CN115992104A - 来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 - Google Patents
来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 Download PDFInfo
- Publication number
- CN115992104A CN115992104A CN202210868807.1A CN202210868807A CN115992104A CN 115992104 A CN115992104 A CN 115992104A CN 202210868807 A CN202210868807 A CN 202210868807A CN 115992104 A CN115992104 A CN 115992104A
- Authority
- CN
- China
- Prior art keywords
- bslpmo10a
- activity
- mlg
- polysaccharide
- lpmo
- 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
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 39
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 39
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 16
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 16
- 102000008109 Mixed Function Oxygenases Human genes 0.000 title abstract description 5
- 108010074633 Mixed Function Oxygenases Proteins 0.000 title abstract description 5
- 150000004676 glycans Chemical class 0.000 title description 2
- 239000000758 substrate Substances 0.000 claims abstract description 43
- 150000004804 polysaccharides Chemical class 0.000 claims abstract description 39
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims abstract description 21
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims abstract description 21
- 229920001221 xylan Polymers 0.000 claims abstract description 19
- 150000004823 xylans Chemical class 0.000 claims abstract description 19
- 230000015556 catabolic process Effects 0.000 claims abstract description 13
- 238000006731 degradation reaction Methods 0.000 claims abstract description 11
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 101710154526 Lytic chitin monooxygenase Proteins 0.000 claims description 48
- 229920002488 Hemicellulose Polymers 0.000 claims description 21
- 229920000161 Locust bean gum Polymers 0.000 claims description 18
- 239000000711 locust bean gum Substances 0.000 claims description 18
- 235000010420 locust bean gum Nutrition 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 240000000731 Fagus sylvatica Species 0.000 claims description 7
- 235000010099 Fagus sylvatica Nutrition 0.000 claims description 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 7
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 claims description 6
- 229920002498 Beta-glucan Polymers 0.000 claims description 6
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 6
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 claims description 5
- 229920002581 Glucomannan Polymers 0.000 claims description 5
- 229920002752 Konjac Polymers 0.000 claims description 5
- 229920002097 Lichenin Polymers 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- 229940046240 glucomannan Drugs 0.000 claims description 5
- 235000010485 konjac Nutrition 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- 244000247812 Amorphophallus rivieri Species 0.000 claims description 4
- 235000001206 Amorphophallus rivieri Nutrition 0.000 claims description 4
- 102000004317 Lyases Human genes 0.000 claims description 4
- 108090000856 Lyases Proteins 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 239000000252 konjac Substances 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 2
- 229960003237 betaine Drugs 0.000 claims description 2
- 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 40
- 102000004190 Enzymes Human genes 0.000 abstract description 34
- 108090000790 Enzymes Proteins 0.000 abstract description 34
- 229920002101 Chitin Polymers 0.000 abstract description 19
- 229920002678 cellulose Polymers 0.000 abstract description 17
- 239000001913 cellulose Substances 0.000 abstract description 17
- 230000002195 synergetic effect Effects 0.000 abstract description 10
- 229920001503 Glucan Polymers 0.000 abstract description 5
- 102000016680 Dioxygenases Human genes 0.000 abstract 1
- 108010028143 Dioxygenases Proteins 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 33
- 230000003197 catalytic effect Effects 0.000 description 19
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 16
- 235000010980 cellulose Nutrition 0.000 description 16
- 238000007254 oxidation reaction Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 239000010902 straw Substances 0.000 description 14
- 230000003647 oxidation Effects 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 229920001542 oligosaccharide Polymers 0.000 description 12
- 150000002482 oligosaccharides Chemical class 0.000 description 12
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 11
- 238000000354 decomposition reaction Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 235000000346 sugar Nutrition 0.000 description 11
- 240000007594 Oryza sativa Species 0.000 description 10
- 235000007164 Oryza sativa Nutrition 0.000 description 10
- 235000009566 rice Nutrition 0.000 description 10
- 229920002230 Pectic acid Polymers 0.000 description 9
- 239000010318 polygalacturonic acid Substances 0.000 description 9
- 150000008163 sugars Chemical class 0.000 description 9
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 8
- 229960005070 ascorbic acid Drugs 0.000 description 8
- 235000010323 ascorbic acid Nutrition 0.000 description 8
- 239000011668 ascorbic acid Substances 0.000 description 8
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 8
- 239000008108 microcrystalline cellulose Substances 0.000 description 8
- 229940016286 microcrystalline cellulose Drugs 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000001814 pectin Substances 0.000 description 7
- 235000010987 pectin Nutrition 0.000 description 7
- 102100032487 Beta-mannosidase Human genes 0.000 description 6
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 108010055059 beta-Mannosidase Proteins 0.000 description 6
- 239000010949 copper Substances 0.000 description 6
- 229930014626 natural product Natural products 0.000 description 6
- 210000004767 rumen Anatomy 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 5
- 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
- 244000068988 Glycine max Species 0.000 description 5
- 235000010469 Glycine max Nutrition 0.000 description 5
- 229920000057 Mannan Polymers 0.000 description 5
- 241000209140 Triticum Species 0.000 description 5
- 235000021307 Triticum Nutrition 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 229910052802 copper Inorganic materials 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 229920005610 lignin Polymers 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 229920003175 pectinic acid Polymers 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- DBTMGCOVALSLOR-DEVYUCJPSA-N (2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](CO)O[C@H](O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-DEVYUCJPSA-N 0.000 description 4
- 235000017060 Arachis glabrata Nutrition 0.000 description 4
- 244000105624 Arachis hypogaea Species 0.000 description 4
- 235000010777 Arachis hypogaea Nutrition 0.000 description 4
- 235000018262 Arachis monticola Nutrition 0.000 description 4
- 240000005979 Hordeum vulgare Species 0.000 description 4
- 235000007340 Hordeum vulgare Nutrition 0.000 description 4
- 229920001543 Laminarin Polymers 0.000 description 4
- 239000005717 Laminarin Substances 0.000 description 4
- 238000012300 Sequence Analysis Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 4
- 230000001590 oxidative effect Effects 0.000 description 4
- 235000020232 peanut Nutrition 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 235000016068 Berberis vulgaris Nutrition 0.000 description 3
- 241000335053 Beta vulgaris Species 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 3
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- 241000511730 Leymus chinensis Species 0.000 description 3
- 238000000692 Student's t-test Methods 0.000 description 3
- LUEWUZLMQUOBSB-ZLBHSGTGSA-N alpha-maltotetraose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-ZLBHSGTGSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- FYGDTMLNYKFZSV-ZWSAEMDYSA-N cellotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-ZWSAEMDYSA-N 0.000 description 3
- 239000007857 degradation product Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 235000021472 generally recognized as safe Nutrition 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- LUEWUZLMQUOBSB-GFVSVBBRSA-N mannan Chemical class O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-GFVSVBBRSA-N 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000007974 sodium acetate buffer Substances 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012353 t test Methods 0.000 description 3
- 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 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 2
- 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 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 240000006439 Aspergillus oryzae Species 0.000 description 2
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 235000018185 Betula X alpestris Nutrition 0.000 description 2
- 235000018212 Betula X uliginosa Nutrition 0.000 description 2
- 241001436672 Bhatia Species 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- 101100068867 Caenorhabditis elegans glc-1 gene Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 108010022172 Chitinases Proteins 0.000 description 2
- 102000012286 Chitinases Human genes 0.000 description 2
- 241000207199 Citrus Species 0.000 description 2
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 108010001682 Dextranase Proteins 0.000 description 2
- 102000002090 Fibronectin type III Human genes 0.000 description 2
- 108050009401 Fibronectin type III Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 229920001706 Glucuronoxylan Polymers 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000203780 Thermobifida fusca Species 0.000 description 2
- JVZHSOSUTPAVII-UHFFFAOYSA-N Xylotetraose Natural products OCC(OC1OCC(OC2OCC(OC3OCC(O)C(O)C3O)C(O)C2O)C(O)C1O)C(O)C(O)C=O JVZHSOSUTPAVII-UHFFFAOYSA-N 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
- 230000009286 beneficial effect Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 235000020971 citrus fruits Nutrition 0.000 description 2
- 229910001431 copper ion Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 235000014304 histidine Nutrition 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 229940027941 immunoglobulin g Drugs 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 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 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- PGNXLDQQCINNPZ-BURFUSLBSA-N n-methyl-n-[(2s,3r,4r,5r)-2,3,4,5,6-pentahydroxyhexyl]undecanamide Chemical compound CCCCCCCCCCC(=O)N(C)C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO PGNXLDQQCINNPZ-BURFUSLBSA-N 0.000 description 2
- 239000006225 natural substrate Substances 0.000 description 2
- OIPPWFOQEKKFEE-UHFFFAOYSA-N orcinol Chemical compound CC1=CC(O)=CC(O)=C1 OIPPWFOQEKKFEE-UHFFFAOYSA-N 0.000 description 2
- 229920001277 pectin Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000000698 schizophrenic effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 101150034227 xyl1 gene Proteins 0.000 description 2
- KPTPSLHFVHXOBZ-BIKCPUHGSA-N xylotetraose 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@H](O[C@H]3[C@@H]([C@@H](O)C(O)OC3)O)OC2)O)OC1 KPTPSLHFVHXOBZ-BIKCPUHGSA-N 0.000 description 2
- DJMVHSOAUQHPSN-VXDFXQCISA-N (2r,3r,4r,5r)-4-[(2s,3r,4r,5s,6r)-5-[(2s,3r,4r,5s,6r)-5-[(2s,3r,4r,5s,6r)-5-[(2s,3r,4r,5s,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,4 Chemical compound O[C@@H]1[C@@H](O)[C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](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@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)[C@@H](CO)O3)O)[C@@H](CO)O2)O)[C@@H](CO)O1 DJMVHSOAUQHPSN-VXDFXQCISA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- 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 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
- 241000203069 Archaea Species 0.000 description 1
- 241000235349 Ascomycota Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000193388 Bacillus thuringiensis Species 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 101100338269 Caenorhabditis elegans his-41 gene Proteins 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000949473 Correa Species 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 101100373011 Drosophila melanogaster wapl gene Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102100037362 Fibronectin Human genes 0.000 description 1
- 201000005569 Gout Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 101710085938 Matrix protein Proteins 0.000 description 1
- 238000007476 Maximum Likelihood Methods 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920001046 Nanocellulose Polymers 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000611789 Paenibacillus curdlanolyticus Species 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 241000218657 Picea Species 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000187392 Streptomyces griseus Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- FTTUBRHJNAGMKL-UHFFFAOYSA-N Xylohexaose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(OC4C(C(O)C(OC5C(C(O)C(O)OC5)O)OC4)O)OC3)O)OC2)O)OC1 FTTUBRHJNAGMKL-UHFFFAOYSA-N 0.000 description 1
- LFFQNKFIEIYIKL-UHFFFAOYSA-N Xylopentaose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(OC4C(C(O)C(O)OC4)O)OC3)O)OC2)O)OC1 LFFQNKFIEIYIKL-UHFFFAOYSA-N 0.000 description 1
- 241000889608 [Kitasatospora] papulosa Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- PYMYPHUHKUWMLA-VPENINKCSA-N aldehydo-D-xylose Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-VPENINKCSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 229920000617 arabinoxylan Polymers 0.000 description 1
- 238000004630 atomic force microscopy Methods 0.000 description 1
- 229940097012 bacillus thuringiensis Drugs 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- SMDHCQAYESWHAE-UHFFFAOYSA-N benfluralin Chemical compound CCCCN(CC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O SMDHCQAYESWHAE-UHFFFAOYSA-N 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
- 125000000188 beta-D-glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 206010061592 cardiac fibrillation Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012668 chain scission Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 108091011157 chitin binding proteins Proteins 0.000 description 1
- 102000021178 chitin binding proteins Human genes 0.000 description 1
- 239000009194 citrus pectin Substances 0.000 description 1
- 229940040387 citrus pectin Drugs 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 230000002600 fibrillogenic effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 230000008642 heat stress Effects 0.000 description 1
- 150000002411 histidines Chemical group 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229930027917 kanamycin Natural products 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
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000002029 lignocellulosic biomass Substances 0.000 description 1
- 208000012396 long COVID-19 Diseases 0.000 description 1
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000002121 nanofiber Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- 238000007248 oxidative elimination reaction Methods 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 210000004483 pasc Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 235000021251 pulses Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108010038196 saccharide-binding proteins Proteins 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
-
- 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
- C12N9/0004—Oxidoreductases (1.)
-
- 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
- C12P19/02—Monosaccharides
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用,由455个氨基酸残基构成,其氨基酸序列如SEQ ID No.1所示。重组BsLPMO10A在50℃和pH5.0时具有最佳活性。底物特异性研究表明,该酶表现出广泛的活性底物谱。与此同时BsLPMO10A在本研究中首次显示出对阿拉伯聚糖的活性。它与一系列糖苷水解酶的协同作用表明BsLPMO10A显着加速了葡聚糖、木聚糖、纤维素和几丁质的糖化。此外,通过六种天然饲料的降解进一步验证了BsLPMO10A和GHs的协同作用。
Description
技术领域
本发明属于酶学领域,具体涉及一种来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用。
背景技术
植物木质纤维素是自然界中最丰富的生物质来源,年产量约为1011-1012吨(Centore,Hochman,&Zilberman,2014)。其中,非淀粉多糖,如纤维素和半纤维素以及木质素是主要成分,赋予植物刚性和抗昆虫和真菌侵袭的能力。在有效利用之前,这些生物聚合物需要完全分解成单体和低聚物。不幸的是,木质纤维素中高度复杂和顽固的结构成为生物降解的障碍。糖苷水解酶(GHs),包括纤维素酶、木聚糖酶和葡聚糖酶,已广泛用于工业,特别是用于食品/饲料添加剂的开发。然而,仅由于天然多糖结构复杂,尤其是晶体结构中链之间形成的氢键,因此,仅仅使用GHs难以彻底分解天然多糖。
最近发现,裂解性多糖单加氧酶(LPMOs,EC 1.14.99.53-56)作为一种新型氧化酶,可催化顽固多糖的氧化裂解。LPMO是铜离子依赖性酶家族,可提高传统GHs的生物质解聚效率,并通过氧化和裂解多糖底物促进天然碳循环。根据碳水化合物活性酶数据库(CAZy,http://www.cazy.org/),LPMO被归类为辅助活性家族(AAs)(Levasseur,Drula,Lombard,Coutinho,&Henrissat,2013)。迄今为止,在细菌、真菌、病毒、昆虫和古生菌中发现了大量作用于纤维素、几丁质、淀粉和其他多糖底物的LPMO(Lombard,GolacondaRamulu,Drula,Coutinho,&Henrissat,2014)。根据其序列和底物特异性分为8个家族,AA9-11和AA13-17。大多数LPMO为β-折叠拓扑结构,其中某些LPMO为β-折叠通过可变长度的环状结构连接(Hemsworth,Henrissat,Davies,&Walton,2014;Harris et al.,2010;Leggio etal.,2015)。鉴于其平坦的底物结合表面,这些酶适用于结晶多糖的结合和裂解,为GHs的后续水解提供更多的进攻位点(Hemsworth,Johnston,Davies,&Walton,2015;Johansen,2016;Zhang et al.,2019)。
在过去十年中,人们在挖掘和表征纤维素和几丁质活性LPMO方面做出了巨大努力(Forsberg et al.,2014;Nakagawa et al.,2015;Paspaliari,Loose,Larsen,&Vaaje-Kolstad,2015)。相比之下,作用于植物木质纤维素两种主要半纤维素成分催化木聚糖(Correa et al.,2019;Couturier et al.,2018;et al.,2019;Jung,Song,Kim,&Bae,2015;et al.,2022;Zerva,Pentari,Grisel,Berrin,&Topakas,2020)和甘露聚糖(Li et al.,2021a;Limsakul et al.,2020)降解的LPMO较少。
大多数芽孢杆菌属物种是非致病性的,并且一直是异源蛋白质表达的有吸引力的宿主,这主要是因为它们能够分泌功能性酶。在所有芽孢杆菌中,枯草芽孢杆菌被美国食品和药物管理局(FDA)普遍认为是安全的(GRAS)。作为益生菌,枯草芽孢杆菌在人类和动物的胃肠道健康中发挥着重要作用。例如,枯草芽孢杆菌改善了家禽的生长性能、免疫反应、热应激耐受性和抗病性(Wang et al.,2022)。同时,该微生物已被证实可以改善瘤胃发育和新生荷斯坦犊牛的健康状况(Wang et al.,2022)。因此,从枯草芽孢杆菌中挖掘新型LPMO将具有重要意义。
发明内容
本发明所要解决的技术问题为:如何从枯草芽孢杆菌中挖掘新型LPMO。
本发明的技术方案为:一种来自枯草芽孢杆菌的裂解性多糖单加氧酶BsLPMO10A,由455个氨基酸残基构成,其氨基酸序列如SEQ ID No.1所示。
编码上述裂解性多糖单加氧酶BsLPMO10A的基因,由于密码子的简并性,存在多种不同的编码序列。
进一步地,所述基因的核苷酸序列如SEQ ID No.2所示。
一种表达载体,含有上述所述的基因。
一种重组菌,含有上述所述的表达载体。
本发明的裂解性多糖单加氧酶BsLPMO10A在促进半纤维素底物降解上的应用。
进一步地,所述半纤维素底物为地衣多糖、β-葡聚糖、刺槐豆胶、魔芋葡甘露聚糖、山毛榉木木聚糖和甜菜碱阿拉伯糖。
本发明的裂解性多糖单加氧酶BsLPMO10A在促进多糖底物β-1,4糖苷键断裂上的应用。
本发明的裂解性多糖单加氧酶BsLPMO10A在与糖苷水解酶协同促进多糖的分解上的应用。
缩略语和关键术语定义
AA 辅助活性家族
BX 山毛榉木聚糖
CAZy 水化合物活性酶数据库
DNS 3,5-二硝基水杨酸
FDA 美国食品和药物管理局
FNⅢ 纤连蛋白Ⅲ型
GHs 糖苷水解酶
GRAS 普遍认为安全
HPAEC-PAD 脉冲安培检测器离子色谱
HRP 辣根过氧化物酶(HRP
IPTG 异丙基硫代-β-D-吡喃半乳糖苷
KGM 魔芋葡甘露聚糖
LBG 刺槐豆胶
MALDI-TOF-MS 基质辅助激光解析电离飞行时间质谱
MCC 微晶纤维素
MLG-B 大麦β-葡聚糖
MLG-L 冰岛苔藓地衣多糖
PASC 磷酸溶胀纤维素
PGA 柑橘聚半乳糖醛酸
SBA 甜菜阿拉伯聚糖
与现有技术相比,本发明具有以下有益效果:
本发明的源自枯草芽孢杆菌(BsLPMO10A)的新型AA10LPMO,对它进行异源表达和功能特征。重组BsLPMO10A在50℃和pH5.0时具有最佳活性。底物特异性研究表明,该酶表现出广泛的活性底物谱,包括MLG-L、MLG-B、LBG、KGM、BX,特别是对于通过β-1,4糖苷键连接的多糖,包括β-(Glc1→4Glc)、β-(Xyl1→4Xyl)和β-(Man1→4Man)。与此同时BsLPMO10A在本研究中首次显示出对阿拉伯聚糖的活性。HPAEC-PAD和MALDI-TOF-MS分析表明,BsLPMO10A主要从混合连接葡聚糖和山毛榉木聚糖中释放聚合度(DP)为3-5的天然寡糖和从DP3ox到DP6ox的C1氧化寡糖。它与一系列糖苷水解酶的协同作用表明BsLPMO10A显着加速了葡聚糖、木聚糖、纤维素和几丁质的糖化。此外,通过六种天然饲料的降解进一步验证了BsLPMO10A和GHs的协同作用。
附图说明
图1.BsLPMO10A和数据库中其他表征的AA10 LPMO的系统发育树。使用MEGA11对功能表征的LPMO进行多序列分析,然后使用构建/测试最大似然法进行系统发育分析。BsLPMO10A(GenBank编号:QDC27792.1)。
图2.SDS-PAGE和酶谱分析。(A)全长BsLPMO10A和(B)BsLPMO10A(CD);泳道M,蛋白Marker。(C)酶谱分析:Ⅰ、MLG-L;Ⅱ、MLG-B;Ⅲ、LBG;Ⅳ、KGM;Ⅴ、BX;Ⅵ、PASC;Ⅶ、胶体几丁质。
图3.BsLPMO10A的生化特征。(A)最适温度和(B)最适pH值。抗坏血酸(C)和H2O2(D)对BsLPMO10A活性的影响;CK,对照组。数据代表平均值±SD(n=4)。使用t检验对CK和实验组之间进行统计分析。**,P<0.05;***,P<0.001。
图4.BsLPMO10A对葡聚糖的降解。(A)BsLPMO10A从MLG-L和MLG-释放的寡糖的HPAEC-PAD色谱图;(B-C)BsLPMO10A对MLG-L(B)和MLG-B(C)释放的寡糖进行MALDI-TOF-MS分析;(D)BsLPMO10A和葡聚糖酶(IDSGLUC5-38)对MLG-L和MLG-B降解产物的HPAEC-PAD色谱图。插图显示了BsLPMO10A和葡聚糖酶(IDSGLUC5-38)从MLG-L和MLG-B得到的天然和C1氧化产物的总和。G1,葡萄糖;G2,纤维二糖;G3,纤维三糖;G4,纤维四糖;G5,纤维五糖;G6,纤维六糖;G1ox,C1-氧化葡萄糖;G2ox。C1-氧化纤维二糖。
图5.BsLPMO10A对BX的降解。(A)BsLPMO10A从BX释放的寡糖的HPAEC-PAD色谱图;(B)BsLPMO10A和木聚糖酶(IDSXYN11-1)对BX降解产物的HPAEC-PAD色谱图。插图显示了由BsLPMO10A和木聚糖酶(IDSXYN11-1)从BX产生的天然产物和C1氧化产物的总和。X1,木糖;X2,木二糖;X3、木三糖;X4,木四糖;X5,木戊糖;X6,木六糖;X1ox,C1-氧化木糖。
图6.BsLPMO10A对其他多糖降解产物的HPAEC-PAD色谱图。(A)KGM和LBG;(B)SBA;(C)果胶和PGA;(D)PASC;(E)几丁质。
图7BsLPMO10A和GHs对多糖降解的协同作用。(A)BsLPMO10A和葡聚糖酶(IDSGLUC5-38)从MLG-L释放的还原糖;(B)BsLPMO10A和木聚糖酶(TfXYN11-1)从BX释放的还原糖;(C)BsLPMO10A和甘露聚糖酶(BsMAN26-3)从LBG释放的还原糖;(D)BsLPMO10A和纤维素酶(IDSGLUC5-11)从PASC释放的还原糖;(E)BsLPMO10A和几丁质酶(BtCHI18-1)从几丁质释放的还原糖。数据代表平均值±SD(n=4)。使用t检验对协同作用和GH单独作用组之间的进行统计分析。*,P<0.05;**,P<0.05;***,P<0.001。
图8.BsLPMO10A和GHs对天然木质纤维素原料降解的协同作用。(A)花生壳;(B)大豆秸秆;(C)羊草秸秆;(D)水稻秸秆;(E)水稻壳;(F)小麦秸秆。数据代表平均值±SD(n=4)。使用Tukey-Kramer检验进行多重比较。大小写字母分别表示纤维素或半纤维素含量差异(P<0.05)。
图9BsLPMO10A的氨基酸比对和结构预测。(A)BsLPMO10A和其他AA10LPMO的氨基酸比对;(B-C)WebLogo分析“组氨酸支架”中涉及的关键残基;(D)BsLPMO10A的同源建模。
具体实施方式
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为从商业渠道购买得到的。
一、菌株、载体和化学品
枯草芽孢杆菌W800N、大肠杆菌BL21(DE3)和pET-30a(+)为常规商品,本实验室保存。冰岛苔藓地衣多糖(MLG-L)、大麦β-葡聚糖(MLG-B)、刺槐豆胶(LBG)、魔芋葡甘露聚糖(KGM)、山毛榉木聚糖(BX)、甜菜阿拉伯聚糖(SBA)、海带多糖、胶体几丁质、约60%的酯化度柑橘果胶、柑橘聚半乳糖醛酸(PGA),寡糖购自Megazyme(Wicklow,Ireland)。磷酸溶胀纤维素(PASC)由微晶纤维素制备(Zhang,Cui,Lynd,&Kuang,2006)。微晶纤维素(MCC)、培养基、卡那霉素和异丙基硫代-β-D-吡喃半乳糖苷(IPTG)购自Sangon(中国上海)。花生壳、大豆秸秆、羊草、水稻秸秆、水稻壳和小麦秸秆采集自当地农场(中国浙江)。
二、实验方法
(1)序列分析
将各枯草芽孢杆菌菌株的全部基因组序列以FASTA格式上传至在线软件dbCANHMMdb v10.0(https://bcb.unl.edu/dbCAN2/blast.php)进行功能注释(Zhang et al.,2018)。然后将从注释结果中获得的候选基因提交到Pfam数据库(http://pfam.xfam.org/search#tabview=tab0),以预测潜在的域和功能。使用Clustal Omega多序列比对程序(http://www.ebi.ac.uk/Tools/msa/clustalo/)对LPMOs进行氨基酸比对。利用I-TASSER服务器(http://zhanglab.ccmb.med.umich.edu/I-TASSER/download/)生成了BsLPMO10A的结构同源模型。利用MEGA 11邻接法构建系统发育树,采用bootstrap法进行系统发育测试,bootstrap复制次数为1000次。取代类型为氨基酸,模型为Jones-Taylor-Thornton(JTT)模型。结果使用ESPript 3.0渲染(Robert&Gouet,2014)。利用WebLogo3对各位点的氨基酸保守序列进行分析(http://weblogo.threeplusone.com/)。
(2)基因克隆、表达、纯化和凝胶电泳
使用快速细菌基因组DNA分离试剂盒(生工,上海,中国)分离来自枯草芽孢杆菌W800N的基因组DNA,并作为BsLPMO10A基因PCR扩增的模板。BsLPMO10A的全长(SEQ IDNo.2)和催化结构域(SEQ ID No.3)均使用BsLPMO10A-SacI和BsLPMO10A-XhoI/BsLPMO10A(CD)-XhoI引物获得(表1)。将所得片段插入到SacI和XhoI之间的pET-30a(+)中,然后通过热激转化到大肠杆菌BL21(DE3)中。重组菌株BL21/pET-30a(+)/BsLPMO10A和BL21/pET-30a(+)/BsLPMO10A(CD)进行IPTG诱导、超声处理和使用配备HisTrapTM的start蛋白纯化系统和纯化FF柱(GE Healthcare BioSciences,美国)(Zhou等人,2020)进行纯化。纯化的酶通过10-kDa Millipore离心过滤器(Merck Millipore,Burlington,MA,USA)进一步过滤以去除咪唑。随后,对酶进行SDS-PAGE(15%电泳胶和4%浓缩胶)(Laemmli,1970)分析。使用含有0.5%(w/v)的各种多糖底物的分离凝胶进行酶谱分析。电泳完成后,将凝胶用甲醇:水(1:3)溶液复性15min,复性后凝胶浸入0.05M乙酸钠缓冲液(pH5.5)中并在37℃下孵育30min。然后将凝胶用1mg/mL刚果红溶液染色,用水和1M NaCl洗涤,直至透明条带可见。并用乙酸固定。
表1引物列表
注:划线为酶切位点
(3)活性测定
在活性测定之前,BsLPMO10A在冰上于3倍摩尔浓度过量的CuSO4中孵育60min,进行铜饱和预处理。使用10-kDa Millipore离心过滤器(Merck Millipore,Burlington,MA,USA)去除未结合的铜,然后将酶重新悬浮在0.05M乙酸钠缓冲液(pH5.5)中。Bradford方法(Bradford,1976)用于确定酶的浓度。除非另有说明,后续试验使用铜饱和酶用于活性测定。
3,5-二硝基水杨酸(DNS)测定:进行了DNS分析(Bailey,Biely,&Poutanen,1992)以确定BsLPMO10A活性。简而言之,将15μL(~1.5μM)纯化酶与60μL 5mg/mL各种多糖底物(MLG-L、MLG-B、LBG、KGM、BX、SBA、海带多糖、PASC、MCC、胶体几丁质、果胶和PGA)溶解在0.05M乙酸钠缓冲液(pH 5.5)中,在37℃和200rpm下反应24h。然后,加入75μL DNS并将溶液煮沸10min。冷却至室温后,使用分光光度法在540nm处测定吸光度。
(4)生化表征
为了估计BsLPMO10A的最适温度,将15μL(~1.5μM)BsLPMO10A与60μL 5mg/mL LBG溶液在不同温度(30-80℃)下孵育24h,并使用DNS方法。BsLPMO10A的最适pH值是通过将酶在各种pH缓冲溶液(pH 2.2-8.0的柠檬酸盐/磷酸盐缓冲液和pH 8.0-9.0的0.2M Tris-HCl缓冲液)中在37℃孵育24h来确定的。最高活性确定为100%。为了研究抗坏血酸和H2O2对BsLPMO10A的影响,酶在5mg/mL LBG溶液中与不同浓度的抗坏血酸(0.5、1、1.5和3mM)和H2O2(0.5、1、2.5、5、10和50mM)在37℃下孵育24h,并使用DNS方法测量活性。
(5)BsLPMO10A的催化产物
为了探索BsLPMO10A的产物和氧化活性,将1.5μM蛋白溶液与各种含有1.5mM抗坏血酸的多糖底物(MLG-L、MLG-B、LBG、KGM、BX、SBA、昆布多糖、PASC、胶体几丁质、果胶和PGA)在37℃下孵育24h。然后使用带有脉冲安培检测器离子色谱[HPAEC-PAD、ICS3000,分离柱为CarboPac PA200(3×250mm)以及CarboPac PA200保护柱(3×50mm)](Dionex,Thermo),如文献中所述(Westereng et al.,2013)进行测定。
为了估计总氧化产物,先使用BsLPMO10A催化,再用一种GH将未反应完多糖底物有效地转化为它们的单体和二聚体。简而言之,将150μL(~15μM)BsLPMO10A与600μL 5mg/mL的各种多糖底物溶液在37℃下孵育24h,然后通过煮沸5min使酶失活。然后将溶液以12,000rpm离心15min,并使用50μL(~0.8μM)的IDSGLUC5-38或50μL(~0.5μM)的IDSXYN11-1在37℃下进一步水解上清液24h,然后煮沸5min使酶失活。将溶液以12,000rpm离心15min液用于HPAEC-PAD分析,配备CarboPac PA1(3×250mm)和CarboPac PA1(3×50mm)保护柱(Dionex,Thermo)。对于MALDI-TOF-MS分析,将1μL上述上清液与1μL溶解在含有10mM乙酸钠的50%乙腈溶液中的2,5-二羟基苯甲酸基质混合,并在样品板上干燥。然后在线性正离子模式下在300-1800的m/z范围内进行测量,使用60.0Hz的频率(UltrafleXtreme,Bruker,Germany)进行10次累积200次拍摄。按照(Kobayashi,Kamiya,&Enomoto,1996)的描述制备氧化的单糖和二糖,稍作修改。简而言之,将含有14mg碘的15mL甲醇溶液缓慢加入10mL20mg/mL样品溶液(溶于ddH2O)中,并在40℃下孵育15min。然后,在室温(25℃)下加入5mL含有0.2g KOH的甲醇溶液,然后在40℃下加热1h,直到碘颜色褪去。通过加入20mL冰水将混合物冷却,并在N2下进一步干燥。最后,在使用前将样品溶解在2mL ddH2O中。
(6)BsLPMO10A和GHs对多糖和天然木质纤维素原料降解的协同作用
为了研究BsLPMO10A和GHs对多糖的协同作用,将7.5μL(~0.75μM)BsLPMO10A和7.5μL(~0.05μM)我们小组先前获得的各种GHs进行反应,包括瘤胃微生物葡聚糖酶(IDSGLUC5-38,原称Cel5A-h38)(Cao et al.,2021),Thermobifida fusca木聚糖酶(TfXYN11-1,原称TFX)(Wang et al.,2015a),瘤胃微生物木聚糖酶(IDSXYN11-1,原称XYN-LXY)(Wang et al.,2015b),枯草芽孢杆菌甘露聚糖酶(BsMAN26-3,原称BSM)(Gao et al.,2019),瘤胃微生物纤维素酶(IDSGLUC5-11,原称Cel5A-h11)(He,Jin,Cao,Mi,&Wang,2019)和几丁质酶(BtCHI18-1,原称BtCHI1)(Wu et al.,2022),反应体系中含有1.5mM抗坏血酸,于37℃中进行孵育。在2、4、8、12、24、48和72h取等分试样,并使用DNS方法评估还原糖。
天然底物测定:在活性测定之前,将花生壳、大豆秸秆、羊草、水稻秸秆、水稻壳和小麦秸秆的天然木质纤维素原料研磨并用NaOH如前所述方法(Cao et al.,2021)进行处理。为了研究BsLPMO10A或各种GHs对天然木质纤维素分解的影响,使用500μL(~20μM)BsLPMO10A或(~0.5μM)GHs在3mL柠檬酸盐/磷酸盐缓冲液(pH 6.0)中消解0.1g各种预处理的原料样品,并在25℃下反应96h。为了研究BsLPMO10A和GHs对天然木质纤维素分解的协同作用,使用500μL(~20μM)BsLPMO10A和(~0.5μM)GHs在3mL柠檬酸盐/磷酸盐缓冲液(pH6.0)中消化0.1g各种预处理的原料样品,并在25℃下反应96h。反应结束时,将每种混合物以12,000rpm的转速离心15min,弃去上清液。不溶性材料用ddH2O冲洗10次,并根据国家可再生能源实验室(NREL)方法(Sluiter et al.,2011)用于纤维素和半纤维素测定。将干燥的天然木质纤维素添加到10mL离心管中,并添加8mL 72%硫酸。在30℃孵育1h后,将最终硫酸浓度稀释至4%,并将混合物置于121℃高压灭菌锅中,孵育1h。样品中的纤维素和半纤维素被水解成单糖,并分别使用苔黑酚(Albaum,&Umbreit,1947)和蒽酮比色法(Hedge,&Hofreiter,1962)进行量化。
三、结果分析
(1)序列分析
序列分析表明,BsLPMO10A由455个氨基酸残基(SEQ ID No.1)组成,包括一个AA10催化结构域和纤连蛋白Ⅲ型(FNⅢ)模块。SignalP5.0预测表明,N端的前40个氨基酸是信号肽。同源性模型表明,BsLPMO10A含有一个免疫球蛋白G(IgG)样β-三明治折叠和一个含有铜离子的组氨酸支架序列(图9)。两个保守的组氨酸形成一个T形的“组氨酸支架”,His41和His130,位于催化结构域的表面,代表了LPMO的典型特征。“组氨酸支架”和几个附近的芳香族氨基酸残基被确定为底物结合或催化位点相关(Book et al.,2014;Labourel et al.,2020;Span&Marletta,2015;Wu et al.,2013;Zhou,Qi,Huang,&Zhu,2019)。与C1-、C4-或C1/C4-氧化模式的AA9酶不同,AA10 LPMO被认为主要以C1-氧化模式催化底物,并可进一步分类为C1-和C1/C4-氧化亚家族(Li et al.,2021b)。在这项研究中,系统发育树分析表明,BsLPMO10A与来自AA10家族的其他C1氧化LPMO聚集在一起(图1)。尽管BsLPMO10A和枯草芽孢杆菌基因组中的其他12种LPMO共存,但BsLPMO10A与这些酶的相似性相对较低(33.70-62.63%),但与来自芽孢杆菌属的未表征的AA10蛋白的相似性较高(99.53%)(WP_001065159.1)。迄今为止,在CAZy数据库中已经对AA10家族中的约30种酶进行了表征,这些酶主要来源于细菌。与这些功能研究的AA10 LPMO相比,BsLPMO10A与地衣芽孢杆菌DSM13BlLPMO10A(GenBank编号:AAU39477.1)(Courtade et al.,,2020)和解淀粉芽孢杆菌DSM7BaLPMO10A(GenBank编号:CBI42985.1)(Hemsworth et al.,2013)的相似度最高,相似度分别为73.4和63.5%,但这两者都是几丁质活性的LPMO。
(2)BsLPMO10A是一种嗜温酶,由一定量的抗坏血酸或H2O2激活
纯化的BsLPMO10A及其截短的催化结构域[BsLPMO10A(CD)]的分子量分别为~56(图2中A)和~34kDa(图2中B)。两种酶都对MLG-L、MLG-B、LBG、KGM、BX和SBA有活性(图2中C)。值得注意的是,与仅催化结构域相比,包含催化结构域和FNⅢ的全长BsLPMO10A对MLG-L(P<0.001)、MLG-B(P<0.001)、LBG(P<0.05)和KGM(P<0.01)(表2)催化活性更高。FNⅢ结构域广泛存在于细菌、酵母、植物和动物蛋白中。据报道,芽孢杆菌属、蜡状芽孢杆菌(Mutahiret al.,2018)和苏云金芽孢杆菌(Manjeet,Madhuprakash,Mormann,Moerschbacher,&Podile,2019)包含一个或多个FNⅢ结构域。然而,人们对其功能和详细机制知之甚少,有时还存在争议。敲除FNⅢ结构域(也称为X1结构域)并未改变TfAA10B的催化效率(Arfi,&Umbreit,2014;Kruer-Zerhusen et al.,2017)。相比之下,周等人(Zhou,Irwin,Escovar-Kousen,&Wilson,2004)表明没有FNⅢ结构域的截短Cel9A-90的催化活性降低到野生型的43%。令人惊讶的是,两种酶TfAA10B和Cel9A-90均来自褐色嗜热裂孢菌。以往的研究表明,FNⅢ模块有利于稳定酶的构象,提高可溶性和不溶性底物的活性(Han,Shang-Guan,&Yang,2019;Kataeva et al.,2002;Mutahir et al.,2018;Nguyen et al.,2021)。关于在不同底物上的不同性能,我们推测BsLPMO10A可能优先作用于某些由葡萄糖或甘露糖单元组成的多糖,其次是由木糖或阿拉伯糖组成的多糖(表2和图2中C)。在这种情况下,FNⅢ结构域极大地有助于底物结合和/或催化效率。因此,与截短酶相比,野生型BsLPMO10A对“优选”底物(包括冰岛苔藓地衣多糖、大麦β-葡聚糖、刺槐豆胶和魔芋葡甘露聚糖)显示出明显更高的活性(P<0.05)。对于“不太优选”的底物,例如山毛榉木聚糖和甜菜阿拉伯聚糖,去除FNⅢ结构域不会导致催化活性的严重降低,导致与野生型和截短的BsLPMO10A的活性相当。在最近的一项研究中,据报道FNⅢ结构域可能通过提高其对可溶性底物的亲和力(较低的Km值)来增加酶活性(Nguyen et al.,2021)。然而,FNⅢ结构域在酶性能中的详细机制仍有待解决。在本研究研究的多糖中,BsLPMO10A和BsLPMO10A(CD)对PASC、MCC、胶体几丁质、果胶或PGA均无活性。因此,采用全长BsLPMO10A进行进一步研究,因为它在各种多糖的分解中具有高活性。
表2.全长和截短BsLPMO10A的比活性
数据代表平均值±SD(n=4)。ND,未检测到。使用t检验进行BsLPMO10A和BsLPMO10A(CD)之间的统计分析。*,P<0.05;**,P<0.05;***,P<0.001
BsLPMO10A的最适温度和pH分别为50℃和5.0(图3中A和3中B),表明BsLPMO10是一种在中性和弱酸性条件下具有活性的中温酶,这与大多数先前报道的LPMO表现出最佳温度范围为40–60℃(Li et al.,2021a;Li,Dilokpimol,Kabel,&de Vries,2022;Zhang,Chen,Long,&Ding,2021)一致。值得注意的是,观察到源自高温堆肥宏基因组的MgLPMO10显示出最高的熔化温度(Tm=83℃)和高达80℃的催化活性(Tuveng et al.,2020)。更引人注目的是,米曲霉AA17酶LPMO-AoAA17显示出100℃的最佳温度,在121℃和15psi高压灭菌后甚至具有活性(Bhatia&Yadav,2021)。众所周知,铜对LPMO的氧化活性至关重要,使用EDTA螯合去除Cu2+会导致LPMO活性和热稳定性降低(Gusakov,Bulakhov,Demin,&Sinitsyn,2017;Semenova et al.,2019)。因此,在本研究中进行的所有测定之前,对BsLPMO10A进行了铜饱和。随后,确定了抗坏血酸和H2O2对BsLPMO10A活性的影响。图3中C显示抗坏血酸显着促进BsLPMO10A活性(P<0.01),在氧化反应中充当电子供体,这与先前报道的一致(Stepnov etal.,2021)。然而,包括1,2-二羟基苯(Zhang et al.,2021)和单宁酸(Frommhagen et al.,2016)在内的一些还原剂对LPMO活性不敏感。同时,O2被认为是参与LPMO氧化反应的共基质(Vaaje-Kolstad et al.,2010)。令人惊讶的是,最近证明H2O2而不是O2驱动LPMO的共底物(Hegnar et al.,2019)。在本研究中,1或2.5mM H2O2分别显着提高了BsLPMO10A活性70.7%(P<0.001)或49.5%(P<0.01)。然而,当H2O2浓度超过5mM时观察到抑制作用(图3中D)。尽管一定量的H2O2会促进LPMO催化,但添加过量的H2O2会对酶产生负面影响并耗尽电子供体(Calderaro et al.,2020),从而抑制LPMO活性(Bissaro et al.,2017;Kuusk et al.,2018;Petrovic et al.,2018)。
(3)BsLPMO10A从底物中释放天然和C1氧化的寡糖,特别是通过β-1,4糖苷键连接的多糖
根据BsLPMO10A的底物范围,葡聚糖样底物用于HPAEC-PAD和MALDI-TOF-MS分析。结果表明,BsLPMO10A能够从MLG-L或MLG-B中释放C1氧化和天然(非氧化)寡糖(图4中A)。然而,由于存在β-1,3-和β-1,4-糖苷键((Eklof,Shojania,Okon,McIntosh,&Brumer,2013),保留时间为5至17min的洗脱峰对应于聚合度(DP)范围为2至6的天然产物,而在17至25min观察到的峰对应于C1氧化产物(Correa et al.,2019;Stepnov et al.,2021;Zhang etal.,2021)。据报道,C4氧化的LPMO主要在30-35min的保留时间内表现出典型的曲线,在约40min时对应于C1/C4双氧化产物的大峰(Zarah Forsberg et al.,2014;Isaksen et al.,2014;Sun et al.,2020)。在这项研究中,没有检测到对应于双氧化产物的信号,因此,我们推测BsLPMO10A是一种在C1碳上氧化的AA10 LPMO(图4中A和4中B)。为了更多地了解催化产物的组成和裂解位点,使用MALDI-TOF-MS验证了寡糖的分子量。图4中B和4中C显示m/z信号为505([M+H Da]+)或527、689、851和1013([M+Na Da]+)的产物对应于纤维三糖(DP3)、纤维四糖(DP4)、纤维五糖(DP5)和纤维六糖(DP6)。m/z值为543、705、867和1029([M+16+Na Da]+)的信号对应于具有末端C1特异性醛糖酸的氧化纤维三糖(DP3ox)、纤维四糖(DP4ox)、纤维五糖(DP5ox)和纤维六糖(DP6ox)(Phillips,Beeson,Cate,&Marletta,2011;X.Zhang etal.,2021)。结果表明,BsLPMO10A主要释放C1氧化和天然寡糖,DP值为3至6。为了化学计量确定BsLPMO10A的总氧化活性,使用一种瘤胃微生物葡聚糖酶(IDSGLUC5-38),可有效地将葡聚糖底物转化为葡萄糖和纤维二糖(Cao et al.,2021)。反应24h后,MLG-L释放出3127±535μmol/L天然产物和3100±139μmol/LC1氧化产物。值得注意的是,与MLG-L相比,BsLPMO10A从MLG-B中释放出更多的天然产物(645±137μmol/L)和C1氧化产物(1260±58μmol/L)(图4中D)。MLG-B和MLG-L被归类为混合键葡聚糖(MLG),这两种底物的主链由通过(1→4)和(1→3)键连接的β-D-葡萄糖残基组成。然而,这些β-(1→4)和β-(1→3)键的比例和分布在MLG之间有所不同。由于BsLPMO10A对β-(1→3)连接的海带多糖无活性(表2),这推断该酶攻击β-(1→4)键以释放天然和C1-氧化的纤维寡糖。MLG-B和MLG-L的β-(1→4):β-(1→3)键的平均比例分别约为3:1和2:1(https://www.megazyme.com),这与MLG-B比MLG-L释放更多产品一致(图4中D)。
类似地,发现BsLPMO10A从BX中释放天然和C1氧化的低聚木糖(图5中A)。BX是一种支链木聚糖,在主链中包含一个木糖单元,并在侧链中装饰有约13%GlcA-OMe(表2)(https://www.megazyme.com/Xylan-Beechwood-purified)。MALDI-TOF-MS分析表明,BsLPMO10A释放的m/z信号为547([M+H Da]+)或569和701([M+Na Da]+)或759、891和1023([M+GlcA-OMe+Na Da]+)分别对应于直链-/支链-木四糖(DP4)、木戊糖(DP5)和木六糖(DP6)。m/z信号为585([M+16+NaDa]+)、695([M+16+H Da]+)或753([M+GlcA-OMe+16+H Da]+)或511、775、907和1039([M+GlcA-OMe+16+Na Da]+的m/z值)对应于C1氧化的线性/支链-木二糖(DP2)、木四糖(DP4)、木五糖(DP5)和木六糖(DP6)(图5中B)。此外,瘤胃微生物木聚糖酶(IDSXYN11-1)有效地将木聚糖转化为木糖和木二糖(Wang,2015a)都用于BX的分解。反应24h后,产生959±56μmol/L的天然产物和387±161μmol/L C1氧化产物(图5中C)。有趣的是,来自BX的C1氧化/天然产物的比率为0.40,低于从MLG-B(0.99)和MLG-L(1.95)获得的比率(图4中D),这可能是由于它偏爱β-(Glc1→4Glc)其次是β-(Xyl1→4Xyl)(表2)。如上所述,多糖主链内的糖环单元和连接类型与LPMO催化密切相关。同时,被修饰的侧链被认为在氧化反应中起作用。在最近的一项研究中,及其同事(2022)研究了来自子囊菌类Thermothielavioides terrestrisLPH172的六种LPMO(TtLPMO9A、TtLPMO9B、TtLPMO9E、TtLPMO9G、TtLPMO9T和TtLPMO9U)在山毛榉葡萄糖醛酸木聚糖(BeWX)、云杉阿拉伯葡萄糖醛酸木聚糖(AGX)和桦木的乙酰化葡糖醛酸木聚糖(acGX)底物上的生化特性。他们发现六个TtLPMO9中的三个(TtLPMO9A、TtLPMO9E和TtLPMO9G)在BeWX上具有活性,它含有较少的与PASC结合的侧链。对于具有更复杂分支的木聚糖底物,仅测试的两种(TtLPMO9A和TtLPMO9E)或一种LPMO(TtLPMO9G)分别对AGX和acGX有活性。
此外,BsLPMO10A还表现出对KGM和SBA的催化作用,产生天然和C1氧化的寡糖(图6中A和6中B)。然而,该酶对PASC、几丁质、果胶和PGA的活性难以检测(图6中C-E)。据文献报道,AA10 LPMOs能够催化几丁质和/或PASC底物,例如来自地衣芽孢杆菌的BaAA10A(Forsberg et al.,2014),来自单核细胞增生李斯特菌的LmLPMO10(Paspaliari et al.,2015)和来自灰色链霉菌的SgLPMO10F(Nakagawa et al.,2015)。因此,这些LPMO过去被归类为几丁质结合蛋白21(CBP21)或碳水化合物结合模块33(CBM33)。然而,除了几丁质和纤维素氧化外,首次报道来自Kitasatospora papulosa的KpLPMO10A作用于木聚糖,木聚糖由β-(1→4)-D-木糖残基组成(Correa et al.,2019)。更令人惊讶的是,源自Paenibacilluscurdlanolyticus的PcAA10显示出对一系列几丁质、纤维素(Avicel和α-纤维素)和半纤维素(桦木木聚糖、燕麦木聚糖和象牙坚果甘露聚糖)底物的活性(Limsakul et al.,2020)。与先前报道的LPMO不同,本研究中开发的BsLPMO10A显示出对半纤维素分解的强大活性,例如MLG、木聚糖、甘露聚糖和阿拉伯聚糖(表2),表现出比PcAA10更宽的活性底物谱。尽管在AA9LPMO中普遍观察到针对甘露聚糖的活性(Agger et al.,2014;Calderaro et al.,2020;Liu,Olson,Wu,Broberg,&Sandgren,2017),但BsLPMO10A和PcAA10显示出很少见的甘露聚糖降解能力在AA10 LPMO中发现。更有趣的是,据我们所知,BsLPMO10A在本研究中首次显示出对阿拉伯聚糖的活性(表2和图6中B)。除了糖环、糖苷键和修饰的侧链外,多糖的分散状态或聚集体的形成也可能有助于LPMO的催化效率。最近的一项研究报告称,AoLPMO9A/B(Chen,Zhang,Long,&Ding,2021)和MtLPMO9B(Sun et al.,2021)的氧化效应急剧下降,同时结晶度指数增加。BsLPMO10A的催化效率在由β-1,4连接的葡萄糖组成的底物中也有所不同。我们推测BsLPMO10A对可溶性底物(包括冰岛苔藓地衣多糖和大麦β-葡聚糖)的活性更高,其次是部分可溶性PASC和不溶性MCC(表2)。综上所述,BsLPMO10A反之亦然。
(4)BsLPMO10A促进多种多糖和天然木质纤维素生物质的降解
鉴于其对多种底物的广泛催化活性,BsLPMO10A被用于与GH协同降解一系列多糖。尽管仅用BsLPMO10A处理后释放的还原糖可忽略不计,但观察到该酶加速了MLG-L、BX、PASC和几丁质的分解(图7)。共同反应72h后,和单独的GH组相比,协同组MLG-L、BX、PASC和几丁质中的还原糖分别为3176±97、7436±165、649±44和2604±130μmol/L,分别为单独组的1.47、1.56、1.44和1.25倍(P<0.001)。对于LBG的分解,与单独的内切甘露聚糖酶水解相比,BsLPMO10A和内切甘露聚糖酶共反应72h后,还原糖产量增加了11.68±7.85%(P=0.12)(图7中C)。值得注意的是,尽管BsLPMO10A本身并没有从PASC或几丁质中释放出类似的寡糖(图6中D和6中E),但它显着促进了相关GH对这两种底物的分解(图7中D和7中E)。由于它们在多糖解聚中的广泛用途,LPMOs被广泛用于与GHs协同进行底物降解。在过去十年中,来自AA9-11和13-16的LPMO已被证明与多种多糖具有协同作用,包括纤维素(Harris et al.,2010;Xin,Blossom,Lu,&Felby,2022)、几丁质(Nakagawa et al.,2015;Paspaliari etal.,2015)、木聚糖(Correa et al.,2019;Jung et al.,2015)和果胶(Sabbadin et al.,2021)。然而,发现BsLPMO10A显着促进多种多糖,尤其是半纤维素,其中主链包含β-1,4糖苷键,而与糖环种类无关(表2和图6)。
随后,BsLPMO10A与葡聚糖酶IDSGLUC5-38、甘露聚糖酶BsMAN26-3和木聚糖酶TfXYN11-1一起用于饲料的分解。为了消除木质素对催化过程的阻碍作用,我们用2%NaOH对原料进行预处理以去除其中的木质素,并获得含有28-38%纤维素和18-32%半纤维素的碱处理样品(Paritosh et al.,2021)。这些结果表明LPMO对半纤维素表现出强烈的活性,但对纤维素几乎没有活性(图8)。仅用BsLPMO10A处理后,除大豆秸秆外,所有测试基质中的半纤维素组合物均显着下降(P<0.05),如葡聚糖、木聚糖、甘露聚糖和阿拉伯聚糖降解的强大能力所示(表2)。例如,BsLPMO10A和甘露聚糖酶之间的协同作用显着降低(P<0.05)半纤维素组成,在花生壳中降低了10.61±6.18%(图8中A),在水稻壳中降低了8.19±3.85%(P<0.05)。图8中E)。BsLPMO10A和葡聚糖酶的协同作用显着降低(P<0.05)半纤维素组成在拉毛果秸秆(图8中C)和水稻壳中的9.65±4.04%(图8中E)。BsLPMO10A和木聚糖酶之间的协同作用显着降低(P<0.05)半纤维素组成在大豆秸秆中降低了17.67±4.68%(图8中B),在水稻壳中降低了10.00±4.14%(图8中E)和小麦秸秆中的16.16±2.79%(图8中F)。此外,BsLPMO10A、葡聚糖酶、木聚糖酶和甘露聚糖酶的协同作用显着解聚了纤维素和半纤维素(P<0.05)。天然植物来源的原料由纤维素、半纤维素、果胶和木质素组成。由于大豆秸秆、拉毛果秸秆、水稻壳和小麦秸秆中的木聚糖和葡聚糖相对丰富(Balan et al.,2008;Wu etal.,2018),木聚糖酶和葡聚糖酶与甘露聚糖酶处理相比,处理对它们的分解更有效,半纤维素减少了9.65-17.67%(图8中B、8中C、8中E和8中F)。此前有报道称,LPMO会导致底物出现缺口并引发纤维结构的解体,导致链断裂和原始纳米纤维的释放(Li et al.,2021b;Villares et al.,2017;Zhang et al.,2017)。因此,LPMO处理的木质纤维素表现出强烈的原纤化。使用原子力显微镜进行的详细形态观察表明,C1-/C4-氧化的LPMO直接结合到纳米晶纤维素的表面以降解底物(Eibinger,Sattelkow,Ganner,Plank,&Nidetzky,2017;Moreau et al.,2019),为BsLPMO10A分解天然底物提供了详细的证据。与多糖氧化LPMO不同的是,一种源自米曲霉的AA17 LPMO(LPMO-AoAA17)被发现通过切割β-O-4键对木质素起作用(Bhatia&Yadav,2021)。
Claims (9)
1.一种来自枯草芽孢杆菌的裂解性多糖单加氧酶BsLPMO10A,由455个氨基酸残基构成,其氨基酸序列如SEQ ID No.1所示。
2.编码权利要求1所述的裂解性多糖单加氧酶BsLPMO10A的基因。
3.根据权利要求2所述的基因,其特征在于,所述基因的核苷酸序列如SEQ ID No.2所示。
4.一种表达载体,含有权利要求2或3所述的基因。
5.一种重组菌,含有权利要求4所述的表达载体。
6.权利要求1所述的裂解性多糖单加氧酶BsLPMO10A在促进半纤维素底物降解上的应用。
7.根据权利要求6所述的应用,其特征在于,所述半纤维素底物为地衣多糖、β-葡聚糖、刺槐豆胶、魔芋葡甘露聚糖、山毛榉木木聚糖和甜菜碱阿拉伯糖。
8.权利要求1所述的裂解性多糖单加氧酶BsLPMO10A在促进多糖底物β-1,4糖苷键断裂上的应用。
9.权利要求1所述的裂解性多糖单加氧酶BsLPMO10A在与糖苷水解酶协同促进多糖的分解上的应用。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210868807.1A CN115992104B (zh) | 2022-07-22 | 2022-07-22 | 来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 |
PCT/CN2022/111015 WO2024016397A1 (zh) | 2022-07-22 | 2022-08-09 | 来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210868807.1A CN115992104B (zh) | 2022-07-22 | 2022-07-22 | 来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115992104A true CN115992104A (zh) | 2023-04-21 |
CN115992104B CN115992104B (zh) | 2024-01-30 |
Family
ID=85994234
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210868807.1A Active CN115992104B (zh) | 2022-07-22 | 2022-07-22 | 来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN115992104B (zh) |
WO (1) | WO2024016397A1 (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116042549A (zh) * | 2022-09-15 | 2023-05-02 | 中国海洋大学 | 裂解性多糖单加氧酶EbLPMO10A及其应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105713913A (zh) * | 2014-12-02 | 2016-06-29 | 中国科学院大连化学物理研究所 | 氧化水解酶基因BtLPMO10B及氧化水解酶与应用 |
CN106544296A (zh) * | 2016-10-20 | 2017-03-29 | 清华大学深圳研究生院 | 促进冈比亚藻生长、光合作用及分泌雪卡毒素的微生物、方法及试剂盒 |
US20180051305A1 (en) * | 2015-03-11 | 2018-02-22 | Genencor International B.V. | Enzymatic activity of lytic polysaccharide monooxygenase |
WO2018106656A1 (en) * | 2016-12-06 | 2018-06-14 | Danisco Us Inc | Truncated lpmo enzymes and use thereof |
CN109957571A (zh) * | 2017-12-14 | 2019-07-02 | 中国科学院大连化学物理研究所 | 一种多糖裂解单加氧酶编码基因及酶和制备与应用 |
US20190233861A1 (en) * | 2016-09-30 | 2019-08-01 | Norwegian University Of Life Sciences | Process for degrading a polysaccharide employing a lytic polysaccharide monooxygenase |
-
2022
- 2022-07-22 CN CN202210868807.1A patent/CN115992104B/zh active Active
- 2022-08-09 WO PCT/CN2022/111015 patent/WO2024016397A1/zh unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105713913A (zh) * | 2014-12-02 | 2016-06-29 | 中国科学院大连化学物理研究所 | 氧化水解酶基因BtLPMO10B及氧化水解酶与应用 |
US20180051305A1 (en) * | 2015-03-11 | 2018-02-22 | Genencor International B.V. | Enzymatic activity of lytic polysaccharide monooxygenase |
CN108699578A (zh) * | 2015-03-11 | 2018-10-23 | 杰能科国际有限公司 | 裂解性多糖单加氧酶的酶活性 |
US20190233861A1 (en) * | 2016-09-30 | 2019-08-01 | Norwegian University Of Life Sciences | Process for degrading a polysaccharide employing a lytic polysaccharide monooxygenase |
CN106544296A (zh) * | 2016-10-20 | 2017-03-29 | 清华大学深圳研究生院 | 促进冈比亚藻生长、光合作用及分泌雪卡毒素的微生物、方法及试剂盒 |
WO2018106656A1 (en) * | 2016-12-06 | 2018-06-14 | Danisco Us Inc | Truncated lpmo enzymes and use thereof |
CN109957571A (zh) * | 2017-12-14 | 2019-07-02 | 中国科学院大连化学物理研究所 | 一种多糖裂解单加氧酶编码基因及酶和制备与应用 |
Non-Patent Citations (3)
Title |
---|
HALL, NEIL.: "登录号CUB56952", GENBANK * |
HALL, NEIL.: "登录号CYHR01000999", GENBANK * |
姚昌阳等: "裂解多糖单加氧酶在毕赤酵母中的异源表达", 湖北大学学报(自然科学版), vol. 41, no. 6 * |
Also Published As
Publication number | Publication date |
---|---|
CN115992104B (zh) | 2024-01-30 |
WO2024016397A1 (zh) | 2024-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Agrawal et al. | Current understanding of the inhibition factors and their mechanism of action for the lignocellulosic biomass hydrolysis | |
Dimarogona et al. | Cellulose degradation by oxidative enzymes | |
Paramjeet et al. | Biofuels: Production of fungal-mediated ligninolytic enzymes and the modes of bioprocesses utilizing agro-based residues | |
Yeoman et al. | Thermostable enzymes as biocatalysts in the biofuel industry | |
Selig et al. | Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities | |
US20150175980A1 (en) | Novel cell wall deconstruction enzymes of scytalidium thermophilum, myriococcum thermophilum, and aureobasidium pullulans, and uses thereof | |
WO2014059541A1 (en) | Novel cell wall deconstruction enzymes of thermoascus aurantiacus, myceliophthora fergusii (corynascus thermophilus), and pseudocercosporella herpotrichoides, and uses thereof | |
CA2657684A1 (en) | Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose | |
Hua et al. | Characterization of a novel thermostable GH7 endoglucanase from Chaetomium thermophilum capable of xylan hydrolysis | |
Barr et al. | Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass | |
Quiroz-Castañeda et al. | Plant cell wall degrading and remodeling proteins: current perspectives | |
Widyasti et al. | Biodegradation of fibrillated oil palm trunk fiber by a novel thermophilic, anaerobic, xylanolytic bacterium Caldicoprobacter sp. CL-2 isolated from compost | |
Sakamoto et al. | Peculiarities and applications of galactanolytic enzymes that act on type I and II arabinogalactans | |
Guo et al. | Enhancing digestibility of Miscanthus using lignocellulolytic enzyme produced by Bacillus | |
Zhao et al. | Two family 11 xylanases from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing industry | |
CN115992104B (zh) | 来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 | |
Moreira et al. | Enzymology of plant cell wall breakdown: an update | |
Meng et al. | Characterization of a thermostable endo-1, 3 (4)-β-glucanase from Caldicellulosiruptor sp. strain F32 and its application for yeast lysis | |
Quiroz-Castañeda et al. | Proteínas que remodelan y degradan la pared celular vegetal: perspectivas actuales | |
Limsakul et al. | A novel AA10 from Paenibacillus curdlanolyticus and its synergistic action on crystalline and complex polysaccharides | |
Xue et al. | Characterization of the recombinant GH10 xylanase from Trichoderma orientalis EU7-22 and its synergistic hydrolysis of bamboo hemicellulose with α-glucuronidase and α-L-arabinofuranosidase | |
Ticona et al. | Paenibacillus barengoltzii A1_50L2 as a source of plant cell wall degrading enzymes and its use on lignocellulosic biomass hydrolysis | |
Onuma et al. | Purification and characterization of a glycoside hydrolase family 5 endoglucanase from Tricholoma matsutake grown on barley based solid-state medium | |
Suvorov et al. | Novelties of the cellulolytic system of a marine bacterium applicable to cellulosic sugar production | |
Sun et al. | BsLPMO10A from Bacillus subtilis boosts the depolymerization of diverse polysaccharides linked via β-1, 4-glycosidic bonds |
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 |