CN113046248A - Penicillium citrinum XZH-16 and application thereof - Google Patents
Penicillium citrinum XZH-16 and application thereof Download PDFInfo
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Abstract
The invention discloses penicillium citrinum XZH-16 and application thereof. The penicillium citrinum (Penicillium citrinum) XZH-16 strain provided by the invention can secrete high lytic polysaccharide monooxygenase and cellulase, wherein the highest LPMO enzyme activity can reach 15.22U/g substrate. The cellulase and LPMO enzyme combined preparation produced by penicillium citrinum XZH-16 is applied to the enzymolysis of forest wood cellulose, and the enzymolysis efficiency is improved by 14.55 percent. The cellulase and LPMO enzyme combined preparation is an oxidizing hydrolase with the function of synergistically degrading cellulose, can break glycosidic bonds of cellulose through oxidation, and promotes combination of cellulase and a fiber substrate to improve enzymolysis efficiency, so that the enzyme dosage in the enzymolysis process is effectively reduced. Can further reduce the production cost of the cellulosic ethanol and accelerate the industrialization process of the cellulosic ethanol.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to Penicillium citrinum (Penicillium citrinum) XZH-16 and application thereof.
Background
Imbalance of energy supply and increasingly severe ecological environment destruction become potential obstacles for social and economic development. The development and utilization of clean renewable energy sources has become a focus of global attention. The biomass is a green renewable resource with great development potential and has the characteristic of carbon neutralization, and the preparation of the alcohol fuel by taking the biomass as a raw material is an important direction for future development. However, how to saccharify lignocellulose biomass with high efficiency, environmental protection and low cost is the key point for preparing the cellulose alcohol fuel.
Cellulases are not single component enzymes, but a collection of multi-component enzyme systems. The method mainly comprises the following steps: endoglucanases (EG, EC 3.2.1.4), exoglucanases (CBH, EC 3.2.1.91) and β -glucosidases (BGL, EC 3.2.1.21). The synergistic action of at least these 3 enzymes is required for efficient degradation of cellulose. However, the enzymatic efficiency of cellulase on pretreated fiber substrates still needs to be further improved.
The invention content is as follows:
the first purpose of the invention is to provide a Penicillium citrinum (Penicillium citrinum) XZH-16 strain for producing cellulase and lytic polysaccharide monooxygenase, which is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 13 months in 2021, with the address: the preservation number of the institute of microbiology of the Chinese academy of sciences, West Lu No.1, Beijing, Chaoyang, Beijing, and Beicheng district, is: CGMCC No. 21426.
The Penicillium citrinum (Penicillium citrinum) XZH-16 is separated and screened from rotten dead branches in the ecological environment. The taxonomy of the fungi shows that: the strain XZH-16 is cultured on a PDA culture medium at 30 ℃ for 3 days, and is a white nearly circular colony, is petal-shaped, has a slightly velvet surface and relatively neat edge, and is easy to pick (as shown in figure 1); the scanning electron microscope of the mycelium of the strain is shown in FIG. 2, and the mycelium is in a compact long strip shape and is intertwined with each other. According to the morphological structure characteristics of the bacterial colony, the strain XZH-16 can be preliminarily determined to be penicillium citrinum by comparing and analyzing according to the fungal identification handbook and the Chinese funguses.
The taxonomic status of Penicillium citrinum (Penicillium citrinum) XZH-16 of the present invention is determined according to the following method:
extracting total DNA of strain XZH-16 by conventional method, amplifying ITS region rDNA sequence of strain, the sequence is shown as SEQ ID NO.3, BLAST comparing and analyzing the sequence with known sequence in GenBank database, obtaining ITS sequence of related species from database, and constructing phylogenetic tree, as shown in FIG. 3. Through comparative analysis, the strain XZH-16 belongs to the genus Penicillium, is named as Penicillium citrinum (Penicillium citrinum) XZH-16, and is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 13 months in 2021, with the address: the preservation number of the institute of microbiology of the Chinese academy of sciences, West Lu No.1, Beijing, Chaoyang, Beijing, and Beicheng district, is: CGMCC No. 21426.
The second purpose of the invention is to provide the application of the Penicillium citrinum (Penicillium citrinum) XZH-16 in the production of cellulase and/or Lytic Polysaccharide Monooxygenase (LPMO).
According to the determination reaction of LPMOs specific substrates, the Penicillium citrinum XZH-16 can produce higher LPMO, the highest LPMO enzyme activity of an enzyme composition preparation prepared by solid-state fermentation of Penicillium citrinum (Penicillium citrinum) XZH-16 reaches 15.22U/g substrate (LPMO enzyme activity unit is defined in the way that each milliliter of enzyme liquid catalyzes 1 mu mol of hydrogen peroxide to degrade per minute in a reaction system and is defined as one enzyme activity unit), and compared with the currently reported orange Thermoascus aurantiacus which produces LPMO, the LPMO enzyme activity is higher. Therefore, the enzyme combination preparation produced by the Penicillium citrinum (Penicillium citrinum) XZH-16 has higher LPMO enzyme activity, and is a novel high-yield enzyme combination preparation.
The third purpose of the invention is to provide the application of the Penicillium citrinum (Penicillium citrinum) XZH-16 in cellulose hydrolysis.
The enzyme combination preparation prepared from Penicillium citrinum (Penicillium citrinum) XZH-16 can be applied to cellulose hydrolysis, in particular to hydrolysis of forest cellulose. The method comprises the steps of crushing poplar, adding commercial cellulase, and then adding an enzyme combination preparation produced by Penicillium citrinum (Penicillium citrinum) XZH-16 for enzymolysis, so that a highly-crystallized poplar substrate structure tends to be loose, the cellulase is more easily combined with the substrate, and the hydrolysis efficiency of the cellulose is finally improved in a synergistic manner.
The fourth purpose of the invention is to provide the application of the Penicillium citrinum (Penicillium citrinum) XZH-16 in enzymolysis saccharification of lignocellulose raw materials.
The fifth purpose of the invention is to provide the application of the Penicillium citrinum (Penicillium citrinum) XZH-16 in the production of enzyme combination preparations with synergistic cellulose degradation.
The sixth object of the present invention is to provide a method for preparing an enzyme composition preparation with synergistic degradation of cellulose, comprising the steps of: preparing solid fermentation product of Penicillium citrinum (Penicillium citrinum) XZH-16, and adding Na2HPO4And soaking the solid fermentation product in a citric acid buffer solution, collecting the liquid to obtain fermentation enzyme liquid, centrifuging, and taking the supernatant to obtain the enzyme composition preparation with the synergistic degradation of cellulose.
The solid-state fermentation product of Penicillium citrinum (Penicillium citrinum) XZH-16 is prepared by the following method: inoculating activated Penicillium citrinum (Penicillium citrinum) XZH-16 into seed culture medium, culturing at 30 deg.C and 150rpm for 3d to obtain seed culture solution; inoculating the seed culture solution into a solid fermentation culture medium, and fermenting for 5d at 30 ℃ to obtain a solid fermentation product; the seed culture medium is prepared by the following method per liter: weighing 200g peeled potato, boiling in water, filtering to obtain potato extract, and adding glucose20g, adding water after dissolving to fix the volume to 1L; the solid state fermentation culture medium comprises rice straws and nutrient solution, wherein the addition amount of the nutrient solution is 10.5mL of nutrient solution added to every 5g of rice straws; the nutrient salt solution comprises the following components: (NH) per liter of nutrient salt solution4)2SO4 10.0g，KH2PO4 4.0g，MgSO4·7H2O 0.5g，CaCl2·2H20.5g of O and the balance of water.
Preferably, the said is with Na2HPO4Soaking the solid fermentation product in a citric acid buffer solution, collecting a liquid to obtain a fermentation enzyme solution, centrifuging, and taking a supernatant to obtain the enzyme composition preparation with the synergistic degradation of cellulose, wherein the enzyme composition preparation comprises the following components: using pH4.8 Na2HPO4Soaking the solid fermentation product in a citric acid buffer solution, shaking at 30 ℃ and 150rpm for 1h, collecting the liquid, namely fermentation enzyme liquid, centrifuging at 8000rpm and 4 ℃ for 5min, and collecting the supernatant, namely the enzyme composition preparation with the synergistic degradation of cellulose.
The seventh purpose of the invention is to provide the enzyme combination preparation with the synergistic degradation of cellulose prepared by the preparation method.
The eighth purpose of the invention is to provide the application of the enzyme combination preparation with the synergistic cellulose degradation function in cellulose hydrolysis. The Penicillium citrinum XZH-16 screened by the invention has the advantages that the LPMO enzyme activity of an enzyme composition preparation generated by the Penicillium citrinum XZH-16 reaches up to 15.22U/g of substrate, the filter paper enzyme activity of the enzyme composition preparation is 0.75IU/g of substrate, the enzyme composition preparation is an oxidizing hydrolase with the function of synergistically degrading cellulose, the enzyme composition preparation can break the glycosidic bond of the cellulose through oxidation, and the combination of the cellulose and the fiber substrate is promoted to improve the enzymolysis efficiency, so that the enzyme adding amount in the enzymolysis process is effectively reduced.
The Penicillium citrinum (Penicillium citrinum) XZH-16 is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 13 months in 2021, and the address is as follows: the preservation number of the institute of microbiology of the Chinese academy of sciences, West Lu No.1, Beijing, Chaoyang, Beijing, and Beicheng district, is: CGMCC No. 21426.
Drawings
FIG. 1 is a morphological diagram of a colony on the PDA medium of Penicillium citrinum (Penicillium citrinum) XZH-16 of the present invention.
FIG. 2 is an electron microscope scanning image of Penicillium citrinum (Penicillium citrinum) XZH-16 of the present invention, wherein the scale bar for A is 10 μm and the scale bar for B is 5 μm.
FIG. 3 is an 18S rDNA phylogenetic tree of Penicillium citrinum (Penicillium citrinum) XZH-16 of the present invention.
FIG. 4 shows the cellulase and LPMOs enzyme activities of the enzyme combination preparation produced by Penicillium citrinum XZH-16 of the present invention, wherein T2 is the control strain Thermoascus aurantiacus T2(Thermoascus aurantiacas).
FIG. 5 shows the effect of adding the enzyme combination preparation of Penicillium citrinum (Penicillium citrinum) XZH-16 of the present invention on the enzymatic hydrolysis of lignocellulose, wherein T2 is Thermoascus aurantiacus T2(Thermoascus aurantiacas).
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: screening of strains
Harvesting was from highly rotten dead shoots. Placing the collected dead branch sample in a conical flask, placing a plurality of glass beads, adding a proper amount of normal saline, and then placing on a 150rpm shaking table for 1h to shake and mix uniformly; transferring 2mL of the mixed solution into 100mL of enrichment culture solution, and then placing the enrichment culture solution on a shaking table with the temperature of 30 ℃ and the rpm of 150 for shaking culture for 3d to obtain an enrichment culture, wherein the formula of the enrichment culture solution is as follows: NaCl 1.0g, K2HPO4 1.5g，KH2PO4 0.5g，NH4NO3 1.0g，MgSO4·7H20.2g of O, 10.0g of carboxymethyl cellulose (CMC), 2.5g of peptone, 0.5g of yeast extract and 1000mL of distilled water. Directly coating the enrichment culture on a Congo red primary screening culture medium, observing and culturing at 30 ℃ for 2-5 days, wherein the formula of the Congo red primary screening culture medium is as follows: KH is contained in each liter of culture medium2PO4 0.5g，(NH4)2SO4 2.0g，MgSO4·7H20.25g of O, 2.0g of CMC, 0.2g of Congo red, 20g of agar and the balance of water, and the pH value is natural. Picking single colony with larger hydrolytic transparent ringAnd (4) streaking and purifying on a PDA culture medium, and storing the purified strain on a PDA slant.
The bacterial strain is used for producing cellulase and LPMOs activity re-screening: respectively inoculating the purified strains stored on the PDA slant into an equivalent re-screening solid fermentation culture medium, culturing at 30 ℃ and 120rpm for 5d, performing solid culture fermentation on the strains, centrifuging, and taking supernatant to obtain crude enzyme solution. The double-sieving solid fermentation medium consists of 5g of rice straw and 10.5mL of nutrient solution, and the nutrient solution comprises the following components: (NH) per liter of nutrient salt solution4)2SO4 10.0g，KH2PO4 4.0g，MgSO4·7H2O 0.5g，CaCl2·2H2O0.5g and the balance of water. The FPA and LPMOs enzyme activities of the crude enzyme solution produced by the strain are respectively measured by a standard filter paper enzyme activity (FPA) method and an Amplex Red-horseradish peroxidase method. The specific method for measuring by the FPA method comprises the following steps: 15mL of a tube with a stopper were added with 0.25mL of the crude enzyme solution and 1.75mL of pH4.8 Na2HPO4Citric acid buffer solution, 1 × 6cm Xinhua medium speed quantitative filter paper strip (about 50mg), mixing well, putting into 50 deg.C water bath to react for 60 min; then adding 2mL of DNS reagent, and boiling for 10min in a boiling water bath; after cooling, distilled water is used for fixing the volume to 15 mL; after constant volume, the OD value was measured at 550 nm. The crude enzyme solution in the blank is replaced by boiling inactivated crude enzyme solution. After the OD value is measured, the standard curve is checked to calculate the content of glucose in the solution. Definition of enzyme activity unit: under the above measurement conditions, the amount of enzyme required for hydrolysis to produce 1. mu. mol of substrate (glucose 180) per minute per one mL of crude enzyme solution was one enzyme activity unit IU. The specific method of the Amplex Red-horseradish peroxidase method comprises the following steps: reference to
The specification of Red Hydrogen Peroxide/Peroxidase Assay Kit, 50. mu.L of Red reagent is prepared, 20. mu.L of crude enzyme solution and 30. mu.L of 1 × Reaction buffer are added to a Reaction system of 100. mu.L, absorbance data is obtained at the wavelength of 530nm by using an enzyme labeling instrument (EON, BioTek), and after incubation for 30min, the absorbance data is obtained at the wavelength of 560nm by using the enzyme labeling instrument. Background signal was measured in control samples without LPMO and subtracted from the assay. After the reaction was carried out at 30 ℃ in the dark for 1 hour, the absorbance was measured at 560 nm. To do soH2O2And (3) making a standard curve for a substrate, and calculating the content of hydrogen peroxide released by the reaction (LPMO enzyme activity unit is defined in the specification, 1 mu mol of hydrogen peroxide is catalyzed and degraded per minute in each milliliter of enzyme liquid in a reaction system, and the LPMO enzyme activity unit is defined as an enzyme activity unit U). And finally, selecting a strain with higher filter paper enzyme activity and LPMOs, and storing the strain by using a PDA inclined plane.
Screening to obtain 1 strain XZH-16 with high LPMO activity. Extracting total DNA of the strain by adopting an Ezup column type fungus genome DNA extraction kit B518259, selecting a universal primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3' shown as SEQ ID NO. 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3' shown as SEQ ID NO. 2) for amplifying a fungus 18S rDNA sequence, and amplifying an ITS region rDNA sequence of the strain XZH-16 total DNA. 10 XTaq Buffer (with MgCl) was included in a 50. mu.L PCR reaction2) mu.L of dNTP (mix) (10mmol/L each) 2. mu.L, 10. mu.M primer ITS 12. mu.L, 10. mu.M primer ITS 42. mu.L, 0.5. mu.L of Tag enzyme (5U/. mu.L), 1. mu.L of 20 ng/. mu.L of template DNA, and the remaining volume was made up with sterile ultrapure water. The gradient denaturation PCR amplification conditions were: pre-denaturation at 95 ℃ for 4min, denaturation at 94 ℃ for 30sec, annealing at 55-60 ℃ for 30sec, extension at 72 ℃ for 50sec, 35 cycles, and extension at 72 ℃ for 10 min. The PCR amplification product was sequenced by Biotechnology engineering (Shanghai) Inc., and the sequence was shown as SEQ ID NO.1, then BLAST comparative analysis was performed on the sequence and known sequences in GenBank database, and ITS region rDNA sequence of related species was obtained from the database to construct phylogenetic tree, as shown in FIG. 3. Through comparative analysis, the strain XZH-16 belongs to the genus Penicillium, is named as Penicillium citrinum (Penicillium citrinum) XZH-16, and is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 13 months in 2021, with the address: the preservation number of the institute of microbiology of the Chinese academy of sciences, West Lu No.1, Beijing, Chaoyang, Beijing, and Beicheng district, is: CGMCC No. 21426.
Example 2: preparation of enzyme composition preparation and enzyme activity determination thereof
1. Preparation of enzyme combination preparation: selecting Penicillium citrinum (Penicillium citrinum) XZH-16 block stored on PDA slant, inoculating into 50mL PDA liquid culture medium, culturing at 30 deg.C and 150rpm for 3d, and activatingicillium citrinum) XZH-16 was inoculated into 50mL of seed medium at an inoculum size of 50. mu.L, and cultured at 30 ℃ and 150rpm for 3 days to obtain a seed culture solution. The seed culture medium contains 200.0g of potato extract, 20.0g of glucose and the balance of water per liter of culture medium; weighing 200.0g peeled potato, adding appropriate amount of water, boiling for 30min, filtering with gauze to obtain filtrate, i.e. potato extract, adding 20.0g glucose, dissolving, adding water to desired volume of 1L, and sterilizing at 115 deg.C for 30 min. Inoculating the seed culture solution into a solid fermentation culture medium in an inoculation amount of 2mL, and fermenting at 30 ℃ for 5d to obtain a solid fermentation product. The solid fermentation medium consists of 5g of straw and 10.5mL of nutrient solution, and the nutrient solution comprises the following components: (NH) per liter of nutrient salt solution4)2SO4 10.0g，KH2PO4 4.0g，MgSO4·7H2O 0.5g，CaCl2·2H20.5g of O and the balance of water. Then 50mL of pH4.8 Na was used2HPO4Soaking the solid fermentation product in citric acid buffer solution, shaking at 30 deg.C and 150rpm for 1h, collecting liquid as fermentation enzyme solution, freeze-centrifuging at 8000rpm and 4 deg.C for 5min, collecting supernatant as enzyme composition preparation (cellulase and LPMO enzyme composition preparation), and storing at 4 deg.C for use.
2. Filter paper enzyme activity (FPA) assay: 15mL of test tube with plug were added 0.25mL cellulase and LPMO enzyme combination preparation, 1.75mL pH4.8 Na2HPO4Citric acid buffer solution, 1 × 6cm Xinhua medium speed quantitative filter paper strip (about 50mg), mixing well, putting into 50 deg.C water bath to react for 60 min; then adding 2mL of DNS reagent, and boiling for 10min in a boiling water bath; after cooling, distilled water is used for fixing the volume to 15 mL; after constant volume, the OD value was measured at 550 nm. The cellulase and LPMO enzyme combination preparations in the blank were replaced with a boiling inactivated cellulase and LPMO enzyme combination preparation. After the OD value is measured, the standard curve is checked to calculate the content of glucose in the solution. Definition of enzyme activity unit: under the above assay conditions, the amount of enzyme required for hydrolysis to produce 1. mu. mol of substrate (glucose 180) per minute per l mL of cellulase and LPMO enzyme combination preparation is one enzyme activity unit IU. Through determination, the filter paper enzyme activity of the cellulase and LPMO enzyme combined preparation produced by Penicillium citrinum (Penicillium citrinum) XZH-16 obtained in the step 1 reaches 0.75IU/g substrate.
LPMO enzyme activity determination: reference to
The specification of Red Hydrogen Peroxide/Peroxidase Assay Kit, 50. mu.L of Red reagent is prepared, 20. mu.L of cellulase and LPMO enzyme combined preparation and 1 × Reaction buffer 30. mu.L are added, a total of 100. mu.L of Reaction system is obtained, absorbance data is obtained at the wavelength of 530nm by using an enzyme labeling instrument (EON, BioTek), and after incubation for 30min, the absorbance data is obtained at the wavelength of 560nm by using the enzyme labeling instrument. Background signal was measured in control samples without LPMO and subtracted from the assay. After the reaction was carried out at 30 ℃ in the dark for 1 hour, the absorbance was measured at 560 nm. To do H2O2And (3) making a standard curve for a substrate, and calculating the content of hydrogen peroxide released by the reaction (LPMO enzyme activity unit is defined in the specification, 1 mu mol of hydrogen peroxide is catalyzed and degraded per minute in each milliliter of enzyme liquid in a reaction system, and the LPMO enzyme activity unit is defined as an enzyme activity unit U). Through determination, the LPMO enzyme activity of the cellulase and LPMO enzyme combined preparation produced by Penicillium citrinum XZH-16 obtained in the step 1 reaches 15.22U/g substrate, and the enzyme activity is higher.
The result shows that the Penicillium citrinum (Penicillium citrinum) XZH-16 of the invention can produce LPMO with high yield, the highest enzyme activity of LPMO of the cellulase and LPMO enzyme combined preparation prepared by solid-state fermentation of the Penicillium citrinum (Penicillium citrinum) XZH-16 reaches 15.22U/g substrate, and the filter paper enzyme activity of the cellulase and LPMO enzyme combined preparation is 0.75IU/g substrate (see figure 4). The LPMO enzyme activity of the cellulase and LPMO enzyme combination preparation prepared by the same solid-state fermentation mode in the step 1 of Thermoascus aurantiacus (Thermoascus aurantiacus) producing LPMO currently reported is 12.5U/g substrate, and the filter paper enzyme activity is only 0.66U/g substrate (see figure 4). Therefore, the cellulase and LPMO enzyme combined preparation prepared from the Penicillium citrinum (Penicillium citrinum) XZH-16 has higher enzyme activity compared with the cellulase and LPMO enzyme combined preparation prepared from the orange Thermoascus aurantiacus (Thermoascus aurantiaca) reported in the prior art, and the Penicillium citrinum (Penicillium citrinum) XZH-16 is a novel high-yield cellulase and LPMO strain.
Example 3: application of cellulase and LPMO enzyme combined preparation in poplar cellulose enzymolysis
1. Reaction substrate: pulverizing poplar powder to 60 mesh, oven drying at 105 deg.C to constant weight, weighing 2.5g poplar powder, mixing with 2.5g solid acid (see example 3 of patent ZL 201110126178.7), and pretreating at 170 deg.C for 30min to obtain reaction substrate.
2. Weighing 0.5g of the reaction substrate obtained in the step 1, adding 10FPU/g of commercial cellulase into the reaction substrate, and adding 25mg of the cellulase and LPMO enzyme combined preparation prepared in the step 1 in the embodiment 2 to obtain an enzymolysis reaction system; hydrolyzing at 50 ℃ for 72 h. And detecting the content of reducing sugar by HPLC.
The effect of the cellulase and LPMO enzyme combination preparation produced by Penicillium citrinum (Penicillium citrinum) XZH-16 strain on poplar cellulose enzymolysis was studied by comparing the cellulase and LPMO enzyme combination preparation produced by Thermoascus aurantiacus T2(Thermoascus aurantiacus) fermented in the same solid state fermentation manner as in step 1 of example 2 with commercial cellulase under the combined action of the enzymolysis reaction system and reaction conditions of step 2 without adding the cellulase and LPMO enzyme combination preparation of the present invention.
The result is shown in fig. 5, when the cellulase and LPMO enzyme combined preparation prepared by screening Penicillium citrinum (Penicillium citrinum) XZH-16 strain solid state fermentation is applied to poplar cellulose enzymolysis, compared with the cellulose and LPMO enzyme combined preparation, the enzymolysis rate is improved by 14.55%. The cellulase and LPMO enzyme combined preparation enables the highly crystallized poplar substrate structure to tend to be loose, so that cellulase is more easily combined with the substrate, the hydrolysis efficiency of cellulose is finally synergistically improved, and the cellulase and LPMO enzyme combined preparation is particularly suitable for application of forest wood cellulose degradation. The cellulase and LPMO enzyme combined preparation is an oxidizing hydrolase with the function of synergistically degrading cellulose, can break glycosidic bonds of cellulose through oxidation, and promotes combination of cellulase and a fiber substrate to improve enzymolysis efficiency, so that the enzyme dosage in the enzymolysis process is effectively reduced. Has wide development prospect for promoting the industrialization process of the cellulosic ethanol.
The above detailed description is specific to possible embodiments of the present invention, and the embodiments are not intended to limit the scope of the present invention, and all equivalent implementations or modifications that do not depart from the scope of the present invention should be included in the present invention.
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acgctcgagg accggacgcg gtgccgccgc tgcctttcgg gcccgtcccc ccggcggggg 180
ggacggggcc caacacacaa gccgggcttg agggcagcaa tgacgctcgg acaggcatgc 240
cctccggaat accagagggc gcaatgtgcg ttcaaagact cgatgattca ctgaattctg 300
caattcacat tagttatcgc atttcgctgc gttcttcatc gatgccggaa ccaagagatc 360
cgttgttgaa agttttaact aatttcgtta taggtctcag actgcaactt cagacagcgt 420
tcaggggggc cgtcggcggg cgcggggccc gccgaggcaa cataggttcg ggcaacacgg 480
gtgggaggtt gggccccgag gggcccgcac tcggtaatga tccttccgca ggttcaccta 540
cgg 543
Claims (10)
1. Penicillium citrinum (Penicillium citrinum) XZH-16 with the preservation number: CGMCC No. 21426.
2. Use of Penicillium citrinum (Penicillium citrinum) XZH-16 according to claim 1 for the production of cellulase and/or lytic polysaccharide monooxygenase.
3. Use of Penicillium citrinum (Penicillium citrinum) XZH-16 according to claim 1 for the hydrolysis of cellulose.
4. Use of Penicillium citrinum (Penicillium citrinum) XZH-16 according to claim 1 for the enzymatic saccharification of lignocellulosic feedstocks.
5. Use of Penicillium citrinum (Penicillium citrinum) XZH-16 according to claim 1 for the production of an enzyme combination preparation with synergistic cellulose degradation.
6. A method for preparing an enzyme composition preparation for synergistically degrading cellulose, which comprises the following steps: preparing solid fermentation product of Penicillium citrinum (Penicillium citrinum) XZH-16, and adding Na2HPO4And soaking the solid fermentation product in a citric acid buffer solution, collecting the liquid to obtain fermentation enzyme liquid, centrifuging, and taking the supernatant to obtain the enzyme composition preparation with the synergistic degradation of cellulose.
7. The method according to claim 6, wherein the solid fermentation product of Penicillium citrinum (Penicillium citrinum) XZH-16 is prepared by the following steps: inoculating activated Penicillium citrinum (Penicillium citrinum) XZH-16 into seed culture medium, culturing at 30 deg.C and 150rpm for 3d to obtain seed culture solution; culturing seed in culture solutionInoculating into solid fermentation medium, fermenting at 30 deg.C for 5d to obtain solid fermented product; the seed culture medium is prepared by the following method per liter: weighing 200g of peeled potatoes, adding water, boiling, filtering to obtain potato extract, adding 20g of glucose, dissolving, and adding water to a constant volume of 1L; the solid state fermentation culture medium comprises rice straws and nutrient solution, wherein the addition amount of the nutrient solution is 10.5mL of nutrient solution added to every 5g of rice straws; the nutrient salt solution comprises the following components: (NH) per liter of nutrient salt solution4)2SO4 10.0g，KH2PO4 4.0g，MgSO4·7H2O 0.5g，CaCl2·2H20.5g of O and the balance of water.
8. The method according to claim 6, wherein the Na is used2HPO4Soaking the solid fermentation product in a citric acid buffer solution, collecting a liquid to obtain a fermentation enzyme solution, centrifuging, and taking a supernatant to obtain the enzyme composition preparation with the synergistic degradation of cellulose, wherein the enzyme composition preparation comprises the following components: using pH4.8 Na2HPO4Soaking the solid fermentation product in a citric acid buffer solution, shaking at 30 ℃ and 150rpm for 1h, collecting the liquid, namely fermentation enzyme liquid, centrifuging at 8000rpm and 4 ℃ for 5min, and collecting the supernatant, namely the enzyme composition preparation with the synergistic degradation of cellulose.
9. An enzyme composition preparation having synergistic cellulose degradation, prepared by the preparation method according to any one of claims 6 to 8.
10. Use of the enzyme combination preparation with synergistic cellulose degradation according to claim 9 for the hydrolysis of cellulose.
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| CN114164124A (en) * | 2021-12-22 | 2022-03-11 | 中国科学院广州能源研究所 | Preparation method of penicillium flora compound synergistic cellulose degradation complex enzyme |
| CN114276935A (en) * | 2021-12-22 | 2022-04-05 | 中国科学院广州能源研究所 | Penicillium XZH-22 and application thereof |
| CN114657073A (en) * | 2022-03-30 | 2022-06-24 | 中溶科技股份有限公司 | Penicillium citrinum strain capable of producing cellobiase at high yield and application thereof |
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| CN114164124A (en) * | 2021-12-22 | 2022-03-11 | 中国科学院广州能源研究所 | Preparation method of penicillium flora compound synergistic cellulose degradation complex enzyme |
| CN114276935A (en) * | 2021-12-22 | 2022-04-05 | 中国科学院广州能源研究所 | Penicillium XZH-22 and application thereof |
| CN114657073A (en) * | 2022-03-30 | 2022-06-24 | 中溶科技股份有限公司 | Penicillium citrinum strain capable of producing cellobiase at high yield and application thereof |
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