CN113512501A

CN113512501A - Penicillium oxalicum XZH-2 and application thereof

Info

Publication number: CN113512501A
Application number: CN202110419980.9A
Authority: CN
Inventors: 梁翠谊; 亓伟; 王闻; 徐孜晗; 王琼; 王忠铭
Original assignee: Guangzhou Institute of Energy Conversion of CAS
Current assignee: Guangzhou Institute of Energy Conversion of CAS
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2021-10-19
Anticipated expiration: 2041-04-19
Also published as: CN113512501B

Abstract

The invention discloses penicillium oxalicum XZH-2 and application thereof. The Penicillium oxalicum (Penicillium oxalicum) XZH-2 strain provided by the invention can secrete a large amount of cellulase and lytic polysaccharide monooxygenase, wherein the lytic polysaccharide monooxygenase has the highest enzyme activity of 17.17U/g of substrate, and the filter paper enzyme activity of 7.37IU/g of substrate, and the cellulase and LPMO enzyme combined preparation produced by the Penicillium oxalicum XZH-2 is applied to enzymolysis of wood cellulose, and the enzymolysis efficiency is improved by 13.35%. 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

Penicillium oxalicum XZH-2 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to Penicillium oxalicum (Penicillium oxalicum) XZH-2 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 oxalicum (Penicillium oxalicum) XZH-2 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. 21425.

The Penicillium oxalicum (Penicillium oxalicum) XZH-2 is separated and screened from river sludge in the ecological environment. The taxonomy of the fungi shows that: the strain XZH-2 was cultured on PDA medium at 30 deg.C for 3d, as shown in FIG. 3, the initial colony was white and rice-grain-shaped, and after 2 days, the colony surface was powdery with hypha villi, and the hypha became dense, greenish and overgrown on the plate. The scanning electron microscope of mycelium and spore is shown in FIG. 1, the mycelium is slender and has flagella, the surface of the mycelium has concave-convex folds, and a large amount of mycelium is attached to the surface. According to the colony morphology and the structural characteristics of conidium, the strain XZH-2 can be preliminarily determined to be penicillium oxalicum by comparing and analyzing according to a fungal identification manual and a Chinese fungoid legend.

The taxonomic status of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 of the present invention is determined according to the following method:

extracting the total DNA of the strain XZH-2 by a conventional method, amplifying an ITS region rDNA sequence of the strain, wherein the sequence is shown as SEQ ID NO.3, carrying out BLAST comparative analysis on the sequence and known sequences in a GenBank database, obtaining ITS sequences of related species from the database, and constructing a phylogenetic tree, which is shown as a figure 2. Through comparative analysis, the strain XZH-2 belongs to the genus Penicillium, is named as Penicillium oxalicum (Penicillium oxalicum) XZH-2, 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. 21425.

The second purpose of the invention is to provide the application of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 in the production of cellulase and/or Lytic Polysaccharide Monooxygenase (LPMO).

According to the determination reaction of LPMO specific substrates, the Penicillium oxalicum (Penicillium oxalicum) XZH-2 can produce higher LPMO, the highest LPMO enzyme activity of an enzyme combination preparation prepared by solid-state fermentation of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 is as high as 17.17U/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 the filter paper enzyme activity of the enzyme combination preparation is 7.37IU/g substrate. Compared with the currently reported fermentation enzyme production of Neurospora crassa (Neurospora crassa) and Thermoascus aurantiacus (Thermoascus aurantiacus), the enzyme activity of LPMO and the enzyme activity of filter paper are higher than those of a comparison strain through the same solid state fermentation mode. Therefore, the LPMO produced by the Penicillium oxalicum (Penicillium oxalicum) XZH-2 has higher enzyme activity than that of LPMO in the existing report, and is a novel high-yield LPMO-producing strain.

The third purpose of the invention is to provide the application of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 in cellulose hydrolysis.

The enzyme combination preparation prepared by the Penicillium oxalicum (Penicillium oxalicum) XZH-2 can be applied to cellulose hydrolysis, in particular to hydrolysis of forest cellulose. After poplar is pretreated, commercial cellulase is added, and then an enzyme combination preparation produced by Penicillium oxalicum (Penicillium oxalicum) XZH-2 is added for enzymolysis, so that the highly crystallized substrate structure of the poplar 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 oxalicum (Penicillium oxalicum) XZH-2 in enzymolysis saccharification of lignocellulose raw materials.

The fifth purpose of the invention is to provide the application of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 in the production of enzyme combination preparations for synergistically degrading cellulose.

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 oxalicum (Penicillium oxalicum) XZH-2, 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 penicillium oxalicumA solid state fermentation of the bacterium (Penicillium oxalicum) XZH-2 is prepared by the following method: inoculating activated Penicillium oxalicum (Penicillium oxalicum) XZH-2 into a seed culture medium, and culturing at 30 ℃ and 150rpm for 3d to obtain a 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 of peeled potatoes, adding water, boiling, filtering by using a warp cloth 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.

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 screened Penicillium oxalicum XZH-2 can produce a novel LPMO, the highest LPMO enzyme activity of an enzyme combination preparation prepared by solid-state fermentation of the Penicillium oxalicum XZH-2 is as high as 17.17U/g of substrate (LPMO enzyme activity unit is defined in the specification that 1 mu mol of hydrogen peroxide is catalyzed and degraded per minute in each milliliter of enzyme liquid in a reaction system and is defined as one enzyme activity unit), the filter paper enzyme activity of the enzyme combination preparation is as high as 7.37IU/g of substrate, the enzyme combination preparation is an oxidizing hydrolase with the function of synergistically degrading cellulose, and the oxidizing hydrolase can break the glycosidic bond of the cellulose through oxidation, promote the combination of the cellulose and the fiber substrate to improve the enzymolysis efficiency and effectively reduce the enzyme adding amount in the enzymolysis process.

The Penicillium oxalicum (Penicillium oxalicum) XZH-2 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. 21425.

Drawings

FIG. 1 is an electron microscope scan of Penicillium oxalicum (Penicillium oxalicum) XZH-2 mycelium and spores.

FIG. 2 is a phylogenetic tree constructed.

FIG. 3 is the colony morphology of Penicillium oxalicum (Penicillium oxalicum) XZH-2 on PDA medium.

FIG. 4 shows LPMO enzyme activity and filter paper enzyme activity of enzyme composition preparations derived from different strains, wherein XZH-2 is Penicillium oxalicum (Penicillium oxalicum) XZH-2, N1 is Neurospora crassa N1(Neurospora crassa), and T2 is Thermoascus aurantiacus T2(Thermoascus aurantiacus).

FIG. 5 shows the enzymolysis effect of the cellulase and LPMO enzyme combination preparation of the present invention in poplar cellulose enzymolysis, wherein XZH-2 is Penicillium oxalicum (Penicillium oxalicum) XZH-2, N1 is Neurospora crassa N1(Neurospora crassa), T2 is Thermoascus aurantiacus T2(Thermoascus aurantiacus).

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

Collecting the sludge from an ecological system of the river sludge. Placing the collected sludge 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; 2mL of the mixed solution was transferred to 100mL of the enriched culture solution,then placing the mixture 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 for 3d at 30 ℃, wherein the formula of the Congo red primary screening culture medium is as follows: KH is contained in each liter of culture medium₂PO₄0.5g，(NH₄)₂SO₄ 2.0g，MgSO₄·7H₂0.25g of O, 2.0g of CMC, 0.2g of Congo red, 20g of technical agar powder and the balance of water, and the pH value is natural. And (4) selecting a single colony with a large hydrolysis transparent ring, streaking and purifying the single colony on a PDA plate culture medium, and storing the purified strain on a PDA inclined plane.

Re-screening the cellulase and LPMO activity produced by the strain: respectively inoculating purified strains stored on a PDA inclined plane into an equivalent re-screening solid state fermentation culture medium, culturing for 5d at 30 ℃ and 120rpm, wherein the re-screening solid state fermentation culture 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，CaCl₂·2H₂0.5g of O, and the balance 1000mL of water. After solid-state culture and fermentation, the strain is centrifuged to obtain supernatant fluid, namely crude enzyme liquid. The FPA and LPMO 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 find the content of glucose in the solution (enzyme activity unit definition: in the above measurementUnder the fixed condition, the enzyme quantity required for hydrolyzing to generate 1 mu mol of substrate (glucose 180) per minute by each l of crude enzyme solution is 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. With 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 way that each milliliter of crude enzyme solution catalyzes 1 mu mol of hydrogen peroxide to degrade in the reaction system per minute, and is defined as an enzyme activity unit U). And finally, selecting a strain with high LPMO and filter paper enzyme activity and storing the strain by using a PDA inclined plane.

Screening to obtain 1 strain XZH-2 with high LPMOs 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-2 total DNA. 10 XTaq Buffer (with MgCl) was included in a 50. mu.L PCR reaction₂) mu.L, dNTP (mix) (10mmol/L each) 2. mu.L, 10. mu.M primer ITS 12. mu.L, 10. mu.M primer ITS 42. mu.L, Tag enzyme (5U/. mu.L) 0.5. mu.L, 20-50 ng/. mu.L template DNA 1. mu.L, and the rest volume is made up with sterile ultrapure water. The denaturing gradient 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. Sequencing the PCR amplification product by biological engineering (Shanghai) corporation, sequencing to obtain the sequence shown as SEQ ID No.1, BLAST comparing the sequence with known sequence in GenBank database, and obtaining ITS region rDNA sequence of related species from the databaseA phylogenetic tree was built as shown in fig. 2. Through sequence comparison and analysis, the strain XZH-2 belongs to the genus Penicillium, is named as Penicillium oxalicum (Penicillium oxalicum) XZH-2, 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. 21425.

Example 2: preparation of enzyme composition preparation and enzyme activity determination

1. Preparation of enzyme combination preparation: selecting a bacterial block of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 stored on a PDA slant, inoculating the bacterial block into 50mL of PDA liquid culture medium, culturing at 30 ℃ and 150rpm for 3d for activation, inoculating the activated Penicillium oxalicum (Penicillium oxalicum) XZH-2 into 50mL of PDA seed culture medium at the inoculation amount of 50 mu L, and culturing at 30 ℃ and 150rpm for 3d to obtain a seed culture solution. The PDA seed culture medium contains 200.0g of potato extract, 20.0g of glucose and the balance of water per liter of culture medium; the preparation method comprises the following steps: 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 1000mL 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, 1X 6cQuantitatively filtering paper strips (about 50mg) at a medium speed in Xinhua, fully mixing uniformly, and putting into a water bath at 50 ℃ for reacting 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 the Penicillium oxalicum (Penicillium oxalicum) XZH-2 obtained in the step 1 reaches 7.37U/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. With H₂O₂And (3) making a standard curve for a substrate, and calculating the content of hydrogen peroxide released by the reaction (LPMOs enzyme activity unit is defined in the specification, 1 mu mol of hydrogen peroxide is catalyzed and degraded per minute in a reaction system by each milliliter of cellulase and LPMO enzyme combined preparation, and the enzyme activity unit is defined as an enzyme activity unit U). Through determination, the enzyme activity of the cellulase and LPMO enzyme combined preparation produced by the Penicillium oxalicum (Penicillium oxalicum) XZH-2 obtained in the step 1 reaches 17.17U/g of substrate, and the enzyme activity is higher.

The result shows that the Penicillium oxalicum (Penicillium oxalicum) XZH-2 can produce LPMO with high yield, the LPMO enzyme activity of the cellulase and LPMO enzyme combined preparation prepared by solid-state fermentation of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 is as high as 17.17U/g substrate, the filter paper enzyme activity of the cellulase and LPMO enzyme combined preparation is as high as 7.37IU/g substrate, the LPMO orange Thermoascus aurantiacaus (Thermoascus aurantiacaus) and Neurospora crassa (Neurospora crassa) which are reported at present are subjected to the same solid-state fermentation mode in the step 1, wherein the LPMO enzyme activity of the cellulase and LPMO enzyme combined preparation prepared by solid-state fermentation of the Neurospora crassa is 15.45U/g substrate, and the filter paper enzyme activity is 4.68IU/g substrate; the LPMO enzyme activity of the cellulase and LPMO enzyme combined preparation prepared by solid-state fermentation of Thermoascus aurantiacus is 12.50U/g substrate, and the filter paper enzyme activity is only 0.66IU/g substrate (see figure 4). Therefore, compared with the LPMO-producing strain reported in the prior art, the Penicillium oxalicum (Penicillium oxalicum) XZH-2 capable of producing cellulase and LPMO has higher enzyme production capability, and is a novel high-yield cellulase and LPMO-producing strain.

Example 3: application of cellulase and LPMO enzyme combined preparation in poplar cellulose enzymolysis

1. Reaction substrate: the poplar powder is crushed to 60 meshes, dried to constant weight at 105 ℃, weighed and mixed with 2.5g of poplar powder and 2.5g of solid acid (the specific preparation method is shown in example 3 of patent ZL 201110126178.7), and pretreated for 30min at 170 ℃ to obtain a 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 cellulase and LPMO enzyme combined preparation produced by respectively fermenting Neurospora crassa N1(Neurospora crassa) and Thermoascus aurantiacus T2(Thermoascus aurantiacus) in the same solid-state fermentation manner as that of the step 1 in the example 2 without adding the cellulase and LPMO enzyme combined preparation of the invention as a control is compared with commercial cellulase according to the combined action of an enzymolysis reaction system and reaction conditions in the step 2, and the effect of the cellulase and LPMO enzyme combined preparation prepared by solid-state fermentation of Penicillium oxalicum XZH-2 strain on poplar cellulose enzymolysis is researched.

The result is shown in fig. 5, when the cellulase and LPMO enzyme combined preparation prepared by solid state fermentation of the screened Penicillium oxalicum (Penicillium oxalicum) XZH-2 strain is applied to poplar cellulose enzymolysis, the enzymolysis rate is improved by 13.35% compared with the case that the cellulase and LPMO enzyme combined preparation is not added. 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.

Sequence listing

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<120> penicillium oxalicum XZH-2 and application thereof

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Claims

1. A strain of Penicillium oxalicum (Penicillium oxalicum) XZH-2 has a deposit number of: CGMCC No. 21425.

2. Use of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 strain according to claim 1 for the production of cellulase and/or a lytic polysaccharide monooxygenase.

3. Use of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 according to claim 1 for cellulose hydrolysis.

4. Use of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 according to claim 1 in enzymatic saccharification of lignocellulosic feedstocks.

5. Use of the Penicillium oxalicum (Penicillium oxalicum) XZH-2 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 oxalicum (Penicillium oxalicum) XZH-2, 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 oxalicum (XZH-2) is prepared by the following method: inoculating activated Penicillium oxalicum (Penicillium oxalicum) XZH-2 into a seed culture medium, and culturing at 30 ℃ and 150rpm for 3d to obtain a 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 of peeled potatoes, adding water, boiling, filtering by using a warp cloth 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 citric acid buffer solution, shaking at 30 deg.C and 150rpm for 1 hr, and collecting the solutionThe body is fermentation enzyme liquid, the fermentation enzyme liquid is centrifuged for 5min at 8000rpm and 4 ℃, and the supernatant is taken to obtain the enzyme combination 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 of claim 9 in the hydrolysis of forest wood cellulose.