CN115725421A - Penicillium oxalicum genetically engineered bacterium GXUR001 and application thereof in preparation of raw amylase preparation - Google Patents
Penicillium oxalicum genetically engineered bacterium GXUR001 and application thereof in preparation of raw amylase preparation Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Penicillium oxalicum genetically engineered bacterium GXUR001 with a preservation number of CCTCC NO: M2022120, which is classified and named as Penicillium oxalicum (Penicillium oxalicum) TE4-10 delta CxrC:: amyR. Experiments prove that after the penicillium oxalicum GXUR001 is cultured in a liquid, the fermentation liquid has the activity of raw amylase, including the activity of raw cassava amylase, the activity of raw corn amylase, the activity of raw wheat amylase and the activity of raw rice amylase. The fermentation liquor can be used for efficiently hydrolyzing untreated raw cassava starch to generate glucose, and has application potential in raw cassava starch conversion and utilization. Therefore, the penicillium oxalicum GXUR001 can be used for producing raw amylase preparation, and the preparation can efficiently hydrolyze untreated raw cassava starch and raw corn starch for preparing glucose and fuel ethanol.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a penicillium oxalicum genetically engineered bacterium GXUR001 and application thereof in preparation of a raw amylase preparation.
Background
The 21 st century is a global industrialized and urbanized era. In order to implement the concept of harmonious symbiosis and green development of people and nature, the renewable energy can not only meet the increasing energy demand of people, but also effectively solve the problems of environmental pollution and the like. Renewable energy sources mainly include solar energy, wind energy, hydroelectric energy, geothermal energy, and biomass energy. Among them, biomass has a great potential for development. The biomass energy has the advantages of being renewable, cheap in raw materials and the like.
The biofuel ethanol produced by taking biomass as a raw material is an important biomass energy, and can be roughly divided into three generations according to different raw materials: the first generation uses sucrose and starch as main raw materials, the second generation uses lignocellulose as main raw materials, and the third generation uses algae as main raw materials. Because the cost of producing biofuel ethanol by using lignocellulose and algae as raw materials is too high, the first generation of biofuel ethanol is still mainly used in the industry at present.
The starch used for producing biofuel ethanol in China mainly comes from aged grains (such as rice, wheat, corn and the like), cassava starch and the like. In the traditional process, the starch processing is mainly divided into two steps: raw starch is firstly gelatinized and liquefied under the action of alpha-amylase at high temperature (about 100 ℃), and then is hydrolyzed into glucose by saccharifying enzyme at 50-60 ℃. The process flow is complex, the energy consumption is high, and the environment is polluted (Cripwell RA, rose SH, favaro L, et al, construction of industrial Saccharomyces cerevisiae strains for the influencing consistent bioprocessing of raw stage Biotechnol Biofuels.2019, 12. There is a great need to develop more efficient and energy efficient starch processing techniques.
Researchers find that raw amylase can directly hydrolyze raw starch into glucose below the starch gelatinization temperature, so that the steps of high-temperature gelatinization and liquefaction are omitted, energy input is reduced, and the cost is reduced. Raw amylases include primarily raw starch saccharifying enzymes, alpha-raw amylases, and beta-raw amylases, and are primarily derived from filamentous fungi such as Aspergillus and Penicillium. However, the yield of raw amylase of filamentous fungi is low, the cost is high, and the industrial production level is far from being reached. Fungal breeding, including mutation breeding and molecular breeding, is currently the most effective way to increase raw amylase production and reduce costs (Zhao SF, xiaong BY, yang L, et al, genetic modifications of diagnostic protocols, new instructions in general, and expression of raw-static-differential enzyme expression in Penicillium. Biotechnology Biofuels bioprod.2022, 62.
Disclosure of Invention
The invention aims to solve the technical problem of providing a penicillium oxalicum genetic engineering bacterium GXUR001 for producing amylase at high yield and application thereof in preparing a raw amylase preparation.
In order to solve the technical problem, the invention adopts the following technical scheme:
the Penicillium oxalicum genetically engineered bacterium GXUR001 has a preservation number of CCTCC NO: M2022120, and is classified and named as Penicillium oxalicum (Penicillium oxalicum) TE4-10 delta CxrC: amyR.
The penicillium oxalicum genetic engineering bacterium GXUR001 can be applied to hydrolysis of raw starch or raw starch-containing substances.
The raw starch is agricultural organic matter containing raw starch such as raw cassava powder, raw corn powder, raw rice powder, raw potato powder, and raw wheat powder.
The application of the penicillium oxalicum genetically engineered bacterium GXUR001 in preparing raw amylase preparations.
A raw amylase preparation, wherein the active ingredient is derived from the genetically engineered bacterium GXUR001 of Penicillium oxalicum of claim 1 or a culture fermentation product thereof.
The process for producing the raw amylase preparation comprises culturing the genetically engineered Penicillium oxalicum GXUR001 of claim 1 in a liquid fermentation medium at 28 ℃ and 180rpm, and collecting the fermentation product.
The liquid fermentation medium is prepared according to the following steps: the ratio of wheat bran, crystalline Avicel and salt solution is 2g, 3g, 100ml, and each 100mL of salt solution contains 0.4g (NH) 4 ) 2 SO 4 ,0.4g KH 2 PO 4 ,0.06g MgSO 4 ·7H 2 O,0.06g CaCl 2 Tween-80.2mL, 100 uL of microelement mother liquor, and pH 5.5; the microelement mother liquor contains 1L of microelementThere are 5g of FeSO 4 ·7H 2 O,1.6g MnSO 4 ·H 2 O,1.4g ZnSO 4 ·7H 2 O,2.0g CoCl 2 。
The culture conditions were 28 ℃ and 180rpm,8d.
A method for hydrolyzing raw starch or a raw starch-containing substance comprises adding the above raw amylase preparation to raw starch or a raw starch-containing substance to carry out a reaction of hydrolyzing raw starch.
The reaction conditions were pH 4.5 and 65 ℃.
The raw amylase preparation can be used for preparing glucose and fuel ethanol
Aiming at the problems of low yield and high cost of the existing raw amylase, the inventor obtains a Penicillium oxalicum genetic engineering bacterium GXUR001 with the preservation number of CCTCC NO: M2022120 and classified name of Penicillium oxalicum (Penicillium oxalicum) TE4-10 delta CxrC: amyR through separation and screening. The penicillium oxalicum GXUR001 is prepared by 4 rounds of EMS chemical mutagenesis and 2 rounds of Co based on the penicillium oxalicum mutant strain A2-13 60 Mutation strain TE4-10 is obtained by gamma-ray mutagenesis, and then a transcription repressing gene PoxCxrC and an over-expression transcription activating gene PoxMyR are simultaneously knocked out from the strain TE4-10. Experiments prove that after the penicillium oxalicum GXUR001 is cultured in a liquid, the fermentation liquid has the activity of raw amylase, including the activity of raw cassava amylase, the activity of raw corn amylase, the activity of raw wheat amylase and the activity of raw rice amylase. The fermentation liquor can be used for efficiently hydrolyzing untreated raw cassava starch to generate glucose, and has application potential in raw cassava starch conversion and utilization. Therefore, the penicillium oxalicum GXUR001 can be used for producing raw amylase preparations, and the preparations can efficiently hydrolyze untreated raw cassava starch for preparing glucose and fuel ethanol.
Drawings
FIG. 1 is a graph showing a comparison of the amylase yields of mutants obtained during mutagenesis of Penicillium oxalicum.
FIG. 2 is a schematic diagram of the construction of the penicillium oxalicum genetically engineered bacterium GXUR 001.
FIG. 3 is a diagram showing the result of the test of the present invention for constructing a genetically engineered bacterium GXUR001 from Penicillium oxalicum, in which: m:1kb DNA marker,1 ddH 2 O, 2-10,3-5:3 GXUR001 transformants.
FIG. 4 is a graph showing the efficiency of producing amylase-hydrolyzed raw cassava starch by constructing the genetically engineered bacterium of Penicillium oxalicum GXUR001 of the present invention, wherein: a: the concentration of glucose released by hydrolyzing raw cassava flour, b: hydrolysis rate of raw cassava starch, c: concentration of glucose released by hydrolyzed raw corn meal, d: hydrolysis rate of raw corn starch.
Description of preservation information
Penicillium oxalicum (Penicillium oxalicum) TE4-10 delta CxrC: amyR with the preservation number of CCTCC NO: M2022120 and the preservation date: 16/02/2022, with the preservation address: wuhan university, china, zip code 430072, depository: china center for type culture Collection.
Preservation conditions are as follows: freezing at low temperature (-70 deg.C).
Detailed Description
The present invention is described in further detail below with reference to specific examples. Wherein, the experimental methods are all conventional methods unless otherwise specified; the materials, reagents and the like used can be obtained from commercial sources; the testa Tritici is wheat bran with water content below 5%.
Drawing the following glucose standard curve and measuring the amylase activity of the raw cassava according to the following methods:
1. drawing of glucose standard curve
And (3) taking 0.05g of glucose standard substance into a 50mL volumetric flask, and carrying out constant volume through deionized water to obtain a glucose standard solution mother liquor with the concentration of 1 mg/mL. According to the formulation of Table 1, the reagents were added to a 2mL EP tube, mixed well, placed in a boiling water bath for 5 minutes, and the sample was cooled to room temperature. Centrifuging at 12,000rpm for 1min, collecting 200 μ L of upper layer solution, loading into 96-well enzyme label plate, measuring light absorption value at 540nm wavelength, drawing glucose standard curve, and obtaining linear regression equation of y =3.297x-0.0318 and variance R 2 ＝0.9993。
TABLE 1 preparation of glucose Standard Curve solutions
2. Determination of activity of raw natural cassava amylase
(1) Preparing a crude enzyme solution: the fermentation broth was filtered through four layers of gauze, and the filtrate was put into a 10mL EP tube, and centrifuged at 8000rpm,4 ℃ for 10min. Taking the supernatant to obtain the crude enzyme solution. With Na 2 HPO 4 Citrate buffer (pH 4.5) was diluted appropriately as the experimental group enzyme solution.
(2) Preparation of control enzyme solution: taking a certain amount of enzyme solution to be detected into a 2mL centrifuge tube, and boiling water bath for 15min as a control group enzyme solution.
(3) Preparation of hydrolysis substrate: weighing 1g raw cassava starch to 100mL Na 2 HPO 4 -citric acid buffer (pH 4.5) to prepare a 1% raw native tapioca starch solution. Taking 0.45mL to 2.0mL EP tube as hydrolysis substrate, placing in 65 deg.C water bath, and preheating for 5min.
(4) 50 mu L of the enzyme solution of the experimental group is added into the preheated substrate, three are parallel, 50 mu L of the enzyme solution of the control group is added into the control group, and the hydrolysis is carried out for 30min under the condition of 65 ℃ water bath.
(5) And (4) after the step (4) is finished, adding 1mL of DNS, carrying out 5min in boiling water at 100 ℃, and cooling on ice after full color development.
(6) mu.L of the solution obtained in step (5) was put into a 96-well plate, and the absorbance at 540nm was measured. And calculating to obtain the concentration of reducing sugar according to the glucose standard curve equation, and then obtaining the yield of raw amylase by an enzyme yield calculation formula.
Raw amylase production (U/mL) = reaction system volume × glucose concentration in reaction system × dilution factor × 1000/glucose relative molecular mass/reaction time/added enzyme solution volume.
One enzyme activity unit (U) is defined as: the raw native tapioca amylase hydrolyzed raw native tapioca starch at a pH of 4.5 at 65 ℃ in an amount of enzyme required to produce 1 μmoL of reducing sugar per 1 minute.
preparing a citric acid-disodium hydrogen phosphate buffer solution with the pH of 4.5:
0.1M citric acid solution: 19.2g of citric acid were dissolved in 1L of deionized water.
0.2M disodium hydrogen phosphate solution: 28.4g of disodium hydrogen phosphate are dissolved in 1L of deionized water.
Appropriate amounts of 0.1M citric acid solution and 0.2M disodium hydrogen phosphate solution were taken and adjusted to pH 4.5 under a pH indicator.
Example I construction of genetically engineered Penicillium oxalicum GXUR001
1. Obtaining of Penicillium oxalicum mutant TE4-10
The penicillium oxalicum GXUR001 is prepared by taking a penicillium oxalicum mutant strain A2-13 (preservation number: CCTCC No: M2020319) as an initial strain, carrying out chemical mutagenesis by 2 rounds of Ethyl Methyl Sulfonate (EMS) to obtain a mutant strain TE2-23 with the yield of raw amylase improved compared with that of the initial strain A2-13, and then carrying out 2 rounds of Co Co on the TE2-23 60 Gamma-ray irradiation to obtain a mutant strain Co2-12 with higher raw amylase yield. And performing 2 rounds of EMS mutagenesis on the mutant strain Co2-12, and screening to obtain a mutant strain TE4-10.
The method comprises the following specific operations: when the original strain A2-13 is subjected to EMS chemical mutagenesis and treated for 10 hours, the lethality rate is 96.2%. Under the condition, mutation and screening are carried out to obtain a mutant strain TE2-23; when the strain TE2-23 is irradiated by Co 60-gamma rays, selecting a corresponding dose with the lethality of 87.2 percent, and carrying out mutation and screening to obtain a mutant strain Co2-12; and then EMS treatment is carried out on the mutant strain Co2-12 spore suspension, and finally the mutant strain TE4-10 is obtained.
The determination and comparison show that the amylase production of the mutant strain obtained in the mutagenesis process is shown in FIG. 1.
2. Construction of DNA cassette for construction of genetic engineering bacteria of penicillium oxalicum
PoxCxrC and PoxMyR are transcription repressing and activating factors that negatively and positively regulate the production of raw amylase in Penicillium oxalicum (Zhang T, mai RM, fang QQ, et al, regulation function of the novel transcription factor CxrC in Penicillium oxalicum. Molecular Microbiology,2021,116 (6): 1512-1532 Li Z, yao G, wu R, et al, synergistic and dose-controlled regulation of cellular gene expression in Penicillium oxide. PLoS Genetics,2015,11 (9): e 1005509), respectively.
The DNA box for constructing the penicillium oxalicum genetic engineering bacteria sequentially consists of a PoxCxrC homologous left arm, a bleomycin resistance gene Ble, a PoxMyR full-length gene sequence including a promoter region, a development reading frame, a terminator region and a PoxCxrC homologous right arm from upstream to downstream. The homologous left arm of the PoxCxrC is shown as SEQ.NO. ID.1 of the sequence table; the Ble gene is shown as SEQ No. ID.2 of a sequence table; the PoxMyR full-length gene is shown as SEQ No. ID.3 of the sequence table; the homologous right arm of the PoxCxrC is shown as SEQ No. ID.4 of the sequence table.
3. Construction of Penicillium oxalicum Gene engineering Strain GXUR001 (shown in FIG. 2)
(1) mu.L of 100mM spermidine aqueous solution was pipetted into a DNA cassette (20. Mu.g), mixed well and added to 100. Mu.L of mutant TE4-10 protoplast, mixed well and left to stand on ice for 30min.
(2) 1mL of PTC solution was added in step 1, mixed well and allowed to stand at room temperature for 25min.
(3) 2mL of STC solution in the step 2 is uniformly mixed, the mixed solution is added into 20mL of regeneration culture medium and uniformly mixed, and then the mixed solution is poured into sterilized culture dishes (about 5mL of the mixed solution is poured into each culture dish); after complete coagulation, the mixture is placed in an incubator at 28 ℃ for 1 hour, and then 35mL of PDA culture medium with bleomycin resistance (the content of the bleomycin is 100 mu g/mL) is added to cover the surface of the mixture; after complete solidification, putting into an incubator at 28 ℃, and carrying out inverted culture for 3-4d.
(4) The transformed asexual spores were washed with 0.1% Tween-80 solution, subjected to gradient dilution with sterile water, and each dilution was plated on PDA medium plates containing 100. Mu.g/mL bleomycin, and subjected to inverted culture at 28 ℃ for 2-3 days.
(5) After step 4, single colonies on the medium were picked, genomic DNA was extracted, and PCR validation was performed (results are shown in fig. 3).
AmyR is renamed to penicillium oxalicum genetically engineered bacterium GXUR001 (short for penicillium oxalicum GXUR 001).
EXAMPLE II preparation of Penicillium oxalicum GXUR001 raw amylase preparation
1. Preparation of culture medium for liquid culture of penicillium oxalicum GXUR001
Preparation of a culture medium:the ratio of wheat bran, crystalline Avicel and salt solution is 2g, 3g, 100ml, and each 100mL of salt solution contains 0.4g (NH) 4 ) 2 SO 4 ,0.4g KH 2 PO 4 ,0.06g MgSO 4 ·7H 2 O,0.06g CaCl 2 Tween-80.2mL, 100 uL of microelement mother liquor, and pH 5.5; the microelement mother liquor contains 5g FeSO per 1L 4 ·7H 2 O,1.6g MnSO 4 ·H 2 O,1.4g ZnSO 4 ·7H 2 O,2.0g CoCl 2 . The size of the wheat bran particles is less than or equal to 0.18mm.
2. Preparation of spore liquid
The PDA solid culture medium is sterilized for 20min at 121 ℃ to obtain a sterilized PDA plate.
The penicillium oxalicum GXUR001 is inoculated on a PDA plate and is placed in a constant temperature incubator at 28 ℃ for culture for 4 days. Washing Penicillium oxalicum GXUR001 asexual spores with 0.1% Tween-80 solution to a concentration of 1X 10 8 one/mL.
3. Preparation of raw amylase preparation
Inoculating the conidium suspension containing the penicillium oxalicum GXUR001 in the step 2 into a liquid culture medium to a final concentration of 1 × 10 6 one/mL. The culture was carried out at 28 ℃ for 8d on a shaker at 180 rpm.
Filtering the fermentation liquid by using four layers of gauze, placing the filtrate in a 10mL centrifuge tube, centrifuging at 4 ℃ and 8000rpm, collecting supernatant, namely raw amylase preparation, and measuring the activity of the raw amylase.
The results are as follows: the raw cassava amylase yield of the penicillium oxalicum GXUR001 raw amylase preparation is 252.58 +/-6.28U/mL. This enzyme production is currently the highest compared to the reported fungal strains (Table 2).
TABLE 2 comparison analysis of the yield of filamentous fungal raw amylase
| Strain name | Starch substrates | Raw amylase yield (U/mL) | Reference to the literature |
| Penicillium oxalicum GXUR001 | Natural raw cassava flour | 252.58 | Study of the invention |
| Penicillium oxalicum TE4-10 | Natural raw cassava flour | 218.6 | Study of the invention |
| Penicillium oxalicum A2-13 | Natural raw cassava flour | 190.96 | |
| Penicillium oxalicum OXXGA 15A | Natural raw cassava flour | 100.97 | |
| Penicillium oxalicum delta PoxKu70 | Raw cassava flour | 55.1 | |
| Penicillium oxalicum GXU20 | Raw cassava flour | 20 | |
| Geobacillus sp.4J | Raw corn flour | 39.6 | |
| Aspergillus flavus NSH9 | Raw and western rice flour | 10.44 | |
Reference documents:
1、Gu LS,Tan MZ,Li SH,et al.,ARTP/EMS-combined multiple mutagenesis efficiently improved production of raw starch-degrading enzymes in Penicillium oxalicum and characterization of the enzyme-hyperproducing mutant.Biotechnology for Biofuels,2020,13:187.
2、Wang L,Zhao S,Chen XX,et al.,Secretory overproduction of a raw starch-degrading glucoamylase in Penicillium oxalicum using strong promoter and signal peptide.Applied Microbiology and Biotechnology,2018,102:9291–9301.
3、Lin HJ,Xian L,Zhang QJ,et al.,Production of raw cassava starch degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol.Journal of Industrial Microbiology and Biotechnology,2011,38:733–742.
4、Jiang T,Cai M,Huang M,et al.,Characterization of a thermostable raw-starch hydrolyzingα-amylase from deep-sea thermophile Geobacillus sp.Protein Expression and Purification,2015,114:15-22.
5、Karim KMR,Husaini A,Sing NN,et al.,Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase(GA2)derived from Aspergillus flavus NSH9.Protein Expression and Purification,2019,164:105462.
EXAMPLE III hydrolysis of raw starch Using raw Amylase preparation
1. Preparation of raw tapioca starch
Drying the cassava blocks purchased from farmer markets in the air, crushing the cassava blocks by using a crusher, and sieving the crushed cassava blocks by using a 80-mesh sieve; washing the cassava powder with deionized water until the supernatant is transparent and clear; drying the washed cassava powder in an oven at the temperature of 55 ℃ to constant weight; pulverizing, sieving with 80 mesh sieve, packaging with fresh-keeping bag, and placing in dry and ventilated place.
2. Efficiency of penicillium oxalicum GXUR001 raw amylase preparation in hydrolyzing raw starch
The reaction system is 50mL, the concentration of a substrate (raw cassava flour/raw corn flour) is set to be 150g/L, and the addition amount of raw amylase is three gradients of 100, 150 and 250U/g (the activity of the raw amylase is calculated by the activity of the raw cassava amylase).
The specific preparation method of the reaction system comprises the following steps: weighing 7.5g of the treated raw cassava/corn meal to Na 2 HPO 4 Citric acid buffer (pH 4.5) and the raw amylase preparation of example 2. The reaction was carried out at 40 ℃ and 180rpm for 120h. Samples are taken in the reaction of 24 hours, 48 hours, 72 hours and 96 hours respectively, the content of reducing sugar in the reaction system is detected, and the hydrolysis rate of raw starch is calculated.
Raw starch hydrolysis rate = 0.9 x 100 weight of glucose per weight of starch in raw tapioca starch.
The result is shown in figure 4, which shows that when the enzyme addition amount is 250U/g, the released glucose concentration is 117.2g/L and the raw cassava starch hydrolysis rate reaches 93.04% when the raw cassava powder is hydrolyzed for 120 hours; when the raw corn flour is hydrolyzed for 84 hours, the concentration of released glucose is 126.1g/L, and the hydrolysis rate of the raw corn starch is 100%.
Claims (10)
1. The Penicillium oxalicum genetically engineered bacterium GXUR001 has a preservation number of CCTCC NO: M2022120, and is classified and named as Penicillium oxalicum (Penicillium oxalicum) TE4-10 delta CxrC: amyR.
2. The use of the genetically engineered bacterium GXUR001 according to claim 1 for hydrolyzing raw starch or raw starch-containing material.
3. Use according to claim 2, characterized in that: the raw starch is raw cassava powder, raw corn powder, raw rice powder, raw potato powder and raw wheat powder.
4. The use of the genetically engineered bacterium GXUR001 of Penicillium oxalicum of claim 1 for the preparation of raw amylase preparation.
5. A raw amylase preparation characterized in that the active ingredient in the preparation is derived from the genetically engineered bacterium Penicillium oxalicum GXUR001 or a culture fermentation product thereof according to claim 1.
6. A process for preparing a raw amylase preparation of claim 5, wherein: culturing the penicillium oxalicum genetically engineered bacterium GXUR001 of claim 1 in a liquid fermentation medium at 28 ℃ and 180rpm, and collecting the fermentation product.
7. The method of claim 6, wherein:
the liquid fermentation medium is prepared according to the following steps: the ratio of wheat bran, crystalline Avicel to salt solution is 2g, 3g, 100ml, and each 100mL of salt solution contains 0.4g (NH) 4 ) 2 SO 4 ,0.4g KH 2 PO 4 ,0.06g MgSO 4 ·7H 2 O,0.06g CaCl 2 Tween-80.2mL, 100 uL of microelement mother liquor, and pH 5.5; the microelement mother liquor contains 5g FeSO per 1L 4 ·7H 2 O,1.6g MnSO 4 ·H 2 O,1.4g ZnSO 4 ·7H 2 O,2.0g CoCl 2 (ii) a The culture conditions were 28 ℃ and 180rpm,8d.
8. A method of hydrolyzing raw starch or raw starch-containing material, characterized by: adding the raw amylase preparation of claim 5 to raw starch or raw starch-containing material to perform a reaction of hydrolyzing the raw starch.
9. The method of claim 8, wherein: the reaction conditions were pH 4.5, 65 ℃.
10. The raw amylase preparation of claim 5 for use in the production of glucose, fuel ethanol.
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| CN101955887A (en) * | 2010-09-02 | 2011-01-26 | 广西大学 | Raw-starch amylase producing penicillium and raw-starch amylase preparation produced thereby |
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| CN108949590A (en) * | 2018-07-24 | 2018-12-07 | 广西大学 | It is a kind of it is high generate tapioca enzyme penicillium oxalicum engineering bacteria and its application |
| CN112210502A (en) * | 2020-10-28 | 2021-01-12 | 广西大学 | Penicillium oxalicum mutant strain A2-13 and application thereof in preparation of raw amylase preparation and degradation of raw starch |
| US20210292776A1 (en) * | 2020-03-19 | 2021-09-23 | Lallemand Hungary Liquidity Management Llc | Yeast expressing glucoamylase with enhanced starch hydrolysis |
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| CN101955887A (en) * | 2010-09-02 | 2011-01-26 | 广西大学 | Raw-starch amylase producing penicillium and raw-starch amylase preparation produced thereby |
| US20150291984A1 (en) * | 2012-03-30 | 2015-10-15 | Novozymes A/S | Processes for producing fermentation products |
| CN108949590A (en) * | 2018-07-24 | 2018-12-07 | 广西大学 | It is a kind of it is high generate tapioca enzyme penicillium oxalicum engineering bacteria and its application |
| US20210292776A1 (en) * | 2020-03-19 | 2021-09-23 | Lallemand Hungary Liquidity Management Llc | Yeast expressing glucoamylase with enhanced starch hydrolysis |
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