CN116083250A - Aspergillus oryzae strain for producing active lipase, application and fermentation enzyme production method and application - Google Patents
Aspergillus oryzae strain for producing active lipase, application and fermentation enzyme production method and application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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Abstract
The invention discloses an aspergillus oryzae strain for producing active lipase, application, a fermentation enzyme production method and application, wherein the aspergillus oryzae strain is a new strain MASL-01 of aspergillus, and the new strain MASL-01 is preserved in the microorganism strain preservation center of Guangdong province, and the preservation number is GDMCC NO.63015. The lipase activity generated by the novel strain of aspergillus oryzae can reach 378000U/g, the lipase can efficiently catalyze the hydrolysis of fatty glyceride into fatty acid, the hydrolysis rate can reach more than 90%, and the lipase has good sn-1,3 position specificity on glyceride and preferentially catalyzes the reaction of fatty acid at sn-1,3 positions of glycerol; meanwhile, the lipase can also catalyze fatty acid to carry out esterification reaction, and the esterification rate is up to 99%. The optimal reaction temperature of the lipase is 40-45 ℃, the thermal stability of the lipase is good, the lipase can realize faster and more efficient hydrolysis at a higher temperature, and the lipase has better industrial production application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an aspergillus oryzae strain for producing active lipase, application and a fermentation enzyme production method and application.
Background
Lipase (Lipase) is a biocatalyst for catalyzing reactions such as grease hydrolysis, esterification synthesis, transesterification and the like. Compared with a chemical catalyst, the biocatalyst has the advantages of high catalytic efficiency, low energy consumption, green environmental protection and the like, and the advantages are more important in the future social development; the lipase is mainly derived from animals, plants and microorganisms, wherein the lipase in the microorganisms has the advantages of rich varieties, short synthesis period, simple and convenient synthesis method and the like, so that the lipase prepared by the microorganisms becomes a main production synthesis way for preparing the lipase at present; the performances of the lipase include enzyme activity, catalytic specificity, thermal stability, repeated use times and the like, and the advantages and disadvantages of the characteristics determine the application of the lipase in various aspects of catalysis, and the lipase has wide application value in the fields of grease processing, organic synthesis and the like, and particularly in recent years, the industrial production and preparation of the lipase are gradually increased.
Most of the existing lipases have the problems of low enzyme activity, poor catalytic specificity, small catalytic reaction range and the like; so the research on lipase at present in China stays in the research stage of laboratory mostly, wherein patent document CN202010034954.X discloses a lipase and a strain producing the lipase, the strain belongs to candida parapsilosis, the strain is screened by coating a Victoria flat plate, the screened strain is fermented for 65 hours at the temperature of 30 ℃, the enzyme activity of fermentation liquor is 95.1+/-2.58U/mL, the preparation method has simple technological process, short culture period and higher economy, but the enzyme activity of the lipase is not high, no corresponding lipase treatment process exists, and the catalysis of the reaction needs to be high-efficiency so as to be further industrialized. Patent CN20110007349 describes an organic solvent resistant lipase with high yield, wider pH application range, better temperature stability, better tolerance to various organic solvents, capability of reacting in some organic solvents, long half-life, capability of splitting 1-phenethyl alcohol reaction and the like, and good application prospect in chiral alcohol splitting, but low enzyme activity for ester bond catalysis, small conversion rate, small catalytic reaction range and insufficient requirement for industrial application.
The lipase is applied to the field of food processing and has different requirements on grease processing, and the lipase has important application (sn is stereospecific position arrangement) on the sn-1,3 position specificity catalysis of glyceride, for example, after the position structure of fatty acid in glyceride is changed in the aspects of preparing nutrition and health care grease and special grease, the glyceride with more nutritive value of fatty acid at sn-1,3 position has multiple physiological functions, is beneficial to human digestion and is more beneficial to human health. Therefore, the development of a lipase with high activity and high specificity for catalyzing hydrolysis of the sn-1,3 position of glyceride is of great importance.
Disclosure of Invention
In order to solve the technical problems, the novel aspergillus oryzae strain for producing the high-activity lipase is obtained through screening, the novel strain is combined with the optimized enzyme production fermentation process, the lipase which has high activity and is specifically catalyzed on the sn-1,3 position of glyceride can be produced, meanwhile, the lipase can keep the catalytic activity at a higher temperature, the proper catalytic temperature is higher, the novel aspergillus oryzae strain is suitable for catalyzing grease to produce fatty acid at a higher temperature, the production efficiency can be remarkably improved, and the production cost is reduced.
In order to achieve the above object, the present invention provides an aspergillus oryzae strain producing active lipase, wherein the aspergillus oryzae strain is aspergillus oryzae MASL-01 (Aspergillus oryzae MASL-01) and is deposited in the collection of microorganism strains in Guangdong province, the deposit address is building 5, building 59 of the 100 th university of pioneer in Guangzhou, and the deposit number is GDMCC No.63015.
Based on a general inventive concept, the invention also provides an application of the aspergillus oryzae strain for producing the active lipase in catalyzing the hydrolysis of the fatty glyceride.
Based on a general inventive concept, the invention also provides a fermentation and enzyme production method of a novel Aspergillus oryzae strain for producing active lipase, which comprises the following steps:
s1, activating an aspergillus oryzae strain MASL-01 inoculated in a slant culture medium to obtain an activated strain;
s2, inoculating the bevel activated strain into a first-stage seed culture medium to obtain a first-stage seed culture strain; inoculating the first-level seed culture strain into a second-level seed culture medium to obtain a second-level seed strain; inoculating the second-level seed strain into a third-level seed culture tank to obtain a third-level seed culture solution;
s3, inoculating the three-level seed culture solution into a fermentation tank to obtain fermentation liquor, treating and collecting the fermentation liquor to obtain crude enzyme, and purifying and collecting the crude enzyme to obtain the lipase with high activity.
Preferably, the slant culture medium comprises 10g/L of yeast extract powder, 20-30 g/L of beef peptone, 20-40 g/L of glucose and 10-20 g/L of agar.
Preferably, the primary seed culture medium and the secondary seed culture medium comprise 10g/L of yeast extract powder, 20g/L of beef peptone and 20g/L of glucose.
Preferably, the three-stage seed culture tank and the culture medium in the fermentation tank comprise the following components in parts by weight: sucrose 0.1-5%, polypeptone 0-8%, corn flour 0-5%, yeast extract powder 0-5%, tween-80 0-3%, talcum powder 0-2%, naNO 3 0～1％、KH 2 PO 4 0.7％、Na 2 HPO 4 ·12H 2 O 0.25％、MgSO 4 ·12H 2 O 0.1％、CaCl 2 0.05%, olive oil 0-5%, the balance water, pH 5.8.
Preferably, the processing and collecting procedure in the step S3 is as follows: the fermentation broth is subjected to plate-frame filter pressing, mycelium and filtrate are collected, a high-speed bead mill stirrer is used for destroying mycelium cell walls, and the treated mycelium is subjected to salting-out precipitation and purification by adopting 10-30% ammonium sulfate solution; the filtrate is subjected to ultrafiltration concentration by adopting a 32-40 kDa membrane, then the concentrated solution is subjected to ammonium sulfate salting out, the salting-out precipitate with 10-0% saturation degree is collected, and the two salting-out precipitates are mixed to obtain refined crude enzyme.
Preferably, the purification and collection process in the step S3 is as follows: the crude enzyme is subjected to chromatographic purification by a hydrophobic chromatography column, firstly, buffer solution with the concentration of 20-100mM and the pH of 6.0-8.5 is used for eluting, then distilled water is used for eluting, finally, 10-70% ethanol water solution is used for eluting, the eluting component is collected, and after buffer solution replacement, ion exchange chromatography purification is performed, so that the lipase with high purity and high activity is obtained.
Based on a general inventive concept, the invention also provides an application of the lipase prepared by the fermentation enzyme production method in catalyzing hydrolysis of fatty glyceride.
Preferably, the temperature of the lipase catalyzed hydrolysis of the fatty glyceride is 40-45 ℃, and the pH of the lipase catalyzed hydrolysis of the fatty glyceride is 5-6.
Compared with the prior art, the invention has the following beneficial effects:
the lipase activity generated by fermenting the novel Aspergillus oryzae strain Aspergillus oryzae MASL-01 can reach 378000U/g, the lipase can efficiently catalyze the hydrolysis of fatty glyceride into fatty acid, the hydrolysis rate can reach more than 99%, the lipase has good sn-1,3 position specificity on glyceride, the fatty acid at sn-1,3 position is preferentially catalyzed and reacted, and the main hydrolysis route of triglyceride is as follows: firstly hydrolyzing the sn-1,3 position into diglyceride, then hydrolyzing the sn-1,3 position in the diglyceride to obtain monoglyceride, and finally hydrolyzing the sn-2 position in the monoglyceride to obtain glycerin; meanwhile, the lipase can catalyze fatty acid to carry out esterification reaction, and in the same way, the position sn-1,3 of glyceride is preferentially esterified in the esterification process, and the esterification rate is up to 99%;
the optimal reaction temperature of the lipase is 40-45 ℃, and the lipase has good thermal stability, and can keep more than 95% of activity at 30-45 ℃, so that the lipase can stably realize faster and efficient grease hydrolysis at a higher temperature, and has good industrial production application value.
Preservation of biological material:
aspergillus oryzae MASL-01 (Aspergillus oryzae MASL-01) was deposited at the China center for type culture Collection (GDMCC) of Guangdong, 11 and 28, with a deposit number of GDMCC No.63015 on floor 5 of the university of Hirship 100 in Guangzhou.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of Aspergillus oryzae strain initially screened from soil along the river of Hunan province in example 1, wherein FIG. 1A is a graph of Aspergillus oryzae strain with maximum aperture diameter initially screened, and FIG. 1B is a graph of ultraviolet lamp irradiation at 365 nm;
FIG. 2 is a diagram of the novel strain MASL-01 of Aspergillus oryzae obtained after re-screening in example 2 of the invention, wherein FIG. 2A is a diagram of the novel strain MASL-01 of Aspergillus oryzae obtained after re-screening, and FIG. 2B is a fluorescence diagram of the novel strain MASL-01 of Aspergillus oryzae under irradiation of 365nm ultraviolet lamp;
FIG. 3 is a graph showing the relation between the hydrolysis rate of the lipase hydrolyzed crude oil and the ratio of each component in experimental example 1 of the present invention;
FIG. 4 is a graph showing the enzyme activity of lipase at different temperatures and at different times in experimental example 2 of the present invention;
FIG. 5 is a graph showing the effect of different temperatures on lipase activity in experimental example 3 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated; the reagents used in the examples were all commercially available unless otherwise specified.
Example 1
Screening and obtaining Aspergillus oryzae strain
The screening method comprises the following steps:
(1) And (3) primary screening: a bag of 50g of soil sample rich in organic matters is taken along the coast of Hunan river, about 2g of soil sample is weighed and placed in 100mL of sterile water. Diluting into different dilutions by using a dilution coating method after shaking for 10min, coating samples of each dilution on a common flat plate, placing the flat plate in a biochemical incubator at 30 ℃ for culturing for 48 hours, selecting a bacterial colony with Aspergillus oryzae characteristics after the bacterial colony grows, further diluting and coating a plate to separate mixed bacteria, finally diluting and coating the separated Aspergillus oryzae on a rhodamine B flat plate, and selecting a strain with the largest aperture diameter and bacterial colony diameter as an Aspergillus oryzae initial strain.
The colony morphology of the initial strain of the aspergillus oryzae is shown in figure 1, and the result is shown in figure 1, wherein A is an aspergillus oryzae strain diagram with the largest aperture diameter of the primary screen, and is consistent with the colony morphology of the aspergillus oryzae; in the graph B is an ultraviolet lamp irradiation graph of 365nm, in the graph 1B, fluorescence is strong, the diameter of an ultraviolet ring is large, and the screened Aspergillus oryzae initial strain is primarily judged to have excellent reproduction and lipase exocrine ability.
The preparation method of the rhodamine B plate comprises the following steps: adding the formula A solution (glucose 2%, yeast extract 1%, beef peptone 2%, agar 2%) into the formula B solution (mixing olive oil and 2% polyvinyl alcohol at a ratio of 1:3, stirring and emulsifying at 10000 rpm), adding rhodamine B solution according to the amount of 0.1g rhodamine B per 100ml after fully mixing, sterilizing at 116 ℃ for 30min, pouring into a flat plate while the mixture is hot, and cooling to obtain the rhodamine B screening flat plate.
(2) And (3) re-screening: and re-screening the initial Aspergillus oryzae strain obtained by the primary screening by using a rhodamine B plate, and culturing an excellent strain which is the novel Aspergillus oryzae strain MASL-01 by using the rhodamine B plate, wherein the screening result is shown in a figure 2, a figure 2A is a MASL-01 diagram of the novel Aspergillus oryzae strain obtained after the re-screening, and a figure 2B is a MASL-01 fluorescent diagram of the novel Aspergillus oryzae strain irradiated by a 365nm ultraviolet lamp.
After observation of the new Aspergillus oryzae strain MASL-01 obtained by the re-screening, it can be seen that: the diameter of the bacterial colony on a observing culture medium is 45-55 mm, the bacterial colony has flocculent texture and a plurality of conidium structures, the bacterial colony is light yellow green to yellow green initially, and then is light tea brown, exudates are absent, and the reverse side of the bacterial colony is colorless; the specific microscopic morphological characteristics are as follows: conidiophore is spherical, and then is radial, scattered and small in size; conidiophores are produced from matrix or aerial hypha, the sporophore stems are 500-3000 or longer, the top sac is nearly spherical or flask-shaped, 20-45 μm, the whole or three-quarters surface is fertile, the spore-producing structure is single-layer or double-layer, the conidiophores are spherical or nearly spherical, and 5-8.6 μm. The mycelium DNA of the new strain GDMCC No.63015 is extracted, ITS sequence sequencing is performed, the mycelium DNA is identified to be highly homologous with aspergillus Aspergillus oryzae (aspergillus oryzae), and the mycelium DNA is identified in combination morphology and molecular biology, and belongs to the new strain of aspergillus and is named as MASL-01. The strain has been deposited at 28.2022 at 11.times.20 with the collection of microbiological strains (GDMCC) of Guangdong province at floor 5 of the university of Hirscho 100 in Guangzhou, with the deposit number GDMCC No.63015.
Example 2
The application of the novel Aspergillus oryzae strain MASL-01 in catalyzing the hydrolysis of fatty acid glyceride comprises the following steps:
(1) Preparation of high-activity lipase by fermentation of aspergillus oryzae new strain MASL-01
A small amount of the Aspergillus oryzae new strain MASL-01 mycelium prepared in the example 1 is inoculated into a slant culture medium, and the slant culture medium is subjected to activation culture for 24 hours at 28 ℃, wherein the composition of the slant culture medium is as follows: 10g/L yeast extract powder, 20g/L beef peptone, 20g/L glucose and 20g/L agar.
After 24 hours, aspergillus oryzae is cultivated and matured, a small amount of hypha is taken out from Aspergillus oryzae strains to be inoculated into a first-stage seed culture medium with the volume of 250mL and the liquid loading volume of 50mL, and the culture is carried out for 36 hours at 30 ℃ and 180rpm, wherein the first-stage culture medium comprises the following components: yeast extract 10g/L, beef peptone 20g/L, glucose 20g/L.
After the primary seed liquid is cultured, 4-5 mycelium pellets are selected from the primary seed culture medium to 18 secondary seed culture medium with the volume of 2L and the liquid loading amount of 500ml, and the secondary seed culture medium is cultured for 36 hours in a constant temperature shaking table at 30 ℃ and 180rpm and consists of: yeast extract 10g/L, beef peptone 20g/L, glucose 20g/L.
Then the mature second-level seed culture medium strain liquid is fully inoculated into 150L of third-level culture medium with the liquid loading amount of 60L, and is cultured for 24 hours in an environment with the temperature of 30 ℃, the stirring speed of 130rmp, the pH value of 5.8 and the aeration flow rate of 1vvm, wherein the third-level culture medium comprises the following components: 5% of sucrose, 8% of polypeptone, 5% of corn meal, 5% of yeast extract powder, 3% of tween-80, 2% of talcum powder and (NH 4) 2 SO 4 1％、KH 2 PO 4 0.7％、Na 2 HPO 4 ·12H 2 O 0.25％、MgSO 4 ·12H 2 O 0.1％、CaCl 2 0.05%, soybean oil 5% and the balance water.
And finally, transplanting the strain cultured in the three-stage culture medium into a fermentation tank culture medium with the volume of 1.3 cubic meters and the liquid loading capacity of 500L, wherein the composition of the fermentation culture medium is the same as that of the three-stage seed culture medium, and culturing and fermenting for 96 hours in an environment with the temperature of 30 ℃ and the stirring rotation speed 130rmp and the pH value of 5.8 to obtain a fermentation liquid.
The fermentation broth is subjected to plate-frame filter pressing, mycelium and filtrate are collected, a high-speed bead mill stirrer is used for destroying mycelium cell walls, and the treated mycelium is subjected to salting-out precipitation and purification by adopting 10-30% ammonium sulfate solution; ultrafiltering and concentrating the filtrate with 32-40 kDa film, salting out the concentrated solution with ammonium sulfate, collecting 10-30% saturated salting-out precipitate, and mixing the two salting-out precipitates to obtain refined crude enzyme. And (3) performing chromatographic purification on the refined crude enzyme by utilizing a hydrophobic chromatographic column, eluting by using a Tris-HCl buffer solution with the concentration of 20mM and the pH of 6.0, eluting by using distilled water, eluting by using a 70% ethanol water solution, collecting the eluted component, and performing ion exchange chromatography purification after buffer solution replacement to obtain the lipase with high purity and high activity.
(2) Lipase Activity assay
The method for measuring the lipase activity is an acid-base titration method, which uses olive oil and a 2% polyethylene aqueous solution as reaction substrates in a ratio of 1:3, mixes a phosphoric acid buffer solution of 0.1mol/L, pH 8.0.0, then adds lipase, reacts for ten minutes at the water bath temperature of 40 ℃, and terminates the reaction by absolute ethyl alcohol, and then uses a potassium hydroxide ethanol standard solution of 0.05mol/L for titration after the reaction is completed. The enzyme activity units are defined as: under the condition that the temperature is 40 ℃ and the pH=8.0, the method generates more micromoles of fatty acid per gram of lipase per minute through decomposition, is simple and convenient to operate, has quick and stable reaction measurement, and is a common lipase activity measurement method.
The activity of the prepared lipase with high purity and high activity was measured according to the method and the result was 378000U/g, which indicates that the lipase prepared by fermenting the novel strain of Aspergillus oryzae of the invention has extremely high enzyme activity.
(3) The high-purity high-activity lipase prepared by the fermentation catalyzes the hydrolysis of the fatty glyceride
Fatty acid glycerides were mixed with water at 1:0.5, enzyme amount: 100 units/fat, reaction temperature 40 ℃, reaction time 48h, stirring speed 300rpm/min. In the hydrolysis process, an alkali (potassium hydroxide) solution is properly added according to the hydrolysis condition, and after the reaction is finished, the mixture is acidified, stood, centrifuged, washed with water, dehydrated, filtered and evaporated to obtain free fatty acid, wherein the final hydrolysis rate of the fatty glyceride reaches 99.3 percent.
Experimental example 1
Catalytic specificity of Lipase for the sn-1,3 position of glycerides
The raw material crude oil is catalyzed and hydrolyzed at the pH value of 8.0 and the temperature of 40 ℃, and the lipase prepared in the example 2 is added, wherein the hydrolysis rate of the raw material crude oil by the lipase is the ratio of an acid value to a saponification value, the acid value is tested according to the national standard GB/T5530-2005, and the saponification value measuring method is tested according to the national standard GB/T5534-2008; the hydrolyzed oil samples with different hydrolysis rates are analyzed by a liquid analyzer to obtain the ratio of each hydrolyzed fatty acid to each glyceride, and the results are shown in figure 3: the results of fig. 3 show that the triglyceride ratio gradually decreases as hydrolysis proceeds, while the diglyceride ratio increases rapidly from the beginning of hydrolysis, and then the monoglyceride ratio also begins to increase; as the hydrolysis reaches a certain degree (the hydrolysis rate is 40-50%), the glycerol starts to appear and the ratio starts to rise rapidly, the diglyceride ratio starts to appear to decrease, and immediately after the monoglyceride ratio also reaches the highest, the glycerol starts to appear to decrease, and the ratio of the glycerol always rises as the hydrolysis proceeds. From this, it can be determined analytically that the main hydrolysis route of triglycerides is to hydrolyze from the sn-1,3 position to diglycerides, then to hydrolyze from the sn-1,3 position in diglycerides to obtain monoglycerides, and finally to hydrolyze from the sn-2 position in monoglycerides to obtain glycerol, indicating the catalytic specificity of lipase for the sn-1,3 position of glycerides. Meanwhile, the final hydrolysis rate of the crude oil reaches more than 99%, which further verifies the high activity of the lipase, and the lipase has extremely high hydrolysis rate for glyceride.
Experimental example 2
Thermal stability of Lipase
Taking lipase prepared in example 3 at 30 ℃, 40 ℃, 45 ℃, 60 ℃, 70 ℃ respectively in phosphate buffer solution with ph=8.0, shaking the flask for 3 hours, sampling according to different time courses, and respectively measuring enzyme activity values at different temperatures according to the enzyme activity measuring method in example 2, wherein the test results are shown in fig. 4: the results of FIG. 4 show that the relative enzyme activity values maintained at 30℃and 40℃and 45℃for three hours were all maintained at 95% or more, whereas lipase deactivation was rapid at 60℃and 70℃at which the reaction was not suitable for lipase-catalyzed reactions.
Experimental example 3
Influence of temperature on lipase Activity
The lipase obtained in example 2 was taken in a phosphate buffer solution having ph=8, and the activity values of the lipase were measured at 30 ℃, 35 ℃, 40 ℃, 42 ℃, 45 ℃, 50 ℃ and 60 ℃ according to the enzyme activity measurement method of example 2, respectively, and the results are shown in fig. 5: the enzyme activity value reaches the highest 375000u/g-378000u/g at the temperature of 40-45 ℃, and the enzyme activity is greatly reduced after 50 ℃, which is not suitable for the catalytic reaction of the lipase which is the biocatalyst. The result is consistent with the thermal stability of the lipase in experimental example 2, the lipase can have the highest enzyme activity at 40-45 ℃ and can keep more than 95% of the enzyme activity, and the lipase is suitable for rapidly and efficiently hydrolyzing grease at a higher temperature.
Comparative example 1
Comparison of different Lipase Activity
Novozym435 lipase, rhizomucormiehei lipase and Candida sp.99-125 lipase were purchased from the market, and the enzyme activities of Novozym435 lipase, rhizomucormiehei lipase and Candida sp.99-125 lipase were measured according to the lipase activity measurement method of Experimental example 1, respectively, and the specific results are shown in Table 1.
TABLE 1 measurement of different Lipase Activity and related Properties
| Lipase species | Enzyme Activity/U/g |
| Example 2 preparation of Lipase | 378000 |
| Novozym435 lipase | 12100 |
| Rhizomucormiehei lipase | 22500 |
| Candida sp.99-125 Lipase | 8510 |
As shown in the results of Table 1, the lipase activity generated by fermenting the novel Aspergillus oryzae strain MASL-01 obtained by mutagenesis screening is remarkably improved compared with the conventional lipase activity, and the novel Aspergillus oryzae strain MASL-01 has important application value in the enzyme-catalyzed hydrolysis of grease.
Comparative example 2
Catalytic effects of different lipases on hydrolysis of fatty acid glycerides
Mixing fatty glyceride with water according to a ratio of 1:0.5, respectively adding four different lipases in comparative example 1 for hydrolysis, calculating the addition amount of the lipase with different enzyme activities according to the addition amount of 4000u/g/g of lipase, wherein the reaction temperature is 40 ℃, the reaction time is 12h, and the stirring speed is 300rpm/min. In the hydrolysis process, according to the hydrolysis rate, a basic (potassium hydroxide) solution is properly added, after the reaction is finished, hydrolase is separated, and a hydration layer is acidified, kept stand, centrifuged, washed with water, filtered and evaporated to obtain free fatty acid, so that the hydrolysis efficiency of fatty glyceride catalyzed by lipase with different enzyme activities is obtained, and the result is shown in Table 2.
TABLE 2 comparison of the efficiency of hydrolysis of fatty acid glycerides catalyzed by different lipases
As shown in the results of Table 2, the lipase prepared in the example 2 of the invention has high activity, and the addition amount of the lipase is obviously reduced compared with that of the conventional lipase, but the hydrolysis efficiency can still reach more than 99% in 12 hours, the hydrolysis reaction time is reduced while the use amount of the lipase is reduced, and the hydrolysis efficiency is improved.
According to the experimental process, lipase and fatty glyceride are selected to react all the time, the time required for hydrolysis of the glyceride to reach more than 99% when different lipases are used for hydrolysis is calculated respectively, and the oil loss rate obtained by separation and recovered after the glyceride hydrolysis reaches 99% is calculated. The oil loss rate refers to the ratio of the mass of fatty acid lost during the reaction to the mass of fatty acid before the reaction. The fatty acid mass before reaction is calculated by combining liquid chromatography analysis data with theory, and the lost fatty acid is the fatty acid mass obtained by subtracting the fatty acid mass after reaction from the fatty acid mass before reaction.
Oil loss ratio = (total fatty acid mass-obtained fatty acid mass)/total fatty acid mass×100%
Measurement and calculation of oil loss rate firstly, carrying out liquid chromatography analysis on raw materials, calculating the mass content of theoretical fatty acid in the raw materials, carrying out complete hydrolysis treatment on the raw materials, carrying out centrifugal separation on an enzyme-fatty acid mixture, removing an enzyme liquid layer, weighing the obtained fatty acid, and further calculating the oil loss rate.
The P-anisidine value of the hydrolysis reaction solution after 99% hydrolysis was measured:
weigh 2.0g of the oil sample in a 25ml volumetric flask, dissolve with isooctane and fix volume. Accurately weighing 5ml of the oil sample into a 25ml colorimetric tube, accurately adding the 1 mlP-anisole reagent by a pipette, shaking, and standing for 10min. The absorbance Ab of the oil sample was measured using isooctane solvent as a blank. Then, the absorbance AS of the oil sample solution was measured using 5ml of isooctane+1 mlP-anisole reagent (left standing for 10 min) AS a blank.
P-anisole value (PAV) =25 (1.2 AS-Ab)/W
Preparation of an anisole reagent: accurately weighing 0.25-g P-anisole, dissolving in a beaker with glacial acetic acid, washing, injecting into a 100ml volumetric flask, and fixing the volume.
The experimental results are shown in table 3.
TABLE 3 results of experiments with fatty acid glycerides catalyzed by different lipases
As shown in Table 3, the lipase prepared in example 2 of the present invention has higher enzyme activity and catalytic efficiency, and the time required for catalyzing the hydrolysis of the same glyceride to reach more than 99% is the shortest, but oxidation of the glyceride during the hydrolysis process can cause the increase of the P-anisidine value, the longer the reaction time is, the more obvious the oxidation is, and the lipase prepared in example 2 of the present invention also enables the P-anisidine value of the hydrolyzed product to be the lowest due to the significantly shortened time required for the hydrolysis, and the quality of the obtained fatty acid is higher. The other three conventional lipases can cause significant increase of P-anisole value and decrease of fatty acid quality due to long hydrolysis time.
The lipase has the phenomenon that part of oil is difficult to separate after being combined in the reaction process, and can be said to be the oil absorption of the lipase, because of the nature, part of oil combined with the lipase is centrifugally removed together, and the yield loss of fatty acid is caused; the "oil absorption" mass ratio of lipase in the centrifugal separation process is close to 1:1, i.e. the more lipase is added, the more fatty acid loss is caused by hydrolysis. Compared with the conventional lipase, the addition amount of the lipase prepared by the invention is obviously reduced, so that the oil loss rate is only 1.1%.
The above embodiments are only preferred embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalents and modifications according to the technical solution and the concept of the present invention within the scope of the present invention.
Claims (10)
1. An aspergillus oryzae strain producing active lipase, which is characterized in that the aspergillus oryzae strain is aspergillus oryzae MASL-01 (Aspergillus oryzae MASL-01) and is deposited in the collection of microorganism strains in Guangdong province, the deposit address is building 5, 100 th university of pioneer in Guangzhou, and the deposit number is GDMCC No.63015.
2. Use of an active lipase producing aspergillus oryzae strain according to claim 1 for catalyzing the hydrolysis of fatty acid glycerides.
3. A method of producing an enzyme by fermentation of an active lipase producing aspergillus oryzae strain according to claim 1, comprising the steps of:
s1, activating an aspergillus oryzae strain MASL-01 inoculated in a slant culture medium to obtain an activated strain;
s2, inoculating the bevel activated strain into a first-stage seed culture medium to obtain a first-stage seed culture strain; inoculating the first-level seed culture strain into a second-level seed culture medium to obtain a second-level seed strain; inoculating the second-level seed strain into a third-level seed culture tank to obtain a third-level seed culture solution;
s3, inoculating the three-level seed culture solution into a fermentation tank to obtain fermentation liquor, treating and collecting the fermentation liquor to obtain crude enzyme, and purifying and collecting the crude enzyme to obtain the lipase with high activity.
4. The fermentation enzyme production method according to claim 3, wherein the slant culture medium comprises 10g/L yeast extract powder, 20-30 g/L beef peptone, 20-40 g/L glucose and 10-20 g/L agar.
5. The fermentation enzyme production method according to claim 3, wherein the primary seed medium and the secondary seed medium each comprise 10g/L yeast extract, 20g/L beef peptone and 20g/L glucose.
6. The fermentation enzyme production method according to claim 3, wherein the three-stage seed culture tank and the medium in the fermentation tank comprise the following components in parts by weight: sucrose 0.1-5%, polypeptone 0-8%, corn flour 0-5%, yeast extract powder 0-5%, tween-80 0-3%, talcum powder 0-2%, naNO 3 0～1％、KH 2 PO 4 0.7％、Na 2 HPO 4 ·12H 2 O 0.25％、MgSO 4 ·12H 2 O 0.1％、CaCl 2 0.05%, olive oil 0-5%, the balance water, pH 5.8.
7. A fermentation enzyme production method according to claim 3, wherein the process collection in step S3 is: the fermentation broth is subjected to plate-frame filter pressing, mycelium and filtrate are collected, a high-speed bead mill stirrer is used for destroying mycelium cell walls, and the treated mycelium is subjected to salting-out precipitation and purification by adopting 10-30% ammonium sulfate solution; the filtrate is subjected to ultrafiltration concentration by adopting a 32-40 kDa membrane, then the concentrated solution is subjected to ammonium sulfate salting out, the salting-out precipitate with 10-30% of saturation degree is collected, and the two salting-out precipitates are mixed to obtain refined crude enzyme.
8. The method for producing enzyme by fermentation according to claim 3, wherein the purification and collection process in the step S3 is as follows: and (3) performing chromatographic purification on the crude enzyme by utilizing a hydrophobic chromatographic column, eluting by using buffer solution with the concentration of 20-100mM and the pH of 6.0-8.5, eluting by using distilled water, eluting by using ethanol water solution with the concentration of 10-70%, collecting the eluted component, and performing ion exchange chromatography purification after buffer solution replacement to obtain the lipase with high purity and high activity.
9. Use of a lipase prepared by the fermentation enzyme-producing method of any one of claims 3-8 in catalyzing hydrolysis of fatty acid glycerides.
10. The use according to claim 9, wherein the lipase-catalysed hydrolysis of fatty acid glycerides is at a temperature of 40 to 45 ℃ and the lipase-catalysed hydrolysis of fatty acid glycerides is at a pH of 5 to 6.
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