) Pichia pastoris
(1) Turning on the electric rotating instrument in advance to preheat the electric rotating instrument;
(2) transferring 10 μ L of recombinant plasmid pPIC9K-xylm and 100 μ L of competent mixture into 0.2cm electric rotating cup, and ice-cooling for 10min;
(3) 1500V, immediately adding 1mL of sorbitol after 5ms of electric shock, washing the bacterial suspension after electric shock from an electric rotating cup, transferring the bacterial suspension into a 1.5mL precooled sterile EP tube, recovering the bacterial suspension for 1-2h at 30 ℃ and 220rpm, coating the bacterial suspension on a screening plate MD plate, and culturing the bacterial suspension for more than 60h at 30 ℃.
3. Screening of Pichia pastoris high copy transformants
(1) For all transformants on the MD plate, all transformants were spotted with sterilized toothpicks onto YPD solid plates containing G418 at a final concentration of 0.5 mg/mL;
(2) A single colony (larger in colony diameter) of the YPD solid plate with a final concentration of 0.5mg/mL of G418 was selected and spotted with a sterilized toothpick onto the YPD solid plate with a final concentration of 2mg/mL of G418.
(3) Single colonies (larger in colony diameter) on YPD solid plates with a final concentration of 2mg/mL G418 were picked, and PCR-verified by a yeast genome-extracted group.
4. Identification of Pichia high copy transformants
Extracting a pichia pastoris genome:
(1) Centrifuging to collect yeast cells after overnight culture, adding 300 μ L of genome lysate into the yeast cells, and repeatedly blowing and sucking the yeast cells by using a pipette to suspend the yeast cells;
(2) Adding a certain amount of quartz sand and shaking on an oscillator for 25min to fully break the cell wall of the yeast;
(3) Adding 400 mu L of genome lysate, and centrifuging at 12000r/min for 10min;
(4) Transferring the obtained supernatant to another EP tube, adding a mixed solution of equal volume of Tris saturated phenol/chloroform (1), fully mixing, centrifuging at 12000r/min for 15min, and transferring the supernatant to another EP tube;
(5) After repeated extraction for 2 times, extracting for 1 time by using chloroform with the same volume so as to remove residual phenol;
(6) 12000r/min, centrifugal 15min, supernatant transferred to another EP tube and added 0.6 times of volume of isopropanol, inverted several times, placed at-80 ℃ for 20min,12000r/min centrifugal 8min to recover genomic DNA precipitation;
(7) Washing the precipitate with 70% alcohol for 2-3 times;
(8) The EP tube was air-dried for 20-30min, left free of alcohol smell, and 40. Mu.L of sterile water was added to dissolve the precipitate.
PCR validation of positive transformants: PCR amplification reaction was performed using genomic DNA as a template, and the conditions of the PCR reaction were verified to be correct (as shown in lane 1 in FIG. 3) according to example 1, thereby obtaining Pichia pastoris recombinant strain GS115/pPIC9K-xylm.
Example 4: construction of XYL expression recombinant bacteria with improved activity and heat stability of bacillus subtilis enzyme
1. Construction of a thermostable XYL expression plasmid pLY-3-xylm
The S51L mutant gene xylm and a bacillus subtilis expression vector pLY-3 are subjected to double enzyme digestion through BamHI and SmaI, and then are connected to construct a recombinant plasmid pLY-3-xylm, the recombinant plasmid pLY-3-xylm is transformed into escherichia coli JM109 competent cells, positive transformants are selected, the plasmid is extracted for enzyme digestion verification and sequencing, and the successful construction is determined, so that the recombinant expression plasmid pLY-3-xylm is obtained.
2. Expression plasmid pLY-3-xylm transformation of Bacillus subtilis WB600
(1) Activating a bacillus subtilis WB600 strain, scribing in three regions on a non-resistance LB plate, and culturing for 12h;
(2) Picking a single colony, inoculating the single colony into a test tube containing 5mL of LB culture medium, and culturing for 12h at 37 ℃ and 220 rpm;
(3) Inoculating 100 μ L of the seed solution into a test tube containing 5mL of SPI culture medium at 37 deg.C and 220rpm according to the inoculation amount of 2%, and culturing for 3-4h to OD 600 ＝1.2；
(4) Quickly inoculating 200 μ L of the culture medium into 2mL of SPII culture medium, culturing at 37 deg.C and 100rpm for 1.5h;
(5) Adding 20 μ L of 10mM EGTA, culturing at 37 deg.C and 100rpm for 10min;
(6) Adding 1-2 μ L recombinant plasmid, culturing at 37 deg.C and 100rpm for 30min, adjusting rotation speed to 220rpm, and culturing for 1-2 hr;
(7) Transferring the bacterial liquid into a sterilized 1.5mL EP tube, centrifuging at 5000rpm for 5min, discarding the supernatant, reserving 50 mu L of culture solution for resuspending the bacteria, and coating the bacterial liquid on a Kan-containing plate;
(8) Selecting transformants, extracting plasmids, and carrying out enzyme digestion verification (shown as a 2-lane in figure 2) to obtain the recombinant bacillus subtilis strain WB600/pLY-3-xylm.
Example 5: expression and preparation of XYL with improved activity and thermal stability in escherichia coli recombinant bacteria
1. Inoculating Escherichia coli recombinant strain BL21 (DE 3)/pET-22 b-xylm into LB liquid culture medium containing aminobenzyl resistance (100 μ g/mL), culturing at 37 deg.C and 220r/min for 2h to obtain bacterial liquid OD 600 =0.6, adding IPTG with final concentration of 0.5mmol/L, inducing culture at 16 deg.C and 120rpm/min16h。
2. And (3) crushing thalli: collecting the fermentation liquor by using a 500mL centrifugal cup, centrifuging at 8000rpm/min for 15min, discarding the fermentation supernatant, adding an appropriate amount of lysine buffer (20 mL lysine buffer is added to each 250mL of the thallus collected by the fermentation liquor) to resuspend and mix the thallus, and carrying out ultrasonic crushing on the thallus. After the disruption was completed, the solution was collected in a 50mL centrifuge tube and centrifuged at 12000rpm for 30min to obtain a soluble protein supernatant.
3. Resin pretreatment and protein and resin combination: two column volumes of lysine buffer were added to the purification column to wash the Ni while centrifuging 2+ Residual ethanol in the resin to balance Ni 2+ And (3) resin. Adding the crushed supernatant to Ni-containing solution after completion of centrifugation 2+ In the resin container, a small rotor is used for reaction and combination for 1-2h on a magnetic stirrer with 100rpm, and meanwhile, the whole process is kept at low temperature (0-4 ℃) so as not to damage the enzyme activity. After the binding was completed, the binding solution was transferred to a gravity column.
4. Protein purification
(1) Adding the combined solution into a gravity column in batches, filtering the solution through the gravity column completely, cleaning the inner wall of a reaction container by using filtrate, transferring the filtrate containing the resin into the gravity column again, and recycling all the resin as much as possible;
(2) After the combined solution completely flows out, adding 5mL of lysine buffer pre-cooled in advance into the purification column for cleaning the hybrid protein which is not combined with the resin or has weak binding force;
(3) Adding precooled 60mL Wash Buffer for six times after the lysine Buffer is drained, and eluting the hybrid protein bound on the resin;
(4) Finally, precooling the resin eluted by 5mL of Elution Buffer containing 500mmol/L of imidazole, repeating the resin Elution, and collecting the filtrate slowly flowing out of the column at the moment to a 30,000kDa ultrafiltration tube, wherein the target protein is in the solution;
(5) Replacement buffer: since the target protein solution is eluted by an Elution Buffer containing 500mmol/L imidazole, a large amount of imidazole in the solution may affect subsequent experiments, thereby requiring Buffer replacement. After centrifugation at 2500 Xg until the volume of the solution in the tube is about 1mL, 9mL of sodium phosphate Buffer (pH 8.0) was added, and the replacement was repeated twice, thereby completing the replacement of the Buffer solution, namely Elution Buffer, into the sodium phosphate Buffer. Finally, the obtained enzyme solution was transferred to a new EP tube and stored at a low temperature (4 ℃) to obtain a pure enzyme solution of the S51L mutant (SDS-PAGE shown in FIG. 4).
Example 6: expression and preparation of XYL with improved activity and thermal stability in pichia pastoris
1. Plate three-region streaking activated recombinant strain GS115/pPIC9K-xylm;
YPD seed liquid culture: selecting single colony, inoculating in 5mL resistance YPD culture medium containing Kan, and shake culturing at 30 deg.C and 200r/min for 24 hr;
BMGY enrichment culture of bacteria: inoculating into BMGY-containing medium at an inoculum size of 2%, and fermenting at 30 deg.C and 200r/min for 16h.
BMMY induction culture: BMGY thallus centrifugation (4 ℃,5000r/min,8 min); adding BMMY (15 mL) for resuspension and centrifugation, repeating twice, finally adding the thallus for resuspension into a BMMY culture medium, adding 250 mu L of methanol every 12 hours for induction and adding 100 mu L of Kan resistance every 24 hours for 6 days;
5. collecting fermentation supernatant to obtain S51L crude enzyme solution, and measuring enzyme activity.
Example 7: expression and preparation of XYL with improved activity and thermal stability in bacillus subtilis recombinant bacteria
1. Inoculating the recombinant bacillus subtilis WB600/pLY-3-xylm into 5mL LB liquid culture medium containing kanamycin (50 mug/mL), and culturing at 37 ℃ and 220r/min overnight;
2. transferring the strain into 50mL LB culture medium according to the inoculum size of 2%, culturing at 37 ℃ at 220r/min for 48h, centrifugally collecting fermentation supernatant to obtain S51L crude enzyme liquid with improved thermal stability, and measuring enzyme activity.
Example 8: determination of S51L enzyme activity and thermal stability
1.S51L principle of enzyme activity determination
The beta-xylosidase can hydrolyze glycosidic bonds in p-nitrophenyl-beta-D-xyloside (p-NPX) to generate p-nitrophenol (p-NP), wherein the p-nitrophenol is yellow under an alkaline condition, the absorbance at 405nm of the p-nitrophenol is measured by using an enzyme labeling instrument, the content of the corresponding p-nitrophenol is calculated, and the enzyme activity of the beta-xylosidase is further calculated.
2. Standard curve of
Accurately measure 0.0139g of ddH for p-nitrophenol 2 Dissolving O, diluting to 100mL to obtain 1mmol/L p-nitrophenol standard solution, and adding ddH 2 O diluting the standard solutions to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0mmol/L respectively, taking the diluted standard p-nitrophenol solution and water as blank control, adding 100 mu L of 0.4mol/L Na 2 CO 3 The solution was subjected to a color reaction, and the absorbance at 405nm of all the standard samples and the control sample was measured. A standard curve is drawn by taking the light absorption value as an ordinate (y) and the concentration of p-nitrophenol as an abscissa (x) (as shown in FIG. 7, y =2.28173x +0.10514, R is 2 ＝0.994)。
3. Definition of enzyme Activity
The β -xylosidase activity unit (U) is defined as: under certain conditions of temperature and pH (pH 8.0 if not specified, and temperature 60 ℃), the enzyme amount required for releasing 1 mu mol of p-nitrophenol per minute is one enzyme activity unit by using p-nitrophenyl-beta-D-xyloside (p-NPX) as a substrate.
Enzyme activity:
in the formula: u represents an enzyme activity unit;
v represents the total volume (mu L) of the final enzyme activity measuring system;
V 0 represents the volume of the enzyme solution (μ L) taken;
V 1 represents the volume of the total reaction solution (. Mu.L);
V 2 represents the volume (. Mu.L) of the reaction solution taken;
c represents the concentration of p-NP (mmol/L);
t represents reaction time (min);
n dilution factor of enzyme solution.
Method and step for measuring XYL enzyme activity
The reaction system is as follows: 60 μ L of the reaction solution was incubated at 60 ℃ for 1min, and 60 μ L of enzyme solution (V) was added 0 ) The total reaction volume was 120. Mu.L (V) 1 ) Reaction at 60 ℃ for 10min under pH =8.0, and 100. Mu.L of the reaction solution (V) was aspirated 2 ) Adding the mixture into 100 mu L of reaction stopping solution to stop reaction, wherein the total volume of the final enzyme activity measuring system is 200 mu L (V); OD was measured at 405nm using a microplate reader. The samples contained 3 sets of replicates.
Note: reaction solution: 60 μ L of p-nitrophenyl- β -D-xylopyranoside (1 mg/mL);
reaction termination solution: 100 μ L of 0.4mol/L Na 2 CO 3 And (3) solution.
5. The following table shows the results of enzyme activity assay (using the pure enzyme solution prepared in example 5, the crude enzyme solutions of S51L prepared in examples 6 and 7, and the crude enzyme solution of wild type XYL prepared in the same manner as above as experimental subjects):
note: in the preparation of a wild-type XYL pure enzyme solution or crude enzyme solution, a wild-type enzyme recombinant strain was first constructed in the same manner as in examples 2, 3 and 4, and then a wild-type enzyme crude enzyme solution was prepared in the same fermentation manner as in examples 5, 6 and 7.
Specific enzyme activity = enzyme activity (U/mL)/crude enzyme solution protein concentration (mg/mL).
6. Optimum temperature
Enzyme solutions of the Wild Type (WT) and the mutant (S51L) (the enzyme solution used was obtained in example 5, and the wild type was prepared in the same manner) were subjected to enzyme activity measurement at pH 8.0 at 10 ℃,20 ℃,30 ℃,40 ℃,50 ℃,60 ℃, 70 ℃,80 ℃, 90 ℃ and 100 wild respectively, and the specific activity and relative activity at each temperature were calculated with the highest activity as 100%, with the results shown in fig. 5 and 6, the optimum temperature for the wild type being 60 ℃ and the optimum temperature for the mutant being 50 ℃.
7. Detection of thermal stability
The change of the thermal stability of XYL is reflected by recording the change of the residual enzyme activity of a Wild Type (WT) and a mutant (S51L) at different temperatures and keeping the temperature for a certain time.
Enzyme solutions of Wild Type (WT) and mutant (S51L) (obtained in example 5, wild type was prepared in the same manner) were incubated at 60 ℃ for 10, 20, 30, 40, 50, 60min, respectively, and the residual enzyme activity was measured once at each time point. The measurement method was carried out in accordance with step 4. The enzyme activity without treatment was taken as 100%, and the residual enzyme activity after treatment was calculated, and the result is shown in fig. 8.
The experimental record shows that the temperature is kept for 30min at 60 ℃, the wild type is basically inactivated, and the residual enzyme activity of the S51L mutant is about 85.7 percent.
Through the comparison, the enzyme activity and the heat stability of the S51L mutant are improved compared with those of the wild type XYL.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the patent. It should be noted that, for those skilled in the art, the above embodiments can be modified, combined and improved without departing from the patent idea, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
Claims (10)
1. The beta-xylosidase mutant is characterized in that the mutant is S51L, and the amino acid sequence is shown as a sequence table SEQ ID NO. 3.
2. A gene encoding the β -xylosidase mutant according to claim 1.
3. The gene encoding β -xylosidase according to claim 2, wherein the gene is represented by SEQ ID No.4 of the sequence Listing.
4. A recombinant vector or recombinant strain comprising the encoding gene of claim 2.
5. The recombinant vector or recombinant strain of claim 4, wherein the expression vector is pET-22b, and the host cell is e.coli BL21 (DE 3); the expression vector is pPIC9K, and the host cell is Pichia pastoris GS115; the expression vector is pLY-3, and the host cell is Bacillus subtilis WB600.
6. Use of the recombinant vector or the recombinant strain according to claim 4 for the production of β -xylosidase.
7. Use of the β -xylosidase mutant according to claim 2.
8. Use according to claim 7, for the preparation of xylose by degradation of xylo-oligosaccharides.
9. Use according to claim 7, for the preparation of xylose from alkali-extracted xylan from wheat straw, rice straw, corn stover, corn cobs or sugar cane bagasse.
10. Use according to claim 7, in the food industry, in the pharmaceutical industry, in the paper industry and in the field of energy production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210992314.9A CN115717135A (en) | 2022-08-18 | 2022-08-18 | Heat-resistant xylosidase mutant and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210992314.9A CN115717135A (en) | 2022-08-18 | 2022-08-18 | Heat-resistant xylosidase mutant and preparation thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115717135A true CN115717135A (en) | 2023-02-28 |
Family
ID=85253967
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210992314.9A Pending CN115717135A (en) | 2022-08-18 | 2022-08-18 | Heat-resistant xylosidase mutant and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115717135A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116396953A (en) * | 2022-11-22 | 2023-07-07 | 天典(广东)生物科技有限公司 | Xylanase mutant and application thereof, and recombinant bacillus subtilis |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994014965A1 (en) * | 1992-12-24 | 1994-07-07 | Gist-Brocades N.V. | Cloning and expression of xylanase b |
| WO2005116271A2 (en) * | 2004-05-25 | 2005-12-08 | The Trustees Of Dartmouth College | Selection of microorganisms with growth dependent upon extracytoplasmic enzymes |
| US20080063774A1 (en) * | 2003-11-19 | 2008-03-13 | Wolfgang Aehle | Multiple mutation variants of serine protease |
| US20150225706A1 (en) * | 2005-11-02 | 2015-08-13 | Battelle Energy Alliance, Llc | Alteration and modulation of protein activity by varying post-translational modification |
| KR20160004673A (en) * | 2014-07-03 | 2016-01-13 | 주식회사 씨티씨바이오 | Mutifunctional beta-Xylosidases |
| CN112111472A (en) * | 2020-09-27 | 2020-12-22 | 天津科技大学 | Novel beta-xylosidase and preparation thereof |
-
2022
- 2022-08-18 CN CN202210992314.9A patent/CN115717135A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994014965A1 (en) * | 1992-12-24 | 1994-07-07 | Gist-Brocades N.V. | Cloning and expression of xylanase b |
| US20080063774A1 (en) * | 2003-11-19 | 2008-03-13 | Wolfgang Aehle | Multiple mutation variants of serine protease |
| WO2005116271A2 (en) * | 2004-05-25 | 2005-12-08 | The Trustees Of Dartmouth College | Selection of microorganisms with growth dependent upon extracytoplasmic enzymes |
| US20150225706A1 (en) * | 2005-11-02 | 2015-08-13 | Battelle Energy Alliance, Llc | Alteration and modulation of protein activity by varying post-translational modification |
| KR20160004673A (en) * | 2014-07-03 | 2016-01-13 | 주식회사 씨티씨바이오 | Mutifunctional beta-Xylosidases |
| CN112111472A (en) * | 2020-09-27 | 2020-12-22 | 天津科技大学 | Novel beta-xylosidase and preparation thereof |
Non-Patent Citations (1)
| Title |
|---|
| 薛业敏;于瑾瑾;戴军;陈尚卫;邵蔚蓝;: "耐热β-木糖苷酶的构建及在木糖制备中的应用", 中国食品学报, no. 06, 30 December 2007 (2007-12-30) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116396953A (en) * | 2022-11-22 | 2023-07-07 | 天典(广东)生物科技有限公司 | Xylanase mutant and application thereof, and recombinant bacillus subtilis |
| CN116396953B (en) * | 2022-11-22 | 2023-12-19 | 天典(广东)生物科技有限公司 | Xylanase mutant and application thereof, and recombinant bacillus subtilis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109576244B (en) | Novel lipase, preparation and application thereof | |
| CN106191083B (en) | The xylanase mutant and its encoding gene and application that a kind of specific enzyme activity improves | |
| CN108018275B (en) | Mutant XYNR of extreme heat-resistant xylanase 1VBR and application thereof | |
| CN112111472B (en) | Novel beta-xylosidase and preparation thereof | |
| WO2015007033A1 (en) | Mutant of xylanase xynas9-m with improved thermal stability and gene and use thereof | |
| WO2021143696A1 (en) | Factor regulating protein expression efficiency of trichoderma reesei, and regulation method and use thereof | |
| JP7459509B2 (en) | Mutant strain of Trichoderma fungus and method for producing protein | |
| CN105695383A (en) | Recombinant strain and application thereof | |
| CN115717135A (en) | Heat-resistant xylosidase mutant and preparation thereof | |
| CN103409458B (en) | Ti-plasmids aspergillus niger gene substitution expression vector and application thereof | |
| CN108034667A (en) | A kind of red monascus alpha-amylase gene, its preparation method and application | |
| CN113106112B (en) | Genetically engineered bacterium for heterologously expressing xanthan endonuclease and application thereof | |
| CN108018274B (en) | Mutant XYNH of extreme heat-resistant xylanase 1VBR and application thereof | |
| CN112522125B (en) | Hyaluronidase engineering bacterium and construction method and application thereof | |
| JP7388195B2 (en) | Trichoderma reesei mutant strain and protein production method | |
| CN103757019B (en) | The recombinant expression system of a kind of promotor and expression foreign protein | |
| EP2062967A1 (en) | Genetically engineered aspergillus | |
| CN109336956A (en) | Functional protein POX01907 and its encoding gene and application | |
| CN113755509A (en) | Lysophospholipase variant, construction method thereof and expression in aspergillus niger strain | |
| CN108949784B (en) | Application of sporulation-related gene sigmaF in enzyme production | |
| CN105524849A (en) | Construction and application of cephalosporin high-yield gene engineering strain independent from methionine | |
| CN108251447B (en) | Plasmid capable of efficiently expressing lipase, construction method and application thereof | |
| CN114686409B (en) | Method for enhancing expression of superoxide dismutase gene and improving glutamine transaminase yield | |
| CN116396953B (en) | Xylanase mutant and application thereof, and recombinant bacillus subtilis | |
| CN108707574A (en) | One plant of yielding lipase engineering bacteria, its construction method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |