CN112210502B

CN112210502B - Penicillium oxalicum mutant strain A2-13 and application thereof in preparation of raw amylase preparation and degradation of raw starch

Info

Publication number: CN112210502B
Application number: CN202011170084.5A
Authority: CN
Inventors: 赵帅; 冯家勋; 顾丽莎; 谈明珠
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2021-10-01
Anticipated expiration: 2040-10-28
Also published as: CN112210502A

Abstract

The invention discloses a penicillium oxalicum mutant strain A2-13 and application thereof in preparation of raw amylase preparation and degradation of raw starch. The invention provides Penicillium oxalicum (Penicillium oxalicum) A2-13, which has a preservation number of CCTCC NO: m2020319. The invention also provides a method for producing raw amylase or raw amylase preparation by using the penicillium oxalicum A2-13, which comprises the following steps: culturing the penicillium oxalicum A2-13 in a basic or optimized fermentation medium for a certain time, collecting a fermentation product, centrifuging and collecting a supernatant to obtain the raw amylase or the raw amylase preparation. Experiments prove that the penicillium oxalicum A2-13 obtained by the invention can produce raw amylase preparation, the preparation can efficiently hydrolyze raw cassava starch and raw corn starch, and the final hydrolysis product is mainly glucose. The preparation can be used for producing alcohol.

Description

Penicillium oxalicum mutant strain A2-13 and application thereof in preparation of raw amylase preparation and degradation of raw starch

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a penicillium oxalicum mutant strain A2-13 and application thereof in preparation of raw amylase preparation and degradation of raw starch.

Background

Amylase is one of the most prominent enzymes in the commercial enzyme market. Depending on the mode of action, these enzymes are mainly classified into α -amylase (α -amylase), glucoamylase (glucoamylase), α -glucosidase (α -glucosidase), and pullulanase (starch debranching enzyme). The alpha-amylase and the saccharifying enzyme are key enzymes for starch processing, and the alpha-amylase randomly cuts alpha-1, 4-glycosidic bonds in a starch molecular chain to generate a large amount of oligosaccharide and a small amount of maltose and glucose. The saccharifying enzyme can hydrolyze alpha-1, 4-glycosidic bond and alpha-1, 6-glycosidic bond, and sequentially release glucose from non-reducing end of starch molecular chain. A few amylase proteins contain Starch-binding domains (Starch-binding domains) that bind directly to the surface of Raw Starch granules, causing amylase enzymes, named Raw-amylases (RSDEs), to hydrolyze Raw Starch below the gelatinization temperature. In nature, raw amylases are produced mainly from Aspergillus (Aspergillus), Penicillium (Penicillium), and the like.

The raw amylase is applied to the starch processing industry, reduces the high-temperature gelatinization and liquefaction processes, saves the cost, reduces the environmental pollution, and has wide application prospect. At present, the yield of raw amylases, particularly raw diastase, of microorganisms mainly penicillium and aspergillus is low, and the large-scale development and utilization of the raw amylases are limited.

Disclosure of Invention

The invention aims to provide a penicillium oxalicum mutant strain A2-13 and application thereof in preparation of raw amylase preparation and degradation of raw starch.

In a first aspect, the invention claims a mutant strain of penicillium oxalicum.

The invention discloses a Penicillium oxalicum mutant strain, in particular to Penicillium oxalicum A2-13, which is obtained by random mutagenesis of a Penicillium oxalicum genetic engineering strain OXPoxGA15A through multiple rounds of Atmospheric and Room Temperature Plasmas (ARTP) and Ethyl Methylsulfonate (EMS).

The penicillium oxalicum mutant strain A2-13 provided by the invention is preserved in China center for type culture Collection (CCTCC for short, address: China, Wuhan university) at 15/07/2020, and the preservation number is CCTCC NO: m2020319.

In a second aspect, the invention claims a bacterial agent.

The active ingredient of the microbial inoculum claimed by the invention is the penicillium oxalicum A2-13.

The microbial inoculum can be the following microbial inoculum 1) or 2):

1) a microbial inoculum for degrading raw starch;

2) a bacterial preparation for degrading raw starch-containing material.

In a third aspect, the invention claims a medium for culturing the aforementioned penicillium oxalicum A2-13.

The culture claimed in the present invention may consist of an organic carbon source and a salt solution. Wherein the ratio of the organic carbon source to the salt solution is 5g to 100 mL; the salt solution is prepared from water and KH₂PO₄Nitrogen source, MgSO₄·7H₂O, Tween 80 and microelement mother liquor, wherein each liter of the saline solution contains KH₂PO₄4.0g of the nitrogen source, 2-8g (e.g., 5g) of the nitrogen source, MgSO₄·7H₂0.6g of O, 802 mL of Tween and 0.1mL of the microelement mother liquor; the microelement mother liquor is composed of solute and solvent, the solvent is water, and the solute and the concentration thereof are respectively FeSO₄·7H₂O 2.5g/L、MnSO₄·H₂O 0.8g/L、ZnSO₄·7H₂O0.7 g/L and CoCl₂ 1.0g/L。

Further, the organic carbon source may be agricultural organic waste, and specifically may be selected from any one or more of the following: wheat bran, crystalline cellulose, corncobs, bagasse, rice straw, raw cassava flour and the like.

Further, the nitrogen source is ammonium nitrate, yeast extract, ammonium sulfate, urea, peptone, sodium nitrate, and/or soybean cake powder.

Further, the pH of the medium may be 3.5-5.5 (e.g., 4.5-5.5).

Further, the agricultural organic waste particles may have a size of 0.25mm or less (moisture content is 5% or less).

In a preferred embodiment of the invention, the agricultural organic waste is wheat bran and crystalline cellulose, and the mass ratio of the wheat bran to the crystalline cellulose in the culture medium is 2: 3.

in a particular embodiment of the invention, the crystalline cellulose is in particular crystalline cellulose Avicel.

In a preferred embodiment of the invention, the pH of the medium is in particular 5.5.

In a fourth aspect, the invention claims any of the following products:

p1), products for the degradation of raw starch prepared with the penicillium oxalicum a2-13 described hereinbefore;

p2), products for degrading raw starch prepared using the above-mentioned bacterial agents;

p3), a raw amylase prepared using the penicillium oxalicum a2-13 described hereinbefore;

p4), a raw amylase prepared using the microbial inoculum described previously.

In P1), the active ingredient of the raw starch degrading product is specifically penicillium oxalicum a2-13 as described hereinbefore. In P2), the active ingredient of the raw starch degrading product is specifically the microbial inoculum described hereinbefore.

Further, P1) the raw starch degrading product may be a raw amylase single preparation or a combined preparation.

The raw amylase single preparation can be prepared by a method comprising the following steps: culturing Penicillium oxalicum A2-13 as described above in a medium as described above at 20-32 deg.C (e.g., 24-32 deg.C, specifically 28 deg.C), and collecting the fermentation product to obtain a raw amylase preparation.

In a preferred embodiment of the present invention, the culture time of said penicillium oxalicum A2-13 is specifically 6 days.

In a preferred embodiment of the inventionThe initial concentration of spores obtained by inoculating the penicillium oxalicum A2-13 into the culture medium is specifically 1X 10⁶one/mL.

Further, the method also comprises the following steps after collecting the fermentation product: and centrifuging the fermentation product and collecting supernatant, wherein the supernatant is the raw amylase single preparation.

The combined preparation consists of the raw amylase single preparation and alpha-amylase.

Further, the enzyme activity ratio of the raw amylase single preparation to the alpha-amylase can be 1: 1.

In a fifth aspect, the invention claims a method of M1) or M2) or M3) below:

m1) a method of degrading raw starch or raw starch-containing material, which may comprise: the raw starch is degraded by reacting the raw amylase as described above either alone or in combination with raw starch or a raw starch-containing material at 40 ℃ in an environment having a pH of 4.5.

Further, when the reaction is carried out, the raw amylase single preparation or the combined preparation is used in an amount of 50-200U/g substrate (e.g., 50-150U/g substrate or 50-100U/g substrate or 100-150U/g substrate). The substrate is raw starch or raw starch-containing material.

Further, the reaction is carried out for a period of time ranging from 48 to 72 hours (e.g., 60 to 72 hours).

In the above method, the environment with pH of 4.5 may be Na with pH of 4.5₂HPO₄-citric acid buffer. The Na having a pH of 4.5₂HPO₄The citric acid buffer is 0.2M Na₂HPO₄Mixing the solution with 0.1M citric acid solution at a ratio of 9.1:10.9, wherein Na is₂HPO₄And the concentrations of citric acid were 0.091M and 0.0545M, respectively.

M2) method for preparing a single preparation of raw amylase as described hereinbefore.

M3) method for producing amylase by culturing penicillium oxalicum a2-13 as described above, comprising culturing penicillium oxalicum a2-13 as described above in a medium as described above at 20-32 ℃ (e.g. 24-32 ℃, particularly 28 ℃).

In a sixth aspect, the invention claims the following applications X1), X2) or X3):

x1) the use of penicillium oxalicum a2-13 as described hereinbefore or of the inoculant described hereinbefore in any one of the following a1) -a 6):

a1) degrading raw starch or raw starch-containing material;

a2) preparing a material for degrading raw starch or raw starch-containing material;

a3) preparing raw amylase;

a4) preparing a product for producing raw amylase;

a5) producing glucose, maltose or ethanol;

a6) preparing products for producing glucose, maltose or ethanol.

X2) use of the medium as described hereinbefore for a2), a4) or a6) above.

X3) use of the product described hereinbefore in any of a1) -a6) above.

In each of the above aspects, the raw starch-containing material may be selected from any one or more of: tapioca, corn, wheat, rice, or a raw meal of these substances.

In a particular embodiment of the invention, the raw starch-containing material is in particular raw tapioca flour or raw corn flour.

Experiments prove that after the penicillium oxalicum A2-13 is cultured by liquid fermentation, the fermentation liquid has the activity of amylase. The use of the alpha-amylase together with commercial alpha-amylase can efficiently hydrolyze raw corn flour and raw cassava flour to produce glucose, and has application potential in conversion and utilization of raw starch.

The penicillium oxalicum A2-13 obtained by the invention can be used for producing raw amylase preparations, and the preparations can efficiently hydrolyze raw corn flour and raw cassava flour. The preparation can be used for producing alcohol.

Deposit description

And (3) classification and naming: penicillium oxalicum (Penicillium oxalicum)

The strain number is as follows: a2-13

The name of the depository: china center for type culture Collection

The preservation unit is abbreviated as: CCTCC (China center for cell communication)

The address of the depository: wuhan university, one of the eight roads in Wuchang district, Wuhan city, Hubei province, postal code: 430072

The preservation date is as follows: year 2020, 07 months and 15 days

Registration number of the preservation center: CCTCC NO: m2020319

Drawings

FIG. 1 shows the hydrolysis loop of the Penicillium oxalicum starting strain OXOXYGA 15A and each mutant strain obtained by mutagenesis of the strain on a culture medium plate containing raw cassava flour.

FIG. 2 shows the raw cassava amylase yields of the strains of Penicillium oxalicum.

FIG. 3 shows the optimum initial pH optimization experimental results of the fermentation medium of Penicillium oxalicum A2-13.

FIG. 4 shows the results of the optimum culture temperature optimization experiment of Penicillium oxalicum A2-13.

FIG. 5 shows the results of experiments for optimizing the optimal carbon source in the fermentation medium of Penicillium oxalicum A2-13.

FIG. 6 shows the results of optimization experiments on the concentration of the optimum combined carbon source in the fermentation medium of Penicillium oxalicum A2-13.

FIG. 7 shows the result of an experiment for optimizing the nitrogen source in the fermentation medium of Penicillium oxalicum A2-13.

FIG. 8 shows the result of an optimization experiment of the optimum concentration of ammonium nitrate as the nitrogen source in the fermentation medium of Penicillium oxalicum A2-13.

FIG. 9 shows the results of an optimization experiment for the optimal concentration of amylase producing inoculated spores by Penicillium oxalicum A2-13.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, the moisture contents of wheat bran, soybean cake flour, rice straw, corn cob, bagasse and raw starch were all 5% (by mass) or less, and the particle sizes of these substances were 0.25mm or less.

The raw cassava flour in the following embodiment is natural raw cassava flour, and the preparation steps are as follows: peeling raw wood potato chips purchased from Nanning city vegetable market, and drying in a 60 ℃ oven; pulverizing with a pulverizer, and sieving with 80 mesh sieve.

The following glucose standard curve was plotted and the amylase activity was measured as follows:

1. drawing of glucose standard curve

0.1g of a constant weight glucose standard was weighed and a 1mg/mL glucose standard solution was prepared with deionized water. Adding various reagents into a 2mL centrifuge tube according to the formula of the table 1, uniformly mixing, carrying out boiling water bath for 10 minutes to develop color, taking out a sample, and cooling to room temperature. Centrifuging at 12,000rpm for 2min, sucking 200 μ L of supernatant into 96-well microplate, measuring absorbance at 540nm wavelength and drawing glucose standard curve to obtain linear regression equation of Y-3.3481X-0.0304 and variance R²0.9996, Y: a light absorption value OD; x: glucose concentration (mg/mL).

TABLE 1 composition of glucose Standard Curve solutions

Numbering	1	2	3	4	5	6	7
								Glucose Standard solution (μ L)	0	50	100	150	200	250	300
Buffer solution (mu L)	500	450	400	350	300	250	200
								Glucose concentration (mg/mL)	0	0.1	0.2	0.3	0.4	0.5	0.6
DNS reagent (mu L)	1000	1000	1000	1000	1000	1000	1000

The buffer in Table 1 was Na at pH 4.5₂HPO₄Citric acid buffer, in which Na₂HPO₄And citric acid concentrations of 0.091M and 0.0545M (i.e., 0.2M Na), respectively₂HPO₄The solution and 0.1M citric acid solution were mixed in a ratio of 9.1: 10.9).

2. Determination of raw amylase activity

Firstly, taking a fermentation system, centrifuging at 4 ℃, and collecting supernatant fluid, namely fermentation supernatant fluid.

② taking fermentation supernatant, adding citric acid-disodium hydrogen phosphate buffer solution (namely 0.2M Na) with pH of 4.5₂HPO₄Mixing the solution and 0.1M citric acid solution according to the proportion of 9.1: 10.9) as a solvent for dilution to obtain an enzyme solution to be detected; boiling the enzyme solution to be tested in boiling water for 10 minutes to inactivate to obtain an inactivated enzyme solution;

③ adding 450 mu L of 1g/100mL of raw natural cassava powder solution (the solvent is citric acid-disodium hydrogen phosphate buffer solution with pH 4.5) into a 2mL centrifuge tube, and preheating for 10min at 65 ℃;

adding 50 mu L of enzyme solution to be detected or 50 mu L of inactivated enzyme solution (negative control), standing at 65 ℃ for 30min for reaction, adding 1mL of DNS solution to terminate the reaction, boiling in water for 10min, cooling to room temperature, centrifuging at 12,000rpm for 10min, collecting supernatant, absorbing 200 mu L of enzyme solution to an enzyme label plate to measure the light absorption value at the wavelength of 540nm, calculating the concentration of reducing sugar according to a glucose standard curve, wherein one enzyme activity unit (U) is defined as the enzyme amount required by hydrolyzing the raw natural cassava starch under the reaction conditions of 65 ℃ and pH 4.5 to generate 1 mu moL of reducing sugar per minute. Then, the yield of the raw cassava amylase in the fermentation supernatant is obtained according to the conversion of the dilution factor.

Example 1 screening of Penicillium oxalicum A2-13

The penicillium oxalicum A2-13 is a strain which is obtained by screening strains with higher amylase activity after multiple rounds of physical and chemical random mutagenesis from original fungal strains in the inventor's laboratory. Penicillium oxalicum (Penicillium oxalicum) genetic engineering strain OXPOGA 15A (CCTCC NO: M2017794; see Chinese patent application No. CN201810816723.7) is used as an original strain, and 3 rounds of Ethyl Methylsulfonate (EMS) mutagenesis is adopted to obtain a mutant strain E3-16 with the activity of raw amylase higher than that of the original strain OXPOGA 15A. Then E3-16 is subjected to normal pressure and normal temperature plasma (mutagenesis to obtain a strain A1-2 with higher amylase activity, and then the strain A1-2 is subjected to ARTP and EMS compound mutagenesis and screening to finally obtain a mutant strain A2-13.

First, preparation of spore suspension

1. The PDA solid medium was sterilized at 121 ℃ for 30min to prepare a PDA plate.

2. Penicillium oxalicum OXXOX GA15A spores cultured on PDA plates for 5 days were washed with 0.1% (v/v) Tween 80 aqueous solution and prepared at a concentration of 1X 10⁸Spore suspension per mL.

Second, EMS mutagenesis

When EMS mutagenesis is carried out on the strain OXOXYGA 15A, spore suspension with the lethality rate of 93.6 percent under the corresponding dose is selected for large-scale separation and screening, and the specific steps are as follows:

1.5 mL of fresh spores suspended in PBS buffer solution are taken, and the concentration of the spores is 10⁸one/mL.

2. Adding an EMS reagent with the same volume as 1.2 percent, shaking and mixing evenly, and placing in a shaking table with the temperature of 28 ℃ and the rpm of 180 for 8 hours.

3. 1mL of mutagenized spore suspension was placed in a 2mL EP tube and 1mL of 5% Na was added₂S₂O₃Terminating the reaction, centrifuging at 12000rpm and 4 deg.C for 10min, pouring off the supernatant, repeating the above steps until no white floccule is present in the tube, centrifuging again, and removing the termination solution Na₂S₂O₃The spores were resuspended in 1mL of sterile water.

4. Diluting the spore liquid 10²-10⁶Double-layer plate coated with 100. mu.L of the extract₂PO₄ 2g,(NH₄)₂SO₄ 1.4g,MgSO₄·7H₂O 0.3g，CaCl₂0.9g, 0.1mL of microelement mother liquor, Trition-1001 mL, 0.5g of ball-milled Avicel, 1g of raw natural cassava powder, 2.0g of agar powder and pH of 5.5; the lower layer: upper media was removed from ball milled Avicel and raw natural tapioca flour) and each gradient was repeated five times. The non-mutagenized spore liquid was taken and diluted and applied in the same manner as a control.

5. Culturing at 28 deg.C for 8d, and screening mutant strain by comparing the ratio of colony hydrolysis ring to colony diameter.

6. Repeat the above steps 3 times.

7. The mutant strain E3-16 was obtained.

Third, ARTP mutagenesis

When the strain E3-16 is subjected to ARTP mutagenesis, spore suspension with the lethality rate of 95.1 percent under the corresponding dose is selected for large-scale mutant strain separation and screening, and the specific steps are as follows:

1.5 mL of fresh spores suspended in 5% glycerol at a concentration of 10⁸Each mL, spore concentration is 10⁶one/mL.

2. 10 μ L of spore liquid was spread evenly on the surface of the sterilized slide.

3. The petri dish with the sample slide was placed in an ARTP mutagen operating room. The instrument power is set at 130w, the air volume is set at 10SLM, and the processing time is 500 s. The slides were transferred to an EP tube containing 1mL of sterile water using sterile forceps.

4. Placing the EP tube on a vortex oscillator, and oscillating for 2min to obtain new spore suspension.

5. Diluting the resuspended spore liquid 10²-10⁶ Taking 100. mu.L of spore liquid to a double-layer primary screening plate (upper layer: KH)₂PO₄ 2g,(NH₄)₂SO₄ 1.4g,MgSO₄·7H₂O 0.3g，CaCl₂0.9g, 0.1mL of microelement mother liquor, Trition-1001 mL, 0.5g of ball-milled Avicel, 1g of raw natural cassava powder, 2.0g of agar powder and pH of 5.5; the lower layer: removal of the upper layer of culture mediumBall milled Avicel and raw natural tapioca flour) were run in five replicates per gradient. The non-mutagenized spore liquid was taken and diluted and applied in the same manner as a control.

Culturing at 6.28 deg.C for 8 days, and screening mutant strain by comparing the hydrolysis loop of bacterial colony with the direct ratio of bacterial colony.

7. The mutant strain A1-2 was obtained.

Four, ARTP-EMS Complex mutagenesis

Selecting the treatment time with the lethality rate of more than 90 percent after the strain A1-2 is treated by the ARTP-EMS combination, carrying out EMS mutagenesis according to the method, carrying out EMS mutagenesis after the termination reaction, carrying out resuspension on the obtained spore suspension by 5 percent of glycerin, carrying out ARTP mutagenesis according to the method, carrying out mass plate coating after the obtained spore liquid is properly diluted, carrying out constant temperature culture at 28 ℃ for 8d, and screening the mutant strain by comparing the direct ratio of the colony hydrolysis ring and the colony.

The results show that: 1616 single colonies were co-isolated by 3 rounds of EMS mutagenesis (table 2). Wherein the mutant strain E3-16 has the largest hydrolysis circle to colony diameter ratio of 1.75 (FIG. 1), and is subjected to ARTP mutagenesis to separate 1120 single colonies, and the ratio of hydrolysis circle to colony diameter is compared to obtain the mutant strain A1-2 with a diameter of 1.78 (FIG. 1). Finally, the strain A1-2 was subjected to ARTP-EMS complex mutagenesis, and 796 single colonies were isolated, in which the diameter ratio of the mutant strain A2-13 was 2.0 at the maximum (FIG. 1).

TABLE 2 mutagenized strains and numbers obtained by high throughput plate prescreening

Mutagenesis	Starting strain	Number of single colony	Obtaining the number of strains through preliminary screening
				1 st round EMS	OXPoxGA15A	441	13
2 nd round EMS	E1-1	352	5
				3 rd round EMS	E2-3	823	20
ARTP	E3-16	1120	35
				ARTP-EMS	A1-2	796	28

Inoculating the mutant strain with higher raw amylase activity obtained by each round of mutagenesis in a liquid enzyme-producing culture medium, culturing for 6d, and determining the raw potato amylase activity of the mutant strain, wherein the raw potato amylase activity of the mutant strain obtained by each round of mutagenesis is gradually increased, as shown in figure 2, wherein A2-13 is the highest and is 162.7 +/-1.44U/mL.

Penicillium oxalicum A2-13 has been deposited at the China center for type culture Collection on month 07 and 15 of 2020, accession number: CCTCC NO: M2020319.

Example 2 optimization of conditions for production of Amylase by liquid fermentation of Penicillium oxalicum A2-13

The experiment is repeated three times, and the specific steps of each repeated experiment are as follows:

first, preparation of spore suspension

2. Penicillium oxalicum A2-13 spores cultured on PDA plates for 5 days were washed with 0.1% (v/v) Tween 80 aqueous solution and prepared at a concentration of 1X 10⁸Spore suspension per mL.

Second, optimization of initial pH of culture medium

1. Preparing fermentation culture media with different pH values

Preparing a basic culture medium: the minimal medium consisted of wheat bran, crystalline cellulose Avicel (PH-101, Sigma-Aldrich #11365) and salt solution. Wherein the ratio of the wheat bran, the crystalline cellulose Avicel and the salt solution is 4.0g to 1.0g to 100 mL. The salt solution is prepared from water and KH₂PO₄、(NH₄)₂SO₄、MgSO₄·7H₂O, Tween 80 and microelement mother liquor, wherein each liter of salt solution contains KH₂PO₄ 4.0g、(NH₄)₂SO₄ 4.0g、MgSO₄·7H₂0.6g of O, 802 mL of Tween and 0.1mL of microelement mother liquor. The microelement mother liquor comprises solute and solvent, wherein the solvent is water, and the content of each solute in each liter of microelement mother liquor is FeSO₄·7H₂O 2.5g、MnSO₄·H₂O 0.8g、ZnSO₄·7H₂O 0.7g、CoCl₂ 1.0g。

The pH of the minimal medium was adjusted to 2.5, 3.5, 4.5, 5.5, 6.5 and 7.5 with 5M aqueous HCl or NaOH, respectively, and autoclaved at 121 ℃ for 30min to obtain a medium with pH 2.5, a medium with pH 3.5, a medium with pH 4.5, a medium with pH 5.5, a medium with pH 6.5 and a medium with pH 7.5, respectively.

2. Fermentation culture

Respectively inoculating the spore suspension obtained in the first step into the culture media with different pH values according to 1% (v/v), and culturing at 28 ℃ and 180rpm for 6 days to obtain a fermentation product; centrifuging (12,000rpm for 15min) and collecting the supernatant, which is the crude enzyme solution.

3. Enzyme activity assay

Collecting the crude enzyme solution, and respectively measuring the activity of the raw amylase of the crude enzyme solution obtained from the culture medium with different pH values.

As a result, the optimum initial pH of the amylase activity of Penicillium oxalicum A2-13 was 5.5.

Thirdly, optimization of culture temperature

1. And optimizing the culture temperature by using the culture medium with the pH of 5.5 in the step two.

2. Inoculating the spore suspension obtained in the first step into the culture medium with the pH value of 5.5 obtained in the second step according to the ratio of 1% (v/v), and respectively placing the culture medium in constant-temperature incubators at different temperatures (20 ℃, 24 ℃, 28 ℃, 32 ℃ and 36 ℃) for culturing for 6 days at 180rpm to obtain a fermentation product; centrifuging (12,000rpm for 15min) and collecting the supernatant, which is the crude enzyme solution.

3. Collecting the crude enzyme solution, and respectively measuring the activity of the raw amylase of the crude enzyme solution obtained by culturing at different temperatures.

As a result, the optimum temperature for the amylase activity of Penicillium oxalicum A2-13 is 28 ℃.

Fourthly, determination of optimal carbon source of culture medium

1. Fermentation culture medium for preparing different carbon sources

And (3) the pH values of the culture mediums of different carbon sources are 5.5, and the culture mediums are sterilized at 121 ℃ for 30min under high pressure, wherein the components and the content of the wheat bran and crystalline cellulose culture medium are the same as those of the basic culture medium in the second step. The wheat bran and bagasse culture medium consists of wheat bran and bagasse and the salt solution obtained in the second step, wherein the ratio of the wheat bran to the bagasse to the salt solution is 4.0g to 1.0g to 100 mL. The wheat bran and raw cassava flour culture medium consists of wheat bran and raw cassava flour and the salt solution obtained in the second step, wherein the ratio of the wheat bran, the raw cassava flour and the salt solution is 4.0g, 1.0g and 100 mL. The wheat bran and bean cake powder culture medium consists of wheat bran and bean cake powder and the salt solution obtained in the second step, wherein the ratio of the wheat bran, the bean cake powder and the salt solution is 4.0g to 1.0g to 100 mL. The wheat bran and rice straw culture medium consists of wheat bran and rice straw and the salt solution obtained in the second step, wherein the ratio of the wheat bran to the rice straw to the salt solution is 4.0g to 1.0g to 100 mL. The wheat bran and corn cob culture medium consists of wheat bran and corn cob and the salt solution obtained in the second step, wherein the ratio of the wheat bran to the corn cob to the salt solution is 4.0g to 1.0g to 100 mL.

2. Fermentation culture

Inoculating the spore suspension obtained in the first step into culture media with different carbon sources obtained in the first step according to 1% (v/v), and culturing for 6 days in a constant-temperature incubator at 28 ℃ at 180rpm to obtain a fermentation product; centrifuging (12,000rpm for 15min) and collecting the supernatant, which is the crude enzyme solution.

3. Collecting the crude enzyme solution, and respectively measuring the activity of the starch-producing enzyme of the crude enzyme solution obtained by culturing in different carbon source culture media.

The results are shown in FIG. 5, wheat bran plus crystalline cellulose Avicel is the best carbon source for amylase production by Penicillium oxalicum A2-13.

Fifthly, determining the optimal concentration of the combined carbon source of the culture medium

1. Preparing culture media with various carbon source combination concentrations:

the culture medium with different mass ratios of wheat bran to crystalline cellulose Avicel consists of wheat bran, crystalline cellulose Avicel and the salt solution obtained in the second step, the pH values are 5.5, and the mixture is autoclaved at 121 ℃ for 30 min. The mass ratio of wheat bran to crystalline cellulose Avicel is set as follows: 1:2,2:2,3:2,1:4,2:3,1:3,3:1,2:1,4:1. The ratio of wheat bran, crystalline cellulose Avicel and salt solution in the culture medium with the mass ratio of wheat bran to crystalline cellulose Avicel being 1:2 is 1.7g:3.3g:100mL, the ratio of wheat bran to crystalline cellulose Avicel and salt solution in the culture medium with the mass ratio of wheat bran to crystalline cellulose Avicel being 2:2 is 2.5g:2.5g:100mL, the ratio of wheat bran to crystalline cellulose Avicel and salt solution in the culture medium with the mass ratio of wheat bran to crystalline cellulose Avicel being 3:2 is 3g:2g:100mL, the ratio of wheat bran to crystalline cellulose Avicel in the culture medium with the mass ratio of 1:4 is 1g:4g:100mL, the ratio of wheat bran to crystalline cellulose Avicel in the culture medium with the mass ratio of 2:3 is 2g:3 mL, and the ratio of wheat bran to crystalline cellulose Avicel in the culture medium with the mass ratio of 1:3 is 3, The ratio of the crystalline cellulose Avicel to the salt solution is 1.25g to 3.75g to 100mL of wheat bran, the ratio of the wheat bran to the crystalline cellulose Avicel to the salt solution in the culture medium with the mass ratio of 3:1 of the wheat bran to the crystalline cellulose Avicel is 3.75g to 1.25g to 100mL of wheat bran, and the ratio of the wheat bran to the crystalline cellulose Avicel to the salt solution in the culture medium with the mass ratio of 2:1 of the wheat bran to the crystalline cellulose Avicel is 3.3g to 1.7g to 100mL of wheat bran; the ratio of wheat bran to crystalline cellulose Avicel to salt solution in the culture medium with the mass ratio of wheat bran to crystalline cellulose Avicel being 4:1 is 4g:1g:100 mL.

2. Fermentation culture

Inoculating the spore suspension obtained in the first step into culture media with different carbon source combination concentrations obtained in the step 1 according to 1% (v/v), and culturing for 6 days in a constant-temperature incubator at 28 ℃ at 180rpm to obtain a fermentation product; centrifuging (12,000rpm for 15min) and collecting the supernatant, which is the crude enzyme solution.

3. Collecting the crude enzyme solution, and respectively measuring the activity of the starch-producing enzyme of the crude enzyme solution obtained by culturing in culture media with different carbon source combination concentrations.

The results are shown in FIG. 6, where a wheat bran to crystalline cellulose Avicel mass ratio of 2:3 is the optimum combined carbon source concentration for amylase production by Penicillium oxalicum A2-13.

Sixthly, determination of optimal nitrogen source of culture medium

1. Preparing culture media containing different nitrogen sources:

and (3) performing high-pressure sterilization on the culture media with different nitrogen sources at the temperature of 121 ℃ for 30min, wherein the pH of the culture media with different nitrogen sources is 5.5, the carbon source in the ammonium sulfate culture medium is wheat bran and crystalline cellulose, the mass ratio is 2:3, and the components and the content of the salt solution are the same as those of the basic culture medium in the second step and are used as a reference culture medium. The urea culture medium is (NH) in ammonium sulfate culture medium₄)₂SO₄4.0g of the resulting medium was replaced with 4.0g of urea. The peptone medium is (NH) in ammonium sulfate medium₄)₂SO₄4.0g of the resulting medium was replaced with peptone 4.0 g. The sodium nitrate culture medium is (NH) in ammonium sulfate culture medium₄)₂SO₄4.0g of the resulting medium was replaced with 4.0g of sodium nitrate. The ammonium nitrate culture medium is (NH) in ammonium sulfate culture medium₄)₂SO₄4.0g of the resulting medium was replaced with 4.0g of ammonium nitrate. The bean cake powder culture medium is prepared by mixing ammonium sulfate(NH) in culture Medium₄)₂SO₄4.0g of the resulting medium was replaced with 4.0g of soybean cake powder.

2. Fermentation culture

Inoculating the spore suspension obtained in the step one into the culture medium containing different nitrogen sources obtained in the step 1 according to 1% (v/v), and culturing for 6 days in a constant-temperature incubator at 28 ℃ at 180rpm to obtain a fermentation product; centrifuging (12,000rpm for 15min) and collecting the supernatant, which is the crude enzyme solution.

3. Collecting crude enzyme liquid, and respectively measuring the activity of the raw amylase of the crude enzyme liquid obtained by culturing under different nitrogen sources.

Results FIG. 7 shows that ammonium nitrate is the best nitrogen source for amylase production by Penicillium oxalicum A2-13.

Seventhly, determination of optimal nitrogen source concentration

1. Preparing nitrogen source culture media with different concentrations:

and (3) nitrogen source culture media with different concentrations, wherein the carbon source is wheat bran and crystalline cellulose Avicel, the mass ratio is 2:3, the salt solution components and the content are the same as those of the basic culture medium in the step two, the nitrogen source is ammonium nitrate, the pH values are 5.5, and the medium is autoclaved at 121 ℃ for 30 min. The concentration of ammonium nitrate is set as follows: 1,2, 3, 4, 5, 6, 7 and 8 g/L.

2. Fermentation culture

Respectively inoculating the spore suspension obtained in the first step into the culture media with different nitrogen sources in different concentrations obtained in the step 1 according to 1% (v/v), and culturing at 28 ℃ and 180rpm for 6 days to obtain fermentation products; centrifuging (12,000rpm for 15min) and collecting the supernatant, which is the crude enzyme solution.

3. Enzyme activity assay

Collecting crude enzyme liquid, and respectively measuring the activity of the raw amylase of the crude enzyme liquid obtained from the culture medium with different concentrations of nitrogen sources.

As a result, the optimum concentration of ammonium nitrate as a nitrogen source for amylase production by Penicillium oxalicum A2-13 was 5g/L as shown in FIG. 8.

Eighthly, determination of optimal spore inoculation concentration

1. Spore suspensions of different concentrations were prepared

Preparing the spores obtained in the step oneThe concentration is 1X 10⁹Spore mother liquor of each/mL, respectively diluted to 1 × 10⁹1X 10 units/mL⁸1X 10 units/mL⁷1X 10 units/mL⁶1X 10 units/mL⁵1X 10 units/mL⁴one/mL of inoculated spore suspension.

2. Fermentation culture

Respectively inoculating the prepared spore suspensions with different concentrations into 100mL of the optimal culture medium obtained in the seventh step according to 1% (v/v), and culturing at 28 ℃ and 180rpm for 6 days to obtain fermentation products; centrifuging (12,000rpm for 15min) and collecting the supernatant, which is the crude enzyme solution.

3. Enzyme activity assay

Collecting the crude enzyme solution, and respectively measuring the activity of the raw amylase of the crude enzyme solution obtained by inoculating different spores with different concentrations.

As a result, the optimum concentration for the amylase-producing spore inoculation of Penicillium oxalicum A2-13 is 1X 10⁸one/mL.

EXAMPLE 3 preparation of raw Amylase preparation Using Penicillium oxalicum A2-13

1. Preparing fermentation medium

The optimal medium for step seven of example 2 was prepared.

2. Preparation of spore liquid

Preparation example 2 step one concentration of 1X 10⁸one/mL of spore inoculum. The above-mentioned optimal medium was inoculated with an inoculum size of 1% (v/v).

3. Preparation of raw amylase preparation

Placing the fermentation medium of the penicillium oxalicum A2-13 spore suspension inoculated in the step 2 in a constant-temperature incubator at 28 ℃ for standing culture for 6 days to obtain a fermentation product; centrifuging (12,000rpm for 15min), and collecting supernatant, wherein the supernatant is the raw amylase preparation.

4. The raw amylase activity of the raw amylase preparation was determined.

The results are as follows: the raw amylase activity of the raw amylase preparation is 191.0 +/-1.5U/mL.

The comparison reports and the test group of penicillium amylase-producing activities are shown in Table 3, which shows that penicillium oxalicum A2-13 produces the highest amylase-producing activity under the conditions of the seven-step optimal culture medium and the eight-step optimal spore inoculation concentration in example 2.

TABLE 3 comparison of amylase activities produced by fermentation of Penicillium strains

Reference documents: lin HJ, Xian L, Zhang QJ, et al 2011.production of raw cassava stage-mapping enzyme by Penicillium and its use in conversion of raw cassava flow to ethanol.J.Ind.Microbiol.Biotechnol.38,733-742.

Example 4 hydrolysis of raw starch Using raw Amylase preparation

First, raw starch is hydrolyzed by raw amylase preparation alone

1. Preparation of raw starch:

peeling potato chips and corn kernels purchased from farmer market (Nanning), oven drying at 60 deg.C, pulverizing, and sieving with 80 mesh sieve.

2. Raw starch hydrolysis

The reaction system is 100mL, the concentration of the substrate (raw cassava flour or raw corn flour) is set to be 100g/L, and the loading amount of the raw amylase preparation is set to be 50U/g of the substrate.

The specific preparation method of the reaction system comprises the following steps: to pH 4.5Na₂HPO₄-adding raw tapioca flour or raw corn flour to the citric acid buffer with the raw amylase preparation of example 3.

The reaction conditions were 40 ℃ and 180rpm, and the reaction was carried out for 72 hours. Samples are taken in the reaction time of 0, 6, 12, 24, 36, 48 and 72 hours respectively, and the content of reducing sugar and the conversion rate of raw starch in the reaction system are detected.

Raw starch conversion 0.9 × 100/weight of raw starch in raw tapioca flour or corn flour.

The results are shown in Table 4, which shows that the starch conversion of raw corn and raw cassava was 87.6% and 62.5%, respectively, at 72 hours of reaction at 50U/g enzyme addition.

TABLE 4 conversion (%), of the amylase preparation from Penicillium oxalicum A2-13 to raw starch

II, raw amylase preparation and commercial alpha-amylase synergistic hydrolysis raw starch

1. Preparation of raw starch:

such as step one.

2. Raw starch hydrolysis

The reaction system is 100mL, the concentration of the substrate (raw cassava flour or raw corn flour) is set to be 100g/L, and the loading amount of the raw amylase preparation is set to be four gradients of 50, 100, 150 and 200U/g substrate

The specific preparation method of the reaction system comprises the following steps: to pH 4.5Na₂HPO₄The raw tapioca flour or raw corn flour was added to the citric acid buffer solution together with the raw amylase preparation of example 3 and the commercial alpha-amylase (product of Beijing Soilebao, Inc.; ratio of addition of raw amylase preparation enzyme activity: commercial alpha-amylase activity ═ 1: 1). One enzyme activity unit (U) of a commercial alpha-amylase is defined as the amount of enzyme required to hydrolyze soluble starch to produce 1. mu. moL of reducing sugars per minute at 65 ℃ and pH 4.5 reaction conditions.

The reaction conditions were 40 ℃ and 180rpm for a total of 72 hours. Samples are taken in the reaction time of 0, 12, 24, 36, 48, 60 and 72 hours respectively, and the content of reducing sugar and the conversion rate of raw starch in the reaction system are detected.

The results are shown in Table 5, which shows that the conversion of raw corn starch reached complete hydrolysis at 60 hours of reaction at an enzyme addition of 100U/g.

TABLE 5 starch conversion (%), of green corn meal hydrolyzed by synergy of Penicillium oxalicum A2-13-producing amylase preparation with commercial alpha-amylase

The results are shown in Table 6, which shows that the conversion of raw cassava starch reached complete hydrolysis at 60 hours of reaction when the enzyme addition was 150U/g.

TABLE 6 starch conversion (%), by Penicillium oxalicum A2-13 production of amylase preparation and Co-hydrolysis of raw cassava flour with commercial alpha-amylase

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. Penicillium oxalicum (B)Penicillium oxalicum) A2-13, wherein the preservation number of the Chinese type culture Collection is CCTCC NO: m2020319.

2. A microbial inoculum, which is characterized in that: the microbial agent comprises the Penicillium oxalicum (Penicillium) of claim 1 as an active ingredientPenicillium oxalicum）A2-13。

3. Culturing the Penicillium oxalicum (Penicillium) of claim 1 (Penicillium oxalicum) The method of A2-13, comprising the steps of: culturing the Penicillium oxalicum of claim 1 in a medium at 20-32 ℃: (Penicillium oxalicum）A2-13；

The culture medium consists of an organic carbon source and a salt solution; wherein the ratio of the organic carbon source to the salt solution is 5g to 100 mL; the salt solution is prepared from water and KH₂PO₄Nitrogen source, MgSO₄·7H₂O, Tween 80 and microelement mother liquor, wherein each liter of the saline solution contains KH₂PO₄4.0g of nitrogen source, 2-8g of nitrogen source and MgSO₄·7H₂0.6g of O, 802 mL of Tween and 0.1mL of the microelement mother liquor; the microelement mother liquor is composed of solute and solvent, the solvent is water, and the solute and the concentration thereof are respectively FeSO₄·7H₂O 2.5 g/L、MnSO₄·H₂O 0.8 g/L、ZnSO₄·7H₂O0.7 g/L and CoCl₂1.0 g/L。

4. The method of claim 3, wherein: the organic carbon source is agricultural organic waste.

5. The method of claim 4, wherein: the agricultural organic waste is selected from any one or more of the following: wheat bran, crystalline cellulose, corn cob, bagasse and rice straw.

6. The method of claim 3, wherein: the nitrogen source is ammonium nitrate, yeast extract, ammonium sulfate, urea, peptone, sodium nitrate and/or soybean cake powder.

7. The method of claim 3, wherein: the pH value of the culture medium is 3.5-5.5.

8. The method of claim 4, wherein: the particle size of the agricultural organic waste is 0.25 mm.

9. The method of claim 5, wherein: the agricultural organic waste is wheat bran and crystalline cellulose, and the proportion of the wheat bran to the crystalline cellulose in the culture medium is 2g:3g of the total weight.

10. The method of claim 7, wherein: the pH of the medium was 5.5.

11. Penicillium oxalicum (Penicillium) of claim 1 (Penicillium oxalicum) The use of A2-13 or the microbial inoculum of claim 2 in any one of the following a1) -a 6):

a1) degrading raw starch or raw starch-containing material;

a2) preparing a product for degrading raw starch or raw starch-containing material;

a3) preparing raw amylase;

a4) preparing a product for producing raw amylase;

a5) producing glucose, maltose or ethanol;

a6) preparing a product for the production of glucose, maltose or ethanol.

12. The application of the culture medium in a2), a4) or a6) is as follows:

a4) preparing a product for producing raw amylase;

a6) preparing a product for the production of glucose, maltose or ethanol;

in said use, the Penicillium oxalicum (B) of claim 1 is cultured in a mediumPenicillium oxalicum）A2-13；

The culture medium consists of an organic carbon source and a salt solution; wherein the ratio of the organic carbon source to the salt solution is 5g to 100 mL; the salt solution is prepared from water and KH₂PO₄Nitrogen source, MgSO₄·7H₂O, Tween 80 and microelement mother liquor, wherein each liter of the saline solution contains KH₂PO₄4.0g of nitrogen source, 2-8g of nitrogen source and MgSO₄·7H₂0.6g of O, 802 mL of Tween and 0.1mL of the microelement mother liquor; the microelement mother liquor consists of solute and solvent, the solvent is water, the solute and the solventRespectively at a concentration of FeSO₄·7H₂O 2.5 g/L、MnSO₄·H₂O 0.8 g/L、ZnSO₄·7H₂O0.7 g/L and CoCl₂1.0 g/L。

13. Use according to claim 12, characterized in that: the organic carbon source is agricultural organic waste.

14. Use according to claim 13, characterized in that: the agricultural organic waste is selected from any one or more of the following: wheat bran, crystalline cellulose, corn cob, bagasse and rice straw.

15. Use according to claim 12, characterized in that: the nitrogen source is ammonium nitrate, yeast extract, ammonium sulfate, urea, peptone, sodium nitrate and/or soybean cake powder.

16. Use according to claim 12, characterized in that: the pH value of the culture medium is 3.5-5.5.

17. Use according to claim 13, characterized in that: the particle size of the agricultural organic waste is 0.25 mm.

18. Use according to claim 14, characterized in that: the agricultural organic waste is wheat bran and crystalline cellulose, and the proportion of the wheat bran to the crystalline cellulose in the culture medium is 2g:3g of the total weight.

19. Use according to claim 16, characterized in that: the pH of the medium was 5.5.