CN114457059A - 一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用 - Google Patents
一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用 Download PDFInfo
- Publication number
- CN114457059A CN114457059A CN202210069085.3A CN202210069085A CN114457059A CN 114457059 A CN114457059 A CN 114457059A CN 202210069085 A CN202210069085 A CN 202210069085A CN 114457059 A CN114457059 A CN 114457059A
- Authority
- CN
- China
- Prior art keywords
- xylanase
- xylo
- enzyme preparation
- oligosaccharide
- oligosaccharides
- 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.)
- Granted
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 68
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 68
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 title claims abstract description 34
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 8
- 241000228341 Talaromyces Species 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 229940088598 enzyme Drugs 0.000 claims description 62
- 102000057297 Pepsin A Human genes 0.000 claims description 15
- 108090000284 Pepsin A Proteins 0.000 claims description 15
- 229940111202 pepsin Drugs 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 229920001542 oligosaccharide Polymers 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000000967 suction filtration Methods 0.000 claims description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 25
- 229920001221 xylan Polymers 0.000 abstract description 5
- 150000004823 xylans Chemical class 0.000 abstract description 5
- 241000894006 Bacteria Species 0.000 abstract description 4
- 241000235058 Komagataella pastoris Species 0.000 abstract description 4
- 239000000758 substrate Substances 0.000 abstract description 3
- 230000002378 acidificating effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 21
- 239000007788 liquid Substances 0.000 description 8
- 102000004142 Trypsin Human genes 0.000 description 7
- 108090000631 Trypsin Proteins 0.000 description 7
- 239000012588 trypsin Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 238000007865 diluting Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 4
- 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
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 210000004211 gastric acid Anatomy 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 150000002482 oligosaccharides Chemical class 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- FOKISINOENBSDM-WLTAIBSBSA-N Gly-Thr-Tyr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FOKISINOENBSDM-WLTAIBSBSA-N 0.000 description 2
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- TZKPNGDGUVREEB-FOHZUACHSA-N Thr-Asn-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O TZKPNGDGUVREEB-FOHZUACHSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 108010044940 alanylglutamine Proteins 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 108010084389 glycyltryptophan Proteins 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PZUPAGRIHCRVKN-UHFFFAOYSA-N 5-[5-[3,4-dihydroxy-6-[(3,4,5-trihydroxyoxan-2-yl)oxymethyl]-5-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxyoxan-2-yl)oxymethyl]oxan-2-yl]oxyoxan-2-yl]oxy-3,4-dihydroxy-6-[(3,4,5-trihydroxyoxan-2-yl)oxymethyl]oxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound OCC1OC(O)C(O)C(O)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(COC4C(C(O)C(O)CO4)O)O3)O)C(COC3C(C(O)C(O)CO3)O)O2)O)C(COC2C(C(O)C(O)CO2)O)O1 PZUPAGRIHCRVKN-UHFFFAOYSA-N 0.000 description 1
- KQFRUSHJPKXBMB-BHDSKKPTSA-N Ala-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)C)C(O)=O)=CNC2=C1 KQFRUSHJPKXBMB-BHDSKKPTSA-N 0.000 description 1
- YAXNATKKPOWVCP-ZLUOBGJFSA-N Ala-Asn-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O YAXNATKKPOWVCP-ZLUOBGJFSA-N 0.000 description 1
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 description 1
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 1
- AUZAXCPWMDBWEE-HJGDQZAQSA-N Arg-Thr-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O AUZAXCPWMDBWEE-HJGDQZAQSA-N 0.000 description 1
- PSUXEQYPYZLNER-QXEWZRGKSA-N Arg-Val-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PSUXEQYPYZLNER-QXEWZRGKSA-N 0.000 description 1
- VYZBPPBKFCHCIS-WPRPVWTQSA-N Arg-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N VYZBPPBKFCHCIS-WPRPVWTQSA-N 0.000 description 1
- AKEBUSZTMQLNIX-UWJYBYFXSA-N Asn-Ala-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N AKEBUSZTMQLNIX-UWJYBYFXSA-N 0.000 description 1
- KWQPAXYXVMHJJR-AVGNSLFASA-N Asn-Gln-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 KWQPAXYXVMHJJR-AVGNSLFASA-N 0.000 description 1
- OPEPUCYIGFEGSW-WDSKDSINSA-N Asn-Gly-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O OPEPUCYIGFEGSW-WDSKDSINSA-N 0.000 description 1
- JGIAYNNXZKKKOW-KKUMJFAQSA-N Asn-His-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC(=O)N)N JGIAYNNXZKKKOW-KKUMJFAQSA-N 0.000 description 1
- IIQIOFVDFOLCHP-UHFFFAOYSA-N Asn-Pro-Ser-Ser Chemical compound NC(=O)CC(N)C(=O)N1CCCC1C(=O)NC(CO)C(=O)NC(CO)C(O)=O IIQIOFVDFOLCHP-UHFFFAOYSA-N 0.000 description 1
- NJPLPRFQLBZAMH-IHRRRGAJSA-N Asn-Tyr-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCSC)C(O)=O NJPLPRFQLBZAMH-IHRRRGAJSA-N 0.000 description 1
- QQXOYLWJQUPXJU-WHFBIAKZSA-N Asp-Cys-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O QQXOYLWJQUPXJU-WHFBIAKZSA-N 0.000 description 1
- RTXQQDVBACBSCW-CFMVVWHZSA-N Asp-Ile-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RTXQQDVBACBSCW-CFMVVWHZSA-N 0.000 description 1
- USNJAPJZSGTTPX-XVSYOHENSA-N Asp-Phe-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O USNJAPJZSGTTPX-XVSYOHENSA-N 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 1
- 241001608472 Bifidobacterium longum Species 0.000 description 1
- 241000186015 Bifidobacterium longum subsp. infantis Species 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- RGXXLQWXBFNXTG-CIUDSAMLSA-N Gln-Arg-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O RGXXLQWXBFNXTG-CIUDSAMLSA-N 0.000 description 1
- VNTGPISAOMAXRK-CIUDSAMLSA-N Gln-Pro-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O VNTGPISAOMAXRK-CIUDSAMLSA-N 0.000 description 1
- SXFPZRRVWSUYII-KBIXCLLPSA-N Gln-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N SXFPZRRVWSUYII-KBIXCLLPSA-N 0.000 description 1
- ININBLZFFVOQIO-JHEQGTHGSA-N Gln-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O ININBLZFFVOQIO-JHEQGTHGSA-N 0.000 description 1
- HLRLXVPRJJITSK-IFFSRLJSSA-N Gln-Thr-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HLRLXVPRJJITSK-IFFSRLJSSA-N 0.000 description 1
- HNAUFGBKJLTWQE-IFFSRLJSSA-N Gln-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)N)N)O HNAUFGBKJLTWQE-IFFSRLJSSA-N 0.000 description 1
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 1
- XMPAXPSENRSOSV-RYUDHWBXSA-N Glu-Gly-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XMPAXPSENRSOSV-RYUDHWBXSA-N 0.000 description 1
- HVYWQYLBVXMXSV-GUBZILKMSA-N Glu-Leu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HVYWQYLBVXMXSV-GUBZILKMSA-N 0.000 description 1
- UERORLSAFUHDGU-AVGNSLFASA-N Glu-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N UERORLSAFUHDGU-AVGNSLFASA-N 0.000 description 1
- VNCNWQPIQYAMAK-ACZMJKKPSA-N Glu-Ser-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O VNCNWQPIQYAMAK-ACZMJKKPSA-N 0.000 description 1
- GDOZQTNZPCUARW-YFKPBYRVSA-N Gly-Gly-Glu Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O GDOZQTNZPCUARW-YFKPBYRVSA-N 0.000 description 1
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 1
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 1
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 1
- NGBGZCUWFVVJKC-IRXDYDNUSA-N Gly-Tyr-Tyr Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 NGBGZCUWFVVJKC-IRXDYDNUSA-N 0.000 description 1
- JZBVBOKASHNXAD-NAKRPEOUSA-N Ile-Val-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N JZBVBOKASHNXAD-NAKRPEOUSA-N 0.000 description 1
- 241000857964 Ileodictyon cibarium Species 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- PRZVBIAOPFGAQF-SRVKXCTJSA-N Leu-Glu-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O PRZVBIAOPFGAQF-SRVKXCTJSA-N 0.000 description 1
- WQWSMEOYXJTFRU-GUBZILKMSA-N Leu-Glu-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O WQWSMEOYXJTFRU-GUBZILKMSA-N 0.000 description 1
- AMSSKPUHBUQBOQ-SRVKXCTJSA-N Leu-Ser-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N AMSSKPUHBUQBOQ-SRVKXCTJSA-N 0.000 description 1
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000013494 PH determination Methods 0.000 description 1
- NJJBATPLUQHRBM-IHRRRGAJSA-N Phe-Pro-Ser Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CO)C(=O)O NJJBATPLUQHRBM-IHRRRGAJSA-N 0.000 description 1
- BGWKULMLUIUPKY-BQBZGAKWSA-N Pro-Ser-Gly Chemical compound OC(=O)CNC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 BGWKULMLUIUPKY-BQBZGAKWSA-N 0.000 description 1
- BNFVPSRLHHPQKS-WHFBIAKZSA-N Ser-Asp-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O BNFVPSRLHHPQKS-WHFBIAKZSA-N 0.000 description 1
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 1
- MHVXPTAMDHLTHB-IHPCNDPISA-N Ser-Phe-Trp Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 MHVXPTAMDHLTHB-IHPCNDPISA-N 0.000 description 1
- SZRNDHWMVSFPSP-XKBZYTNZSA-N Ser-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N)O SZRNDHWMVSFPSP-XKBZYTNZSA-N 0.000 description 1
- DYEGLQRVMBWQLD-IXOXFDKPSA-N Ser-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CO)N)O DYEGLQRVMBWQLD-IXOXFDKPSA-N 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 241001136494 Talaromyces funiculosus Species 0.000 description 1
- VIBXMCZWVUOZLA-OLHMAJIHSA-N Thr-Asn-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O VIBXMCZWVUOZLA-OLHMAJIHSA-N 0.000 description 1
- JXKMXEBNZCKSDY-JIOCBJNQSA-N Thr-Asp-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O JXKMXEBNZCKSDY-JIOCBJNQSA-N 0.000 description 1
- GXUWHVZYDAHFSV-FLBSBUHZSA-N Thr-Ile-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GXUWHVZYDAHFSV-FLBSBUHZSA-N 0.000 description 1
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 1
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 1
- JNKAYADBODLPMQ-HSHDSVGOSA-N Thr-Trp-Val Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)[C@@H](C)O)=CNC2=C1 JNKAYADBODLPMQ-HSHDSVGOSA-N 0.000 description 1
- CYCGARJWIQWPQM-YJRXYDGGSA-N Thr-Tyr-Ser Chemical compound C[C@@H](O)[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CO)C([O-])=O)CC1=CC=C(O)C=C1 CYCGARJWIQWPQM-YJRXYDGGSA-N 0.000 description 1
- QNTBGBCOEYNAPV-CWRNSKLLSA-N Trp-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)O QNTBGBCOEYNAPV-CWRNSKLLSA-N 0.000 description 1
- HIZDHWHVOLUGOX-BPUTZDHNSA-N Trp-Ser-Val Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O HIZDHWHVOLUGOX-BPUTZDHNSA-N 0.000 description 1
- QAYSODICXVZUIA-WLTAIBSBSA-N Tyr-Gly-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O QAYSODICXVZUIA-WLTAIBSBSA-N 0.000 description 1
- TYFLVOUZHQUBGM-IHRRRGAJSA-N Tyr-Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 TYFLVOUZHQUBGM-IHRRRGAJSA-N 0.000 description 1
- KRXFXDCNKLANCP-CXTHYWKRSA-N Tyr-Tyr-Ile Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 KRXFXDCNKLANCP-CXTHYWKRSA-N 0.000 description 1
- YLBNZCJFSVJDRJ-KJEVXHAQSA-N Val-Thr-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O YLBNZCJFSVJDRJ-KJEVXHAQSA-N 0.000 description 1
- GUIYPEKUEMQBIK-JSGCOSHPSA-N Val-Tyr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(O)=O GUIYPEKUEMQBIK-JSGCOSHPSA-N 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 229940004120 bifidobacterium infantis Drugs 0.000 description 1
- 229940009291 bifidobacterium longum Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- -1 xylo-oligosaccharide tetrasaccharide Chemical class 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用。所述酶制剂包含5000 U/L‑50000 U/L的木聚糖酶XYNTF0，该酶来源于篮状菌，其氨基酸序列如SEQ ID NO：1所示。本发明利用毕赤酵母工程菌高效表达的木聚糖酶，并制备为酶制剂，在酸性条件下具有很高的活性，且对底物专一性高。所述木聚糖酶酶解木聚糖所得产物中，低聚木糖的含量大于70%，产量高，可被广泛应用于低聚木糖的生产。
Description
技术领域
本发明涉及酶工程技术领域，具体涉及一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用。
技术背景
低聚木糖亦称木寡糖（xylooligosaccharides），是由2~10个D-木糖以β-1,4-木糖苷键结合而成的非均一性多糖，是功能性低聚糖家族中的一个重要成员。低聚木糖有明显的双歧杆菌增殖作用，而且除青春双歧杆菌、婴儿双歧杆菌和长双歧杆菌外，大多数肠道菌对低聚木糖的利用率都比较差。与其他的低聚糖相比，低聚木糖不易被消化水解，不利于其广泛利用。
目前，低聚木糖的生产会采用酶法水解木聚糖，人们多采用细菌和霉菌来源的木聚糖酶，但木聚糖酶来源不同，结构和性质也不尽相同，其酶解木聚糖后释放出的寡糖链长也不相同。细菌和霉菌来源的木聚糖酶产物中木二糖～木四糖的含量比较低，而木糖及其他链长度组分含量较高。因此，现在急需开发出性能更好、专一性更强的木聚糖酶，更广泛的应用于低聚木糖的生产。
发明内容
本发明提供了一种含木聚糖酶的酶制剂及其在用于制备低聚木糖中的应用。所述酶制剂中含有篮状菌来源的木聚糖酶XYNTF0，利用其能够生产高含量的低聚木糖。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一种含木聚糖酶的酶制剂，所述酶制剂中包含5000 U/L-50000 U/L的木聚糖酶XYNTF0。
进一步的，所述木聚糖酶XYNTF0的氨基酸序列如SEQ ID NO：1所示，其核苷酸序列如SEQ ID NO：2所示。
进一步的，所述木聚糖酶XYNTF0来源于篮状菌，其最适pH值为3.0，最适反应温度为40℃。
本发明还提供了所述的含木聚糖酶的酶制剂在用于生产低聚木糖中的应用。
进一步的，所述低聚木糖的生产步骤为：将玉米芯清洗、粉碎，60℃浸泡12h，抽滤烘干后经体积比为0.2%的硫酸溶液120℃预处理1h，降温至40℃-50℃，加入所述酶制剂，搅拌均匀后，在40℃-50℃下，维持酶解1h-8h，过滤收集上清液，脱色处理后得到低聚木糖。
进一步的，所述酶制剂中木聚糖酶的添加量为1000 U/L-50000 U/L。
优选的，所述低聚木糖的生产步骤为：将玉米芯清洗、粉碎，60℃浸泡12h，抽滤烘干后经体积比为0.2%的硫酸溶液120℃预处理1h，降温至40℃-50℃，按10000 U/L的添加量加入所述酶制剂，搅拌均匀后，在40℃-50℃下，维持酶解2h-6h，过滤收集上清液，脱色处理后得到低聚木糖。
进一步的，所述低聚木糖为低聚木二糖、低聚木三糖、低聚木四糖、低聚木五糖、低聚木六糖和低聚木七糖。
进一步的，所述低聚木糖的含量大于70%。
本发明还提供了所述的含木聚糖酶的酶制剂在用于制备医药保健品中的应用。
进一步的，所述酶制剂具有酸耐受性和胃蛋白酶耐受性。
与对比文件相比，本发明具有以下优点和有益效果：
本发明所用木聚糖酶XYNTF0是通过人工基因突变和大量筛选的方法，对篮状菌来源的酸性木聚糖酶进行改良，筛选得到的，其在酸性条件下具有较高活性，最适生长pH值为3.0，具有很好的耐酸性，能够很好的匹配玉米芯提取木聚糖过程中的pH值。除此之外，该酶还具有很好的胃蛋白酶耐受性，能够成为一种内源性酶制剂，且该酶对底物的专一性强，利用其制备成酶制剂，通过酶解作用能够生产含量高的低聚木糖。因此，含木聚糖酶XYNTF0的酶制剂具有应用前景。
附图说明
图1为木聚糖酶蛋白电泳图（泳道：1为蛋白质Marker；2为XYNTF0重组菌株发酵液上清）。
图2为木聚糖酶 XYNTF0的发酵曲线。
图3为木聚糖酶 XYNTF0的pH特性分析。
图4为木聚糖酶 XYNTF0的反应温度分析。
图5为木聚糖酶 XYNTF0的酸耐受性分析。
图6为木聚糖酶 XYNTF0经胃酸和胃蛋白酶处理后的残留率。
图7为木聚糖酶 XYNTF0经模拟肠道消化液后残留率。
具体实施方式
结合以下具体实例对本发明的技术方案作进一步详细的说明。下列实施例中未注明具体条件的实验方法，通常可按常规条件，如J.萨姆布鲁克（Sambrook）等编写的《分子克隆实验指南》中所述的条件，或按照制造厂商所建议的条件运行。
本发明前期通过易错PCR方法构建木聚糖酶突变库，筛选得到一种木聚糖酶突变体XYNTF0，其来源于篮状菌（Talaromyces funiculosus），其氨基酸序列如SEQ ID NO：1所示，并根据毕赤酵母密码子偏好性优化其DNA序列如SEQ ID NO：2所示。
实施例1：木聚糖酶的酶学性质测定
将木聚糖酶XYNTF0基因用EcoRⅠ和NotⅠ双酶切位点连接到pPIC9K质粒上，得到的重组表达载体电转入毕赤酵母中，制得酶制剂，具体步骤为：将活化的毕赤酵母GS115接种到含有20mL YPD液体培养基中，30℃摇瓶培养至OD600为1.2～1.5后低温离心收集菌体，依次用20mL冰冷无菌水和5mL浓度为1mol/L的冰冷山梨醇溶液清洗菌体，最后用1mL山梨醇溶液重悬菌体制得酵母感受态悬液。将100μL酵母感受态与10μL线性化载体混匀后转移到预冷的电转杯中电击转化，电转化的条件为1.5kV，6 msec。电击后加入1mL山梨醇溶液，转移至1.5mL离心管中30℃温育1h，5000rpm离心5min，收集菌体涂布于MD筛选平板上倒置培养，直至长出阳性单克隆（图1）。将阳性单克隆接入LB培养基中，30℃振荡培养18h后，加1%甲醇诱导，继续振荡培养，此后每24h添加培养体积1%的甲醇。诱导表达50 h后，每8-12 h取培养液，离心后，取上清检测酶活。
酶活定义：在37℃、pH为5.5的条件下，每分钟从浓度为5.0 mg/mL的木聚糖溶液中降解释放1 μmol还原糖所需要的酶量为一个酶活力单位（U）。
结果如图2所述，木聚糖酶XYNTF0随着发酵时间的延长，酶活不断增加，但在培养91h时，酶活提高最明显。
1、最适反应pH的测定
分别配制不同pH值（pH 3.0 ~ 8.0）的乙酸乙酸钠缓冲液，同时用上述缓冲液分别配制不同pH值底物，将所测酶稀释到合适倍数，按照木聚酶酶活检测方法测定各pH条件下的酶活。以最高酶活所在pH为100%，其它条件下测得的酶活占此酶活的百分数即为该酶在此条件下的酶活存留率。
结果如图3所示，木聚糖酶XYNTF0最适反应pH值为3.0，其为酸性木聚糖酶。
2、最适反应温度的测定
在pH 5.5条件下，分别测定30℃、40℃、50℃、60℃、70℃、80℃温度时发酵上清液的木聚糖酶活力，以最高酶活为100%，计算相对酶活，做温度-相对酶活曲线。
结果如图4所示，木聚糖酶XYNTF0最适反应温度为40℃。
3、pH耐受性的测定
将木聚糖酶用pH 2.0 ~ pH 11.0进行稀释（稀释比例约为最终测定酶活稀释倍数10~20倍），在37 ℃水浴中处理60 min。再次以pH 5.5缓冲液进行稀释，以在4℃保存所测得的酶活作为100%，其他pH值条件下所测得酶活占此酶活的百分比即为该酶在此条件下的酶活存留率。
结果如图5所示，木聚糖酶XYNTF0具有很好的酸耐受性，其在最适pH=3条件下，酶活释放率达到200%。
4、内源酶抗性测定（酶制剂对于内源性酶耐受性试验）
酶液的配制：将酶制剂进行稀释，稀释20倍，并分别用1M HCl溶液调节至pH 2.0及pH 6.8；
胃蛋白酶和胰蛋白酶酶液的配制：称取胃蛋白酶和胰蛋白酶进行溶解，配成5%浓度的胃蛋白酶液和5%胰蛋白酶液，2500 rpm/min离心5分钟。并分别将胃蛋白酶液用盐酸调节pH至2.0，胰蛋白酶液用碱调节pH至6.8；
胃蛋白酶及胰蛋白酶抗性测定步骤：
酶液(pH 2.0)：胃蛋白酶液=4:1，将混合好的胃蛋白酶液环境酶液37℃环境下保温120 min。
酶液(pH 6.8)：胰蛋白酶液=4:1，将混合好的胰蛋白酶液环境在37 ℃环境下保温240 min。
保温结束，酶液置于室温，用缓冲液进行稀释同时根据需要调节到所需的测定pH值，进行酶活的检测。
结果如图6和7所示，木聚糖酶XYNTF0经胃酸处理后残留率为75.27%，经胃蛋白酶处理后，残留率为74.50%，经胃蛋白酶处理后残留率为80.34%，说明其具有良好的胃酸及胃蛋白酶耐受性，且对肠道pH的耐受性都很好，进一步说明木聚糖酶XYNTF0能够成为一种内源性酶制剂。
实施例2：木聚糖酶在生产低聚木糖中的应用
将玉米芯进行清洗、粉碎，60℃浸泡12h，抽滤烘干后经0.2%硫酸溶液120℃预处理1h，降温至40-50℃后，分别按5000 U/L、10000 U/L、20000 U/L、30000 U/L、50000 U/L的添加量加入木聚糖酶XYNTF0，搅拌均匀后，在40-50℃下，维持约7小时，进行充分酶解。酶解结束后，将蒸煮液升温煮沸10min，滤纸过滤取上清液。将得到的上清液进行活性炭脱色处理，按照国标《QBT 2984-2008低聚木糖》所述方法进行HPLC测定。
测定结果如表1所示，由于木聚糖酶生产低聚木糖的要求是酶解产物中低聚木糖含量高，而单糖含量越低越好，因此，10000 U/L为最佳使用添加量，利用木聚糖酶XYNTF0水解玉米芯后得到的产物中，木单糖在酶解2-6h后含量仅为26.03%。
表1：木聚糖酶酶解玉米芯后所得产物HPLC分析。
表中，X2-7：低聚木二糖～低聚木七糖；X2-4：低聚木二糖～低聚木四糖；X7为低聚木七糖；X6为低聚木六糖，以此类推。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
序列表
<110> 青岛尚德生物技术有限公司
<120> 一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用
<141> 2022-01-20
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 206
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Phe Pro Ser Glu Leu Ala Gln Arg Ala Ala Gly Asp Leu Ser Lys Arg
1 5 10 15
Gln Ser Ile Thr Thr Ser Gln Thr Gly Thr Asn Asn Gly Tyr Tyr Tyr
20 25 30
Ser Phe Trp Thr Asn Gly Gly Gly Glu Val Thr Tyr Thr Asn Gly Asp
35 40 45
Asn Gly Glu Tyr Ser Val Thr Trp Val Asp Cys Gly Asp Phe Thr Ser
50 55 60
Gly Lys Gly Trp Asn Pro Ala Asn Ala Gln Thr Val Thr Tyr Ser Gly
65 70 75 80
Glu Phe Asn Pro Ser Gly Asn Ala Tyr Leu Ala Val Tyr Gly Trp Thr
85 90 95
Thr Asp Pro Leu Val Glu Tyr Tyr Ile Leu Glu Ser Tyr Gly Thr Tyr
100 105 110
Asn Pro Ser Ser Gly Leu Thr Ser Leu Gly Gln Val Thr Ser Asp Gly
115 120 125
Gly Thr Tyr Asp Ile Tyr Ser Thr Gln Arg Val Asn Gln Pro Ser Ile
130 135 140
Glu Gly Thr Ser Thr Phe Asn Gln Tyr Trp Ser Val Arg Thr Glu Lys
145 150 155 160
Arg Val Gly Gly Thr Val Thr Thr Ala Asn His Phe Ala Ala Trp Lys
165 170 175
Ala Leu Gly Leu Glu Met Gly Thr Tyr Asn Tyr Met Ile Val Ser Thr
180 185 190
Glu Gly Tyr Glu Ser Ser Gly Ser Ser Thr Ile Thr Val Ser
195 200 205
<210> 2
<211> 621
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ttcccttcag aattggctca aagagctgca ggtgacttgt ctaagaggca atccattacc 60
acttcccaaa caggaacaaa taacggatac tattattcat tctggactaa cggaggtgga 120
gaagttacat acactaacgg agataatggt gaatactctg ttacatgggt tgattgtgga 180
gatttcacct ccggtaaggg atggaaccca gccaatgcac aaaccgtcac ttactccggt 240
gagttcaacc catctggtaa tgcttattta gcagtttacg gttggacaac tgatccatta 300
gtcgaatact atatcttaga atcctacggt acttataacc cttcctcagg tctgacatct 360
ttaggacaag tcacctcaga tggtggtaca tacgatattt actccacaca acgtgtcaac 420
caaccatcca tcgaaggtac ttctaccttt aaccaatatt ggtccgttcg tacagaaaag 480
cgtgtcggag gaactgtcac caccgcaaat catttcgctg cctggaaggc cctgggtctt 540
gaaatgggaa catataatta tatgatagtc tctaccgaag gttatgaatc atccggttcc 600
tcaactatca ctgtctcatg a 621
Claims (10)
1.一种含木聚糖酶的酶制剂，其特征在于，所述酶制剂中包含5000 U/L-50000 U/L的木聚糖酶XYNTF0。
2.根据权利要求1所述的含木聚糖酶的酶制剂，其特征在于，所述木聚糖酶XYNTF0的氨基酸序列如SEQ ID NO：1所示，其核苷酸序列如SEQ ID NO：2所示。
3.根据权利要求1所述的含木聚糖酶的酶制剂，其特征在于，所述木聚糖酶XYNTF0来源于篮状菌，其最适pH值为3.0，最适反应温度为40℃。
4.权利要求1-3任一项所述的含木聚糖酶的酶制剂在用于生产低聚木糖中的应用。
5.根据权利要求4所述的应用，其特征在于，所述低聚木糖的生产步骤为：将玉米芯清洗、粉碎，60℃浸泡12h，抽滤烘干后经体积比为0.2%的硫酸溶液120℃预处理1h，降温至40℃-50℃，加入所述酶制剂，搅拌均匀后，在40℃-50℃下，维持酶解1h-8h，过滤收集上清液，脱色处理后得到低聚木糖。
6.根据权利要求5所述的应用，其特征在于，所述酶制剂中木聚糖酶的添加量为1000U/L -50000 U/L。
7.根据权利要求3所述的应用，其特征在于，所述低聚木糖为低聚木二糖、低聚木三糖、低聚木四糖、低聚木五糖、低聚木六糖和低聚木七糖。
8.根据权利要求7所述的应用，其特征在于，所述低聚木糖的含量大于70%。
9.权利要求1-3任一项所述的含木聚糖酶的酶制剂在用于制备医药保健品中的应用。
10.根据权利要求9所述的应用，其特征在于，所述酶制剂具有酸耐受性和胃蛋白酶耐受性。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210069085.3A CN114457059B (zh) | 2022-01-21 | 2022-01-21 | 一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210069085.3A CN114457059B (zh) | 2022-01-21 | 2022-01-21 | 一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114457059A true CN114457059A (zh) | 2022-05-10 |
| CN114457059B CN114457059B (zh) | 2024-03-19 |
Family
ID=81408882
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210069085.3A Active CN114457059B (zh) | 2022-01-21 | 2022-01-21 | 一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114457059B (zh) |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992013942A1 (fr) * | 1991-02-01 | 1992-08-20 | Institut National De La Recherche Agronomique (Inra) | Xylanase, souches de bacillus productrices de xylanase et leurs utilisations |
| US6534101B1 (en) * | 1998-05-06 | 2003-03-18 | Aveatis Animal Nutrition S.A. | Enzymes mixture obtained from Penicillium funiculosum |
| CN1403591A (zh) * | 2001-09-05 | 2003-03-19 | 中国农业大学 | 低聚木糖的制备方法 |
| KR20050097878A (ko) * | 2004-04-05 | 2005-10-10 | 산동 롱라이브 바이오-테크놀로지 컴퍼니 리미티드 | 자일로올리고당의 제조 방법 |
| CN104293747A (zh) * | 2008-12-23 | 2015-01-21 | 杜邦营养生物科学有限公司 | 具有木聚糖酶活性的多肽 |
| CN104560920A (zh) * | 2015-01-26 | 2015-04-29 | 青岛蔚蓝生物集团有限公司 | 一种酸性木聚糖酶突变体及其应用 |
| CN104593446A (zh) * | 2014-12-25 | 2015-05-06 | 山东龙力生物科技股份有限公司 | 一种纯低聚木糖粉的制备方法 |
| CN106381273A (zh) * | 2015-07-29 | 2017-02-08 | 青岛蔚蓝生物集团有限公司 | 一株高产木聚糖酶的黑曲霉菌株 |
| CN109997970A (zh) * | 2019-03-07 | 2019-07-12 | 青岛红樱桃生物技术有限公司 | 一类酶活和耐热性提高的酸性木聚糖酶突变体及其编码基因和应用 |
| CN110607291A (zh) * | 2018-06-14 | 2019-12-24 | 青岛蔚蓝生物集团有限公司 | 耐热木聚糖酶突变体 |
| CN110724645A (zh) * | 2018-07-16 | 2020-01-24 | 青岛蔚蓝生物集团有限公司 | 一种毕赤酵母突变菌株及其应用 |
| CN111100853A (zh) * | 2018-10-25 | 2020-05-05 | 中国农业大学 | 木聚糖酶xyn11A及其编码基因与应用 |
| CN111492053A (zh) * | 2017-12-20 | 2020-08-04 | 帝斯曼知识产权资产管理有限公司 | 动物饲料组合物及其用途 |
-
2022
- 2022-01-21 CN CN202210069085.3A patent/CN114457059B/zh active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992013942A1 (fr) * | 1991-02-01 | 1992-08-20 | Institut National De La Recherche Agronomique (Inra) | Xylanase, souches de bacillus productrices de xylanase et leurs utilisations |
| US6534101B1 (en) * | 1998-05-06 | 2003-03-18 | Aveatis Animal Nutrition S.A. | Enzymes mixture obtained from Penicillium funiculosum |
| CN1403591A (zh) * | 2001-09-05 | 2003-03-19 | 中国农业大学 | 低聚木糖的制备方法 |
| KR20050097878A (ko) * | 2004-04-05 | 2005-10-10 | 산동 롱라이브 바이오-테크놀로지 컴퍼니 리미티드 | 자일로올리고당의 제조 방법 |
| CN104293747A (zh) * | 2008-12-23 | 2015-01-21 | 杜邦营养生物科学有限公司 | 具有木聚糖酶活性的多肽 |
| CN104593446A (zh) * | 2014-12-25 | 2015-05-06 | 山东龙力生物科技股份有限公司 | 一种纯低聚木糖粉的制备方法 |
| CN104560920A (zh) * | 2015-01-26 | 2015-04-29 | 青岛蔚蓝生物集团有限公司 | 一种酸性木聚糖酶突变体及其应用 |
| CN106381273A (zh) * | 2015-07-29 | 2017-02-08 | 青岛蔚蓝生物集团有限公司 | 一株高产木聚糖酶的黑曲霉菌株 |
| CN111492053A (zh) * | 2017-12-20 | 2020-08-04 | 帝斯曼知识产权资产管理有限公司 | 动物饲料组合物及其用途 |
| CN110607291A (zh) * | 2018-06-14 | 2019-12-24 | 青岛蔚蓝生物集团有限公司 | 耐热木聚糖酶突变体 |
| CN110724645A (zh) * | 2018-07-16 | 2020-01-24 | 青岛蔚蓝生物集团有限公司 | 一种毕赤酵母突变菌株及其应用 |
| CN111100853A (zh) * | 2018-10-25 | 2020-05-05 | 中国农业大学 | 木聚糖酶xyn11A及其编码基因与应用 |
| CN109997970A (zh) * | 2019-03-07 | 2019-07-12 | 青岛红樱桃生物技术有限公司 | 一类酶活和耐热性提高的酸性木聚糖酶突变体及其编码基因和应用 |
Non-Patent Citations (7)
| Title |
|---|
| FURNI SS, C. S. 等: "RecName: Full=Endo-1 , 4-beta-xylanase C; Short=Xylanase C; AltName: Full=1 , 4-beta-D-xylan xylanohydrolase C; Flags: Precursor", GENEBANK DATABASE, pages 9 * |
| JEAN-GUY BERRIN等: "Substrate and product hydrolysis specifi city in family 11 glycoside hydrolases: an analysis of Penicillium funiculosum and Penicillium griseofulvum xylanases", APPL MICROBIOL BIOTECHNOL, vol. 74, pages 1001 - 54 * |
| ZHIZHUANG XIAO 等: "Cloning and characterization of the f irst GH10 and GH11 xylanases from Rhizopus oryzae", APPL MICROBIOL BIOTECHNOL, vol. 98, pages 8211 * |
| 周文美;王照波;徐子婷;胡晓瑜;: "球毛壳菌ACCC30566木聚糖酶生产低聚木糖的研究", 贵州农业科学, no. 12, 15 December 2009 (2009-12-15), pages 126 - 129 * |
| 肖凯军;李娜;: "不同原料酶法制备低聚木糖的研究及成分分析", 中国食品添加剂, no. 04, 15 August 2010 (2010-08-15), pages 126 - 129 * |
| 邵 静等: "饲用木聚糖酶在人工消化液中的 稳定性评价", 饲料研究, no. 12, pages 47 - 49 * |
| 黄家骥, 许正宏, 陶文沂: "以Bacillus pumlis.木聚糖酶水解玉米芯制备木寡糖", 食品与发酵工业, no. 05, 30 May 2004 (2004-05-30), pages 5 - 9 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114457059B (zh) | 2024-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8551751B2 (en) | BX11 enzymes having xylosidase activity | |
| Cannio et al. | A xylan-degrading strain of Sulfolobus solfataricus: isolation and characterization of the xylanase activity | |
| Yan et al. | Properties of a xylanase from Streptomyces matensis being suitable for xylooligosaccharides production | |
| WO2017197546A1 (zh) | 一种β-甘露聚糖酶mRmMan5A及其编码基因与应用 | |
| CN111534503B (zh) | Haloferula sp.β-N-乙酰氨基己糖苷酶在合成人乳寡糖中的应用 | |
| CN109679864B (zh) | 一种产转糖基活性β-半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法 | |
| CN109988714B (zh) | 一种里氏木霉及其应用 | |
| CN105018448B (zh) | 一种真菌来源的耐热酸性纤维素酶及其基因和应用 | |
| CN104975039B (zh) | 一种重组质粒及其在降解纤维素原料中的应用 | |
| CN107164398A (zh) | 一种重组α‑半乳糖苷酶基因、载体、工程菌及其应用 | |
| WO2015021930A1 (zh) | 一种内切木聚糖酶及其编码基因和应用 | |
| CN107586767B (zh) | 一种耐热内切型木聚糖酶EpXYN1及其编码基因和应用 | |
| DK164070B (da) | Fremgangsmaade til fremstilling af et sammensat cellulase/xylanase-enzympraeparat | |
| CN109679938B (zh) | 一种几丁质酶Chit46及其表达纯化方法与应用 | |
| WO2021218171A1 (zh) | 一种β-甘露聚糖酶的制备方法及在制备部分水解甘露聚糖中的应用 | |
| CN110117586B (zh) | 一种超耐热的木聚糖酶XynGold及基因与应用 | |
| CN103614303B (zh) | 一种表达糖化酶的里氏木霉菌株 | |
| CN105154417B (zh) | 一种真菌来源的酸性纤维素酶及其基因和应用 | |
| CN114457059A (zh) | 一种含木聚糖酶的酶制剂及其在用于生产低聚木糖中的应用 | |
| CN101481696B (zh) | 一种适冷内切β-木聚糖酶的基因XynA及应用 | |
| CN113151055B (zh) | 产果胶酶芽孢杆菌及其发酵方法、以及胡椒脱皮的方法 | |
| CN107236692B (zh) | 白蚁溶纤维类芽孢杆菌NP1、木聚糖酶PtXyn1及其编码基因和应用 | |
| CN115216460B (zh) | 一种米黑根毛霉糖苷水解酶12家族木葡聚糖酶的制备方法及应用 | |
| JP6319907B2 (ja) | 耐熱性β―キシロシダーゼ | |
| CN102732496B (zh) | 一种木聚糖酶及其应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |