CN115011571B - Schizolysis type polysaccharide monooxygenase and application thereof - Google Patents
Schizolysis type polysaccharide monooxygenase and application thereof Download PDFInfo
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- CN115011571B CN115011571B CN202210683411.XA CN202210683411A CN115011571B CN 115011571 B CN115011571 B CN 115011571B CN 202210683411 A CN202210683411 A CN 202210683411A CN 115011571 B CN115011571 B CN 115011571B
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- polysaccharide monooxygenase
- amylase
- starch
- oxygen bleaching
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- 102000008109 Mixed Function Oxygenases Human genes 0.000 title claims abstract description 39
- 108010074633 Mixed Function Oxygenases Proteins 0.000 title claims abstract description 39
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 38
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 38
- 150000004676 glycans Chemical class 0.000 title claims abstract 7
- 102000004190 Enzymes Human genes 0.000 claims abstract description 40
- 108090000790 Enzymes Proteins 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 38
- 230000008569 process Effects 0.000 claims abstract description 25
- 239000004753 textile Substances 0.000 claims abstract description 13
- 229940088598 enzyme Drugs 0.000 claims description 36
- 239000007788 liquid Substances 0.000 claims description 30
- 150000001875 compounds Chemical class 0.000 claims description 20
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 18
- 229910052760 oxygen Inorganic materials 0.000 claims description 18
- 239000001301 oxygen Substances 0.000 claims description 18
- 102000004139 alpha-Amylases Human genes 0.000 claims description 16
- 108090000637 alpha-Amylases Proteins 0.000 claims description 16
- 229940024171 alpha-amylase Drugs 0.000 claims description 16
- 239000004744 fabric Substances 0.000 claims description 16
- 229920000742 Cotton Polymers 0.000 claims description 15
- 238000004061 bleaching Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 238000009990 desizing Methods 0.000 claims description 13
- 108010059892 Cellulase Proteins 0.000 claims description 10
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 10
- 229940106157 cellulase Drugs 0.000 claims description 10
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- 239000001110 calcium chloride Substances 0.000 claims description 9
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 9
- 239000004094 surface-active agent Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 239000003755 preservative agent Substances 0.000 claims description 6
- 230000002335 preservative effect Effects 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000002738 chelating agent Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 230000000149 penetrating effect Effects 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 108010059820 Polygalacturonase Proteins 0.000 claims description 2
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000000643 oven drying Methods 0.000 claims description 2
- 238000010923 batch production Methods 0.000 claims 2
- 239000007844 bleaching agent Substances 0.000 claims 1
- 238000009987 spinning Methods 0.000 claims 1
- 229920002472 Starch Polymers 0.000 abstract description 34
- 239000008107 starch Substances 0.000 abstract description 34
- 235000019698 starch Nutrition 0.000 abstract description 34
- 230000000694 effects Effects 0.000 abstract description 22
- 239000004382 Amylase Substances 0.000 abstract description 14
- 102000013142 Amylases Human genes 0.000 abstract description 14
- 108010065511 Amylases Proteins 0.000 abstract description 14
- 235000019418 amylase Nutrition 0.000 abstract description 14
- 239000000758 substrate Substances 0.000 abstract description 13
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 238000003766 bioinformatics method Methods 0.000 abstract description 3
- 230000000593 degrading effect Effects 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 238000012512 characterization method Methods 0.000 abstract description 2
- 230000003197 catalytic effect Effects 0.000 abstract 1
- 150000004804 polysaccharides Chemical class 0.000 description 29
- 101710154526 Lytic chitin monooxygenase Proteins 0.000 description 8
- 230000015556 catabolic process Effects 0.000 description 8
- 238000006731 degradation reaction Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000002002 slurry Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 238000005065 mining Methods 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 241001149711 Aspergillus lentulus Species 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 2
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 2
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 229940072107 ascorbate Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 238000009412 basement excavation Methods 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004134 energy conservation Methods 0.000 description 2
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- 230000006698 induction Effects 0.000 description 2
- 238000007639 printing Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- PKYCWFICOKSIHZ-UHFFFAOYSA-N 1-(3,7-dihydroxyphenoxazin-10-yl)ethanone Chemical compound OC1=CC=C2N(C(=O)C)C3=CC=C(O)C=C3OC2=C1 PKYCWFICOKSIHZ-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 241000219196 Armoracia Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- KIHFFINCXFGABP-UHFFFAOYSA-N Cl.[I] Chemical compound Cl.[I] KIHFFINCXFGABP-UHFFFAOYSA-N 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical class [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 241000683814 Sericus Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 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 1
- 238000009940 knitting Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
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- 230000001737 promoting effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 101150057627 trxB gene Proteins 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
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
- C12N9/0004—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
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06L—DRY-CLEANING, WASHING OR BLEACHING FIBRES, FILAMENTS, THREADS, YARNS, FABRICS, FEATHERS OR MADE-UP FIBROUS GOODS; BLEACHING LEATHER OR FURS
- D06L1/00—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods
- D06L1/12—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods using aqueous solvents
- D06L1/14—De-sizing
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
- D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
- D06M2101/02—Natural fibres, other than mineral fibres
- D06M2101/04—Vegetal fibres
- D06M2101/06—Vegetal fibres cellulosic
Abstract
According to the invention, the family members of the polysaccharide monooxygenase AA13 are excavated and cracked from a public database through bioinformatics analysis, the sequence of the polysaccharide monooxygenase with starch activity is obtained, and protein expression and enzyme activity characterization are carried out on the sequence, so that the sequence can efficiently cooperate with amylase to decompose starch substrates, and the catalytic efficiency of degrading the substrates by the amylase can be effectively improved. The enzyme is applied to a textile cold-reactor process, and the cold-reactor process treatment time of the enzyme added with the schizolysis polysaccharide monooxygenase is 20-30 ℃ within the room temperature range, so that the cold-reactor treatment time can be effectively shortened, the cost is saved, and the production efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of textile, and relates to application of a schizolysis polysaccharide monooxygenase in pretreatment of textile printing and dyeing.
Background
The cleaving polysaccharide monooxygenases (lytic polysaccharide monooxygenases, LPMOs) are a recently discovered class of copper ion-dependent oxidases that, unlike traditional glycoside hydrolases, selectively cleave the C1 and/or C4 glycosidic linkages of recalcitrant polysaccharide substrates such as cellulose, chitin, xylan, starch, etc., with their unique oxidative mechanism, tending to loosen their structure and facilitating the catalysis of subsequent glycoside hydrolases. LPMOs currently comprise a total of seven families, AA9, AA10, AA11, AA13, AA14, AA15 and AA 16. Among them, the AA13 family is the only one found so far that can act on and only act on starch and its related substrates, and is therefore also called starch-active cleavage polysaccharide monooxygenase, which can degrade various starch substrates such as corn starch, amylose, amylopectin, etc., does not show any activity on cellulose and chitin, and has been widely used in various fields of food processing, pharmaceutical production, textile applications, etc. The traditional textile industry is an industry with heavy pollution, high energy consumption and large water consumption. The pollution problem prevention, control, energy conservation and emission reduction are sustainable development production routes advocated in China at present.
The pretreatment of printing and dyeing is used as an important link in the knitting industry chain, wherein the energy conservation, the emission reduction and the environmental protection are important, the energy consumption is reduced, the production efficiency is improved, and the method is a necessary way for sustainable development. The biological enzyme method cold-heap pretreatment process is a typical process for efficiently saving energy of cotton knitted fabrics, and most important in the biological enzyme method is amylase, and most of researches at present are focused on improving the activity of the amylase so as to achieve the effect of the cold-heap treatment process.
CN 105970633B discloses a biological complex enzyme preparation for textile, which uses a compounding process of alpha-amylase, cellulase, xylanase and pectase to make cotton fabric treated by cold-stacking process for 6-12h under room temperature condition, so as to effectively degrade the sizing agent on the surface of the fabric, and achieve desizing effect.
In summary, in the field of the lyase-polysaccharide monooxygenase, the addition of the lyase-polysaccharide monooxygenase can loosen starch substrates more loosely, so that the efficiency of degrading starch by amylase is effectively and synergistically promoted. The AA13 family sequence has few enzyme activity characterization, and the application of the AA13 family starch active schizolysis polysaccharide monooxygenase which is more effective in excavation to the textile field is a novel mode for effectively reducing the energy consumption of the dyeing pretreatment.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a schizolysis polysaccharide monooxygenase which can more effectively improve the process efficiency of a cold reactor.
The invention firstly provides application of a cleaved polysaccharide monooxygenase in starch degradation, wherein the amino acid sequence of the cleaved polysaccharide monooxygenase is shown as SEQ ID NO: 1. More preferably, the cleaving polysaccharide monooxygenase is used in conjunction with an alpha-amylase to degrade starch.
In a specific embodiment, the starch is a soluble starch.
The invention also provides application of the cleaved polysaccharide monooxygenase in a textile cold-stacking process, which is characterized in that the cleaved polysaccharide monooxygenase is compounded with amylase to improve the degradation efficiency of starch slurry, and the amino acid sequence of the cleaved polysaccharide monooxygenase is shown as SEQ ID NO: 1.
The invention further provides a biological enzyme compound preparation used in the textile cold-stacking process, which is characterized by comprising a lyase polysaccharide monooxygenase, alpha-amylase, cellulase, xylanase and pectinase.
Preferably, calcium chloride, preservative and surfactant are also included.
More preferably, the amounts of the components are as follows:
1mg/L to 10mg/L of lycan polysaccharide monooxygenase
Alpha-amylase 800-2000U/ml
Cellulase 800-1000U/ml
Xylanase 8000-12000U/ml
Pectase 400-1200U/ml
2-8g/L of calcium chloride
Preservative 5-15g/L
50-100g/L of surfactant
Finally adding water to fix the volume to 1L;
preferably pH 6.0 to 7.0.
The invention further provides a method for desizing all-cotton bad cloth by adopting the biological enzyme compound preparation, which is characterized in that,
(1) Padding biological compound enzyme liquid: padding the all-cotton grey cloth in the biological compound enzyme liquid at the temperature of 30-50 ℃;
(2) Cooling the reactor for 4-12h at 20-30 ℃; preferably 6 to 12 hours, more preferably 8 to 12 hours;
(3) And carrying out a subsequent oxygen bleaching process.
Specifically, the oxygen bleaching process is as follows:
washing with hot water at 60-80deg.C for 2-3 times, washing with water at normal temperature for 1-2 times, oxygen bleaching with oxygen bleaching liquid, washing with hot water at 60-80deg.C for 2-3 times again, and oven drying;
preferably, the oxygen bleaching solution comprises the following components: 10g/L oxygen bleaching auxiliary, 15g/L hydrogen peroxide, 8g/L chelating agent and 2g/L penetrating agent, the liquid carrying rate is 100%, and the mixture is steamed for 30min under saturated steam at 100 ℃.
The invention has the beneficial effects that
Most of cotton cloth slurry is mainly starch slurry, so the main key enzyme in biological enzyme desizing is amylase. The cleavable polysaccharide monooxygenase disclosed by the invention firstly acts on the C1 or C4 glycosidic bond of starch, so that after the starch slurry is in a loose state, the amylase is easier to continuously and rapidly degrade the starch, namely the monooxygenase which can be expressed and has higher enzyme activity is excavated, and an unexpected effect is obtained when the monooxygenase disclosed by the invention is applied to a cold-stacking process. Therefore, the cleavable polysaccharide monooxygenase promotes the enzyme activity of amylase, effectively improves the starch decomposition efficiency and reduces the cost. The addition of the cleavable polysaccharide monooxygenase promotes the improvement of the degradation efficiency of the starch slurry, thereby shortening the action time of the amylase on the starch slurry in the cotton fabric. Therefore, the cold reactor treatment time can be shortened under the condition that the reaction temperature is as low as 20-30 ℃, the limit of the cold reactor technology on the environmental temperature in the process of degrading the slurry by amylase is further broken through, and the method is favorable for wide application and popularization. Specifically, with the addition of the schizolysis polysaccharide monooxygenase in the biological enzyme method cold-stacking process, the cold-stacking time in the prior art can be reduced from 6-12h to 6-10h, the treatment time of the cold-stacking process is shortened, and the production efficiency is improved.
Drawings
Fig. 1: SDS-PAGE after AILPMO expression. Wherein M: marker 1: AILPMO 2: control group.
Fig. 2: the effect of AILPMO on the synergistic degradation of substrates with amylase.
Detailed Description
EXAMPLE 1 sequence mining of the lytic polysaccharide monooxygenase
And carrying out bioinformatics induction analysis on the existing members of the AA13 family recorded in the CAZy database, and determining the sequence and structural characteristics of the family. Secondly, constructing a technical route for mining potential members of the AA13 family from NCBI massive sequence data of a public database based on a hidden Markov model HMMER (http:// www.hmmer.org /) according to a bioinformatics analysis result. Finally, candidate expression members that match the AA13 family characteristics were screened from similar sequences obtained by mining by domain annotation (Batch CD-Search: https:// www.ncbi.nlm.nih.gov/cdd /) and homology modeling (SWISS-MODEL: https:// swissmodel. Expasy/interactive).
EXAMPLE 2 lytic polysaccharide monooxygenase expression
(1) The bioinformatics analysis was mined to obtain a new starch derived from strain Aspergillus lentulus
The cleaved polysaccharide monooxygenase sequence of the active LPMO (gene sequence number GAQ05913.1, the encoded amino acid sequence of which is shown as SEQ ID NO: 1) is inserted into the multiple cloning site of the PET32a vectorEcoRI andNotbetween I, E.coli BL21 (trxB) was transformed for expression, and the expressed protein was designated AILPMO.
(2) Induction of expression: the activated bacteria liquid was aspirated from the corresponding tubes according to 1% inoculum size and added to baffle flasks that had been filled with 800 mL TB liquid medium containing both ampicillin and kana resistances, and the baffle flasks were placed on a shaker at 37 ℃, at 220 rpm, for cultivation at about 2h. Then 1 ml bacterial liquid is sucked from the conical flask to measure the OD value, when the OD600nm is between 0.6 and 0.8, the IPTG inducer with the final concentration of 0.2 mM is immediately added into the bacterial liquid, the temperature is 16 ℃, the rpm is 220, and 24 h expression is induced, and then the bacterial liquid is collected. The ultrasonication and centrifugation were performed to obtain 10ul SDS-PAGE for verification, and PET32a empty vector was used as an unconditioned group, as shown in FIG. 1.
Protein purification using AKTA purifier 10 (GE) protein purifier, selection of HisTrapTMHP
(GE) purification by affinity chromatography, purification to AILPMO, freeze drying and subsequent testing.
Example 3 determination of the enzymatic Activity of starch Activity-cleaving polysaccharide monooxygenase and synergistic degradation of substrate Effect with alpha-amylase
Method for detecting oxidation of starch active schizose polysaccharide monooxygenase crude enzyme liquid by AmplexTM Ultra Red method
Activity. The experimental specific operation steps are as follows:
(1) Adding crude enzyme solution of starch active schizolysis polysaccharide monooxygenase into the mixed solution, and measuring the activity system
Armoracia horse radish peroxidase 3.75U/ml, ascorbate 10 -5 M, amplexTM Ultra Red (10-acetyl-3, 7-dihydroxyphenoxazine) 25. Mu.M, 20 mmol/L sodium phosphate buffer was adjusted to pH 6.0.
(2) The excitation wavelength of the detector was set at 560 nm, the emission wavelength at 590 nm, the detection temperature at 30 c,
the detection time was 30min, and starch was reflected in fluorescence enhancement rate (Slope, rfu/s) by continuous measurement
Oxidative activity of active lytic polysaccharide monooxygenases. In general, the higher the oxidative activity of the enzyme protein, the fluorescence
The greater the enhancement rate, the positive correlation, the measured AILPMO enzyme activity was 5X 10 5 rfu/s。
The pH of the buffer was set to 5.0 with 2% soluble starch as substrate, the temperature of the metal bath was set
The temperature was 50 ℃. The experimental group was first charged with 0.3mg of the cleaved polysaccharide monooxygenase obtained by purification in example 2 and 10 -5 M ascorbate solution was incubated with soluble starch in a metal bath 1 h. After the incubation was completed, 100. Mu.L of 5-fold diluted alpha-amylase was added to the reaction system, and the reaction was allowed to proceed in a metal bath for 5 minutes, immediately after which a color reaction was performed with dilute hydrochloric acid-iodine solution, and the absorbance at OD660nm was measured. The control group was not added with any starch-activated cleavage polysaccharide monooxygenase, only 100. Mu.L of 5-fold diluted alpha-amylase was added, and the reaction was carried out in a metal bath for 5 minutes, and the chromogenic reaction was carried out in the same way and the absorbance value was measured. The percentage of the soluble starch converted by two groups of experiments is calculated according to the enzyme activity of the alpha-amylase, and the effect of the starch activity to crack the polysaccharide monooxygenase and the alpha-amylase to cooperatively degrade the starch substrate is estimated. The starch substrate conversion rate is shown in figure 2 of the specification, and the result shows that: AILPMO can improve amylase degradationEfficiency of the substrate: namely, under the condition of the same substrate concentration, the reaction time is 5min, and the starch degradation rate is 86.5% in the control group without AILPMO; in the experimental group with AILPMO, the starch degradation rate increased to 91.4%. The results show that AILPMO expressed by the excavation has the function of promoting the degradation efficiency of amylase.
Example 4 biological enzyme Complex in Cold pile Process and application Effect comparison in textile Cold pile Process
The biological compound enzyme liquid comprises the following components:
AILPMO 1mg/L-10mg/L was obtained in example 2.
Alpha-amylase 800-2000U/ml
Cellulase 800-1000U/ml
Xylanase 8000-12000U/ml
Pectase 400-1200U/ml
2-8g/L of calcium chloride
Preservative 5-15g/L
50-100g/L of surfactant
Finally adding water to fix the volume to 1L.
1. Preliminary comparison of application Effect
(1) Padding biological compound enzyme liquid: padding all-cotton grey cloth (specification 20 multiplied by 16) in biological compound enzyme liquid at 40 ℃, wherein the liquid carrying rate is 100% after one padding; wherein the biological compound enzyme liquid comprises the following components in percentage by weight: 800U/mL of alpha-amylase, 900U/mL of cellulase, 10000U/mL of xylanase, 800U/mL of pectase, 2g/L of calcium chloride and 80g/L JFC of surfactant, and 0, 1mg, 3mg, 5mg, 8mg, 10mg and pH of AILPMO purified enzyme solution prepared in example 2 are respectively added without changing the above components; adding water to constant volume to 1L.
(2) Cold stacking for 2h at 30 ℃.
(3) 2 passes were rinsed with hot water at 70 c, 1 pass with cold water (i.e., warm water), and then oxygen rinsed with an oxygen rinse solution comprising the following components: 10g/L oxygen bleaching auxiliary, 15g/L hydrogen peroxide, 8g/L chelating agent and 2g/L penetrating agent, wherein the liquid carrying rate is 100%, steaming for 30min under saturated steam at 100 ℃, washing for 2 times with hot water at 70 ℃ again, and drying. The desizing rate of the cotton cloth pretreated by the method is measured by iodine solution, and the desizing rate is considered to be good when the cotton cloth is treated by adopting a TEGEWA standard color card in Germany, wherein the color card is divided into 9 grades, the 1 grade is the worst, and the 9 grades are the best, and generally reaches more than 7 grades. The results are shown in Table 1.
| AILPMO addition mg/L | 0 | 1 | 3 | 5 | 8 | 10 |
| Desizing rate | Level 1 | Level 1 | Level 2 | 3 grade | Grade 4 | Grade 4 |
It shows that the AILPMO can reach grade 4 when the AILPMO is added in an amount of 8mg/L under the condition of cooling to a cold reactor at 30 ℃ for 2 hours. The subsequent trial AILPMO addition was considered to increase the cold pack time appropriately at lower levels.
2. Application test one
(1) Padding biological compound enzyme liquid: padding all-cotton grey cloth (specification 20 multiplied by 16) into the biological compound enzyme liquid at 40 ℃, wherein the liquid carrying rate is 100% after one padding; wherein the biological compound enzyme liquid comprises the following components in percentage by weight: 1.0mg of the prepared schizochy polysaccharide monooxygenase, 1200U/mL of alpha-amylase, 900U/mL of cellulase, 10000U/mL of xylanase, 800U/mL of pectase, 2g/L of calcium chloride, 10g/L of preservative and 80g/L of surfactant, and the pH value is 6.0-7.0;
(2) Cold stacking for 6 hours at 30 ℃;
(3) The desizing rate reaches 9 stages as before in the oxygen bleaching process.
It can be seen that the desizing rate reaches 9 levels after 6 hours under the cold pile condition of 30 ℃.
3. Application test II
(1) Padding biological compound enzyme liquid: padding all-cotton grey cloth (specification 20 multiplied by 16) into the biological compound enzyme liquid at 40 ℃, wherein the liquid carrying rate is 100% after one padding; wherein the biological compound enzyme liquid comprises the following components in percentage by weight: 1.5mg of a schizolysis polysaccharide monooxygenase, 1200U/mL of alpha-amylase, 900U/mL of cellulase, 10000U/mL of xylanase, 800U/mL of pectase, 2g/L of calcium chloride, 80g/L JFC surfactant, and the pH value is 6.0-7.0;
(2) Cold stacking for 8 hours at 25 ℃;
(3) The desizing rate reaches 9 stages as before in the oxygen bleaching process.
Compared with the second test, when the AILPMO addition amount is properly increased (namely 1.5 mg), the cold reactor temperature can be reduced to 25 ℃, and the desizing rate can reach the effect of 9 stages only by 8 hours.
4. Application test three
(1) Padding biological compound enzyme liquid: padding the all-cotton grey cloth (specification 20 multiplied by 16) at 40 ℃ into the biological composite
In enzyme liquid, the liquid carrying rate is 100% after soaking and rolling; wherein the biological compound enzyme liquid comprises the following components in percentage by weight: 2.0mg of a schizolycra polysaccharide monooxygenase, 1200U/mL of alpha-amylase, 900U/mL of cellulase, 10000U/mL of xylanase, 800U/mL of pectase, 2g/L of calcium chloride, 80g/L of JFC surfactant, and the pH value is 6.0-7.0;
(2) Cold stacking for 10 hours at 20 ℃;
3) The desizing rate reaches 9 stages as before in the oxygen bleaching process.
Compared with the second or third test, when the AILPMO addition amount is properly increased (namely 2 mg), the cold reactor temperature can be reduced to 20 ℃, and the desizing rate can reach the effect of 9 stages only by 10 hours.
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> a lytic polysaccharide monooxygenase and uses thereof
<160> 1
<170>PatentIn version 3.5
<210> 1
<211> 251
<212> PRT
<213> Aspergillus sericus (Aspergillus lentulus)
<400> 1
MKAFTTLTVLTLTTCVAGHGYLYIPSSRTRLGNEAGVDSCPECTILEPVSSWPNLDSAPVSRSGPCGYNARDSIDYNQPTSNWGTKPVATYTAGQEVEVQWCVDHNGDHGGMFSYRICQDQSIVDKLLDSSYLPTQAEKQAAEDCFEAGLLPCTDVNGQECGYSPDCAQGQACWRNDWFTCNGFQAAERPKCQGVDNAPLNSCYTSIAGGYTVTKKVKIPDYVSNHTLLSFKWNSFQTGQIYLSCADISIS 251
Claims (4)
1. A method for desizing an all-cotton gray fabric, which is characterized by comprising the step of padding the all-cotton gray fabric by using a biological enzyme compound preparation used in a textile cold-pad-batch process, wherein the biological enzyme compound preparation used in the textile cold-pad-batch process comprises a cleaved polysaccharide monooxygenase, alpha-amylase, cellulase, xylanase, pectinase, calcium chloride, preservative and surfactant, and the amino acid sequence of the cleaved polysaccharide monooxygenase is shown as SEQ ID NO:1 is shown in the specification;
the biological enzyme compound preparation used in the textile cold-stacking process comprises the following components in parts by weight:
1mg/L to 10mg/L of lycan polysaccharide monooxygenase
Alpha-amylase 800-2000U/ml
Cellulase 800-1000U/ml
Xylanase 8000-12000U/ml
Pectase 400-1200U/ml
2-8g/L of calcium chloride
Preservative 5-15g/L
50-100g/L of surfactant
Finally adding water to fix the volume to 1L;
pH 6.0～7.0。
2. the method of claim 1, comprising the steps of
(1) Padding the all-cotton grey cloth in the biological enzyme compound preparation used in the spinning cold-stacking process at the temperature of 30-50 ℃;
(2) Cooling the reactor for 4-12h at 20-30 ℃;
(3) And carrying out a subsequent oxygen bleaching process.
3. The method according to claim 2, characterized in that the oxygen bleaching process is in particular as follows: washing with hot water at 60-80deg.C for 2-3 times, washing with water at normal temperature for 1-2 times, oxygen bleaching with oxygen bleaching liquid, washing with hot water at 60-80deg.C for 2-3 times again, and oven drying.
4. A method according to claim 3, wherein the oxygen bleach liquor comprises the following components: 10g/L oxygen bleaching auxiliary, 15g/L hydrogen peroxide, 8g/L chelating agent and 2g/L penetrating agent, the liquid carrying rate is 100%, and the mixture is steamed for 30min under saturated steam at 100 ℃.
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