CN116731898A - Two strains producing feruloyl esterase - Google Patents

Two strains producing feruloyl esterase Download PDF

Info

Publication number
CN116731898A
CN116731898A CN202310038026.4A CN202310038026A CN116731898A CN 116731898 A CN116731898 A CN 116731898A CN 202310038026 A CN202310038026 A CN 202310038026A CN 116731898 A CN116731898 A CN 116731898A
Authority
CN
China
Prior art keywords
enzyme activity
culture medium
burkholderia
strain
fermentation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310038026.4A
Other languages
Chinese (zh)
Inventor
童文华
李佳伟
何萍
黄志久
王洪禹
车经纬
黄丹
罗惠波
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zui Qingfeng Liquor Co ltd
Sichuan University of Science and Engineering
Original Assignee
Zui Qingfeng Liquor Co ltd
Sichuan University of Science and Engineering
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zui Qingfeng Liquor Co ltd, Sichuan University of Science and Engineering filed Critical Zui Qingfeng Liquor Co ltd
Priority to CN202310038026.4A priority Critical patent/CN116731898A/en
Publication of CN116731898A publication Critical patent/CN116731898A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01073—Feruloyl esterase (3.1.1.73)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Abstract

The invention discloses two strains for producing feruloyl esterase, which are separated from white spirit Daqu, in particular to enterococcus faecium L-7 and Burkholderia BK100, wherein the strains are preserved in China Center for Type Culture Collection (CCTCC) with the preservation numbers of CCTCC No. M20221555 and CCTCC No. M20221553 respectively. The optimal fermentation conditions are shown as follows by optimizing the fermentation enzyme production conditions of enterococcus faecium L-7: when the strain is cultured for 96 hours at 32 ℃ and 160r/min under the condition of the inoculation amount of 6%, the enzyme activity of feruloyl esterase produced by the strain is highest and can reach 44.68U/L, and the enzyme activity is improved by 68.92% compared with 26.45U/L before the strain is not optimized. Burkholderia BK100 has the characteristics of high enzyme activity, high temperature resistance and the like of producing ferulic acid esterase, is suitable for Daqu preparation and white wine brewing, can improve the ferulic acid content and improve the quality of white wine.

Description

Two strains producing feruloyl esterase
Technical Field
The invention relates to the technical field of bioengineering, and relates to two strains for producing feruloyl esterase, in particular to enterococcus faecium L-7 and Burkholderia BK100.
Background
Daqu is a saccharification starter in Chinese white spirit brewing, and is rich in microorganisms, hydrolytic enzymes and flavor substances. Daqu can provide stable microbial flora for brewing white wine and also can provide nutrition for the growth of microorganisms. Meanwhile, biological enzymes generated by various microorganisms in the Daqu are beneficial to the decomposition of raw materials in the fermentation process of the white spirit. Daqu plays a key role in white spirit fermentation, wherein ' the yeast is bone of the wine ', and high-quality distiller's yeast is an important precondition for producing high-quality white spirit. The microorganisms in the Daqu and the biological enzymes produced by the microorganisms provide important guarantee for the subsequent production of the qu liquor, and have important influence on the unique style of the formed various aromatic white spirits.
Ferulic acid (Ferulic acid) is a phenolic acid substance, has strong effects of resisting oxidation, resisting radiation, preventing and treating coronary heart disease, inhibiting liver injury, etc., and can be widely used in industries such as medicine, cosmetics, food, etc. Ferulic acid is used as an organic phenolic acid, has strong fragrance-producing effect, and is one of fragrance-producing substances in wine. Ferulic acid can also be used as precursor of fragrance in wine, and can be converted into vanillin, 4-EG, 4-vinyl guaiacol, etc. under the action of microorganism, which makes an important contribution to forming unique style of Chinese liquor. Ferulic acid is widely present in plant cell walls, forms a network structure with polysaccharide and lignin through ether bonds or ester bonds, and limits the utilization of cellulose and the degradation of plant fiber materials by microorganisms.
Ferulic acid esterase belongs to carboxylesterase subclasses, and can catalyze and hydrolyze ester bonds or ether bonds between ferulic acid and polysaccharide, cellulose and lignin to generate ferulic acid. Feruloyl esterase can also be used for synthesizing esters, such as hydroxycinnamate, etc. Fungi, bacteria and yeasts all secrete feruloyl esterases. The feruloyl esterases from different sources have great differences in enzymatic properties, sequence homology and spatial structure. The reported feruloyl esterase mainly originates from microorganisms, but the enzyme activity of the feruloyl esterase separated from the feruloyl esterase is generally low, the microbial strains are mainly screened from soil, sea water, human and animal intestinal tracts, and the strains for producing the feruloyl esterase are relatively few from white spirit brewing raw materials. The feruloyl esterase has wide application value in various fields of food, feed, medicine, paper making, textile and the like, and can accelerate the degradation of cell walls of raw materials and enable microbial transformation reaction in Daqu to be quicker and more thorough when being applied to the white wine brewing process. In addition, in the process of brewing white spirit, the acidity and the ethanol concentration in a fermentation system are gradually increased along with the fermentation, and most commercial strains cannot tolerate the adverse conditions such as high acid and high ethanol concentration.
Currently, a comparison document (CN 109468232A) discloses Xie Wasan cyst bacteria producing feruloyl esterase and application thereof in the production of white spirit Daqu. The research shows that enterococcus faecium is widely existed in fermented food, not only has influence on the aroma, flavor and quality of the food, but also has the probiotic effects of regulating intestinal flora, improving immunity of organisms and the like, but has no report of producing feruloyl esterase and ferulic acid.
In addition, burkholderia has been searched for producing lipase, ACC deaminase and cellulase, but no report of producing feruloyl esterase is made, and patent publication No. CN109468232A has been searched for, and the technology discloses a strain which is separated from Daqu and can produce feruloyl esterase, and the strain powder of the strain is proportionally added into the production of Daqu liquor. The patent with the publication number of CN113308424A is searched, and the technology discloses that the enzyme activity of the feruloyl esterase produced by fermenting bacillus pumilus can be maintained to be more than 80 percent at the temperature of 45-60 ℃. However, the above prior art does not mention whether the screened bacteria are still suitable for growth during brewing, nor the optimisation of the assay conditions and the use of crude enzyme solutions of feruloyl esterase-producing strains.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide two strains for producing feruloyl esterase. Enterococcus faecium L-7 has the characteristics of acid and alkali resistance, salt resistance, ethanol resistance and the like, can be used in the brewing process of white spirit, improves the content of ferulic acid in the white spirit, enhances the health care function of the white spirit, and BK100 of Burkholderia has the characteristics of high temperature resistance and alkali resistance, and is suitable for the metabolic engineering transformation of the later-stage high-yield ferulic acid.
The technical purpose of the invention is realized by the following technical scheme:
in a first aspect, two strains producing feruloyl esterase are provided, in particular enterococcus faecium L-7 and Burkholderia BK100; the enterococcus faecium L-7 is preserved in China Center for Type Culture Collection (CCTCC) No. M20221555, and the preservation date is 2022, 10 and 10; the Burkholderia BK100 is preserved in China Center for Type Culture Collection (CCTCC) No. M20221553, and the preservation date is 2022, 10 months and 10 days.
Further, the BK100 temperature tolerance range of Burkholderia is 20-57 ℃, and the BK grows well at 25-40 ℃, and the optimal growth temperature is 30 ℃.
Furthermore, the pH tolerance of the enterococcus faecium L-7 is pH 4-11, the enterococcus faecium L-7 grows well at pH 7-10, and the strain grows and grows most rapidly at pH 8.
Furthermore, the enterococcus faecium L-7 can normally grow within the range of 1% -3% of NaCl concentration and 1% -7% of ethanol concentration.
Furthermore, the enterococcus faecium L-7 grows well when the NaCl concentration is 2% and the ethanol concentration is 5%.
In a second aspect, the screening process of enterococcus faecium L-7 comprises the following steps:
and 1) sampling: taking 10g of samples from each corner and the center of the finished Daqu, grinding and mixing uniformly to obtain Daqu samples;
second) enrichment: weighing 2g of the Daqu sample obtained in the step one), filling the Daqu sample into a 250mL triangular flask, adding 100mL of sterile physiological saline, shaking uniformly, and carrying out shaking culture for 30min at 37 ℃ under the condition of 180r/min to prepare bacterial suspension;
third), screening: serial gradient dilution is carried out on the bacterial suspension prepared in the step 2) by using sterile physiological saline, and 100 mu L of dilution factors are respectively taken to be 10 -3 、10 -4 、10 -5 、10 -6 Coating on a screening culture medium, making three gradients in parallel, and inversely culturing the coated culture dish in a 37 ℃ incubator for 72 hours;
fourth), purification: observing and screening the colony and the transparent ring size on the culture medium, picking the colony with larger transparent ring, streaking on the LB solid culture medium, culturing for 24 hours in a 37 ℃ incubator, repeating for three times to obtain a single colony of the target strain; the single colonies obtained were inoculated into LB liquid medium and cultured overnight. Inoculating 1ml of bacterial liquid into a fermentation medium, culturing for 72 hours, ending fermentation, centrifuging, taking supernatant, filtering to obtain a liquid phase vial, measuring the ferulic acid content in the fermentation liquid by using a high performance liquid chromatography, and preserving strains producing ferulic acid esterase in a glycerol tube;
Wherein, the screening culture medium in the steps three) and four) is as follows: 2g/L of sodium nitrate, 1g/L of dipotassium hydrogen phosphate, 0.5g/L of potassium chloride, 0.5g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate, 20g/L of agar, 1L of ultrapure water and sterilization at 121 ℃ for 20min; cooling to 70deg.C, adding 1.5% (V/V) ethyl ferulate (10% W/V in N, N-dimethylformamide solution), and sterilizing by filtration through 0.22um sterile filter membrane;
the LB solid medium in the fourth step) is: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L agar, 1L ultrapure water, natural pH, 121 ℃ and sterilization for 20min;
the LB liquid medium in the fourth step) is: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 1L ultrapure water, natural pH, 121 ℃ and sterilization for 20min;
fermentation medium in step four): wheat bran 10g/L, yeast extract 1g/L, potassium chloride 0.5g/L, magnesium sulfate heptahydrate 0.5g/L, dipotassium hydrogen phosphate 0.1g/L, ultrapure water 1L, pH nature, 121 ℃, sterilization for 20min, and independent sterilization of magnesium sulfate heptahydrate and addition of the magnesium sulfate heptahydrate into a culture medium.
In a third aspect, the screening process of burkholderia BK100 comprises the following:
and 1) enrichment: fermenting in certain winery for 1-30 days, storing Daqu Qu Yang for one month and two months, pulverizing, mixing to obtain yeast powder, placing in-80 refrigerator, adding 10g yeast powder into 90mL enriched culture medium, and making into 10 -1 Daqu enrichment of (2), and diluting to obtain 10 -2 -10 -7 Daqu dilution of (2);
two) screening of strains with high feruloyl esterase yield
1) And (3) primary screening: inoculating four Daqu dilutions with different gradients on a primary screening culture medium plate, inversely culturing for 3d at 30 ℃, purifying and culturing single bacterial colonies on the culture medium, inoculating the culture medium to the primary screening culture medium for 5d based on the culture at 30 ℃, observing whether transparent rings are formed, and inoculating bacterial colonies with the transparent rings to the primary screening culture medium for purifying and separating again to obtain pure bacterial colonies;
2) And (3) re-screening: a. the strain which can generate more transparent circles is obtained in the step 1), and the strain is separated and purified for multiple times, wherein the transparent circles of Burkholderia are the largest. B, inoculating Burkholderia BK100 into 100mL of LB medium, and placing the LB medium at 30 ℃ for culturing for 48 hours; b. inoculating Burkholderia BK100 into 100ml of rescreen culture medium with an inoculum size of 2% by volume, culturing at 30 ℃ for 72 hours at 180r/min, and then measuring the ferulic acid content in the fermentation broth by using high performance liquid chromatography to obtain a strain with the enzyme activity of producing ferulic acid esterase of 41.6U/L, and preserving the strain in a glycerol tube;
the enrichment medium in step one) is: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L glucose, 1L distilled water, natural pH, and sterilizing at 121deg.C for 20min;
The primary screening culture medium in the step 1) is as follows: potassium chloride 0.5g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate heptahydrate 0.5g/L, sodium nitrate 2.0g/L, ferrous sulfate heptahydrate 0.01g/L, agar 20g/L, sucrose 30g/L, distilled water 1L, pH nature, sterilization at 121 ℃ for 20min. Cooling to 60 ℃ and adding 10mL of ethyl ferulate (10% w/V in N, N-dimethylformamide);
the LB medium in the step 2) is: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, 1L distilled water, natural pH, and sterilizing at 121deg.C for 20min;
the rescreening medium in step 2) is: 10g of yeast powder, 0.5g of potassium chloride, 0.5g of magnesium sulfate heptahydrate, 1.0g of dipotassium hydrogen phosphate, 20g of agar, 1L of distilled water, natural pH, sterilization at 121 ℃ for 20min, cooling to 60 ℃ and adding 10mL of ethyl ferulate (10% W/V in N, N-dimethylformamide solution).
In a fourth aspect, there is provided the use of enterococcus faecium L-7 as described in the first aspect for the fermentative production of feruloyl esterase.
Further, the fermentation conditions for producing feruloyl esterase by enterococcus faecium L-7 fermentation are as follows: the inoculation amount is 3-7%, the rotating speed is 140-220 r/min, the culture temperature is 22-42 ℃, and the culture time is 48-144 h.
Furthermore, the fermentation conditions for producing feruloyl esterase by enterococcus faecium L-7 fermentation are as follows: the inoculation amount is 6%, the rotating speed is 160r/min, the culture temperature is 32 ℃, and the culture time is 96 hours.
In a fifth aspect, there is provided the use of burkholderia BK100 as described in the first aspect for the fermentative production of feruloyl esterase.
Further, the enzyme activity measurement condition of the feruloyl esterase produced by the BK100 fermentation of Burkholderia is that the optimal reaction temperature of the enzyme activity in the crude enzyme liquid is 20-60 ℃, the optimal reaction pH of the enzyme activity in the crude enzyme liquid is 5.0-9.0, the optimal addition amount of the crude enzyme liquid is 250-350 mu L, and the optimal reaction time of the enzyme activity in the crude enzyme liquid is 15-30min.
Furthermore, the enzyme activity measurement condition of the feruloyl esterase produced by the fermentation of Burkholderia BK100 is that the optimal reaction temperature of the enzyme activity in the crude enzyme liquid is 42 ℃, the optimal reaction pH of the enzyme activity in the crude enzyme liquid is 7.1, the addition amount of the crude enzyme liquid is 320 mu L, and the optimal reaction time of the enzyme activity in the crude enzyme liquid is 22min.
The enterococcus faecium L-7 related to the invention is classified and named as: enterococcus faecium Enterococcus faecalis, preserved in China Center for Type Culture Collection (CCTCC) at the address: china, university of Wuhan, preservation number CCTCC No. M20221555, and preservation date of 2022, 10 months and 10 days.
The Burkholderia BK100 of the invention is classified and named as: burkholderia Burkholderia, deposited in China Center for Type Culture Collection (CCTCC), address: china, university of Wuhan, preservation number CCTCC No. M20221553, and preservation date of 2022, 10 months and 10 days.
Compared with the prior art, the invention has the following beneficial effects:
1) The enterococcus faecium L-7 is screened from the Daqu of the white wine, and has the characteristics of acid and alkali resistance, salt resistance, alcohol resistance and the like through the adaptive evolution of the growth environment, so that the strain is beneficial to improving the capability of producing ferulic acid esterase by using molecular biological means such as genetic engineering, metabolic engineering, synthetic biology and the like in the later stage, and further improves the content of ferulic acid in the white wine;
2) The enterococcus faecium L-7 can normally grow in the range of 1% -7% of ethanol concentration, and the enterococcus faecium L-7 can be used for improving the content of a health factor-ferulic acid in white spirit brewing;
3) The enterococcus faecium L-7 is derived from Daqu of white spirit, and the toxicity gene detection and the harmful metabolite experiment result are negative;
4) The enterococcus faecium L-7 can be fermented to generate feruloyl esterase to degrade feruloyl ester bonds in plant cell walls, and the feruloyl ester bonds can be applied to the brewing process of white wine to degrade the cell walls of raw materials more fully, so that microbial transformation reactions in a system are more thorough;
5) The optimal fermentation conditions are shown as follows by optimizing the fermentation enzyme production conditions of enterococcus faecium L-7: when the strain is cultured for 96 hours at 32 ℃ and 160r/min under the condition that the inoculation amount is 6%, the enzyme activity of feruloyl esterase produced by the strain is highest and can reach 44.68U/L, which is improved by 68.92% compared with 26.45U/L before the strain is not optimized;
6) The burkholderia BK100 is obtained by screening the white spirit Daqu, and the burkholderia BK100 has the characteristics of high temperature resistance and alkali resistance due to special Daqu fermentation environment, and is suitable for the metabolic engineering transformation of the later-stage high-yield ferulic acid;
7) Burkholderia BK100 has the characteristics of high enzyme activity, high temperature resistance and the like of producing ferulic acid esterase, is suitable for preparing Daqu and brewing white spirit, can improve the ferulic acid content and improve the quality of the white spirit;
8) Burkholderia BK100 has wide acid-base tolerance range, can grow in the pH range of 5.0-9.0, and has the characteristic of alkali resistance;
9) The enzyme activity measurement conditions of the feruloyl esterase produced by the fermentation of Burkholderia BK100 are optimized, and the result shows that the optimal measurement conditions are as follows: the optimal reaction temperature of the enzyme activity in the crude enzyme liquid is 42 ℃, the optimal reaction pH of the enzyme activity in the crude enzyme liquid is 7.1, the addition amount of the crude enzyme liquid is 320 mu L, and the optimal reaction time of the enzyme activity in the crude enzyme liquid is 22min; the enzyme activity of feruloyl esterase measured under the condition is 85.08U/L, which is 104.52 percent higher than that of the strain before optimization;
10 The method adopts a microbial fermentation method to obtain the high-yield feruloyl esterase, and has the advantages of simple operation, high yield, environmental protection and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments or the description of the prior art will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a phylogenetic tree of L-7 in an embodiment of the invention;
FIG. 2 is a standard graph of ferulic acid in an embodiment of the invention;
FIG. 3 is a chart of a ferulic acid standard (upper) and an enzyme reaction liquid (lower) in an example of the present invention;
FIG. 4 is a graph showing the growth curve of L-7 in the examples of the present invention;
FIG. 5 is an electropherogram of L-7 virulence gene amplification in an embodiment of the present invention;
FIG. 6 is a graph showing the results of an L-7 hemolysis test in the examples of the present invention;
FIG. 7 is a physical diagram of the morphological feature of L-7 in the embodiment of the invention;
FIG. 8 is a gram of L-7 in an example of the invention;
FIG. 9 is a graph showing the effect of pH tolerance of L-7 in the examples of the present invention;
FIG. 10 is a graph showing the salt tolerance effect of L-7 in the examples of the present invention;
FIG. 11 is a graph showing the effect of L-7 on ethanol tolerance in the examples of the present invention;
FIG. 12 is the primary screening results of Burkholderia in the examples of the present invention;
FIG. 13 is a crude enzyme liquid chromatogram of BK100 in an embodiment of the invention;
FIG. 14 is a BK100 phylogenetic tree in an embodiment of the present invention;
FIG. 15 is a BK100 growth curve in an embodiment of the invention;
FIG. 16 is a growth temperature tolerance of BK100 in an embodiment of the invention;
FIG. 17 is a growth pH tolerance of BK100 in an embodiment of the invention;
FIG. 18 is a strain transparent loop of BK100 in an embodiment of the invention;
FIG. 19 is a gram stain result of BK100 in the examples of the present invention;
FIG. 20 is a scanning electron microscope image of BK100 in an embodiment of the invention;
FIG. 21 is a ferulic acid standard curve of BK100 in an embodiment of the invention;
FIG. 22 is a crude enzyme liquid chromatogram of BK100 with ferulic acid standard in an embodiment of the invention;
FIG. 23 is a graph showing the effect of the reaction temperature of BK100 on the enzyme activity measurement in the examples of the present invention;
FIG. 24 is a graph showing the effect of BK100 reaction time on enzyme activity assay in examples of the present invention;
FIG. 25 is a graph showing the effect of BK100 reaction pH on enzyme activity assay in examples of the present invention;
FIG. 26 shows the effect of the addition amount of BK100 crude enzyme solution on the measurement of enzyme activity in the examples of the present invention.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the present invention is further described in detail below with reference to fig. 1 to 26 and embodiments 1 to 7.
Example 1: isolation and screening of Strain L-7
First) sampling, enrichment and screening
Taking 10g of sample from each corner and center of the finished Daqu, grinding and mixing uniformly to obtain Daqu sample; weighing 2g of Daqu sample, filling into a 250mL triangular flask, adding 100mL of sterile physiological saline, shaking uniformly, and shake culturing at 37deg.C under 180r/min for 30min to obtain bacterial suspension; serial gradient dilution of enriched bacterial suspension with sterile physiological saline solution, and dilution ratio of 100 μl of the bacterial suspension to 10 -3 、10 -4 、10 -5 、10 -6 Coating on a screening culture medium, making three gradients in parallel, and inversely culturing the coated culture dish in a 37 ℃ incubator for 72 hours;
screening media in g/L: 1L of the medium contains 2g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate heptahydrate, 0.01g of ferrous sulfate heptahydrate, 20g of agar and 1L of ultrapure water, sterilizing for 20min at 121 ℃, cooling to about 70 ℃, adding 1.5% (V/V) of ethyl ferulate (10% W/V is dissolved in N, N-dimethylformamide solution), and filtering and sterilizing by a 0.22 mu m sterile filter membrane).
Two) separation and purification
Observing the size of the colony and the transparent ring on the culture medium, picking 5 colonies with larger transparent ring, streaking on LB solid culture medium, culturing in a 37 ℃ incubator for 24 hours, repeating for three times, and obtaining single colony of target strain. The single colonies obtained were inoculated into LB liquid medium, respectively, and cultured overnight. Inoculating 1ml of bacterial liquid into a fermentation culture medium, culturing for 72 hours in parallel, ending fermentation, centrifuging, taking supernatant, passing through a 0.22 mu m filter membrane into a liquid phase vial, measuring the ferulic acid content in the fermentation liquid by using a high performance liquid chromatography, preserving the strain with the highest ferulic acid yield in a glycerol tube, naming the strain as L-7, and subsequently optimizing enzyme production conditions by taking enterococcus faecium L-7 as a starting strain.
LB solid medium is calculated in g/L: the 1L culture medium contains tryptone 10g, yeast extract 5g, sodium chloride 10g, agar 20g, pH is natural, and sterilization is carried out for 20min at 121 ℃.
LB liquid medium is calculated in g/L: the 1L culture medium contains 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride, natural pH, 121 ℃ and sterilization for 20min.
Fermentation medium is calculated in g/L: the 1L culture medium contains wheat bran 10g, yeast extract 1g, potassium chloride 0.5g, dipotassium hydrogen phosphate 0.1g, ultrapure water 1L, pH is natural, 121 ℃ and sterilization is carried out for 20min, and then 0.5g of magnesium sulfate heptahydrate is added into the culture medium after independent sterilization.
Example 2: l-7 related 16S rDNA identification and biological characterization study
First) authentication
Firstly, the bacterial strain is preliminarily judged to be gram-positive bacteria through morphological characteristic observation, then, the sequence comparison is carried out between the sequencing result identified by 16S rDNA molecular biology and NCBI database, and a phylogenetic tree is constructed by utilizing MEGA7.0.26 software, so that the bacterial strain is determined to be enterococcus faecium and named Enterococcus faecalis L-7, and the bacterial strain is shown in figure 1.
Two) drawing ferulic acid standard curve
1.0mg of ferulic acid standard substance is accurately weighed, 50% methanol is used for preparing ferulic acid standard mother liquor with the concentration of 0.1g/L, and then mother liquor is used for preparing ferulic acid standard solutions with the concentrations (g/L) of 0.04, 0.03, 0.02, 0.01, 0.007, 0.004 and 0.001 respectively, and peak areas are measured by using a high performance liquid chromatograph. And drawing a standard curve by taking the concentration (g/L) of the standard solution as an abscissa and the peak area as an ordinate, and performing a linear regression equation. As shown in fig. 2, the regression equation y=32281x+24.844, the correlation coefficient R 2 =0.9997, which indicates that the two are linearly related well.
Three) determination of ferulic acid content
Selecting single colony of target strain, inoculating into 15mL centrifuge tube containing 5mL LB liquid culture medium, culturing overnight, inoculating into 100mL fermentation culture medium with 1% inoculum size, inoculating 3 groups of strains in parallel at 37 deg.C in 180r/min, culturing for 72 hr, centrifuging at 12000r/min for 10min after fermentation, filtering supernatant with 0.22 μm filter membrane, and performing high performance liquid chromatography detection analysis, wherein the culture medium without bacteria is used as blank control. The strain with highest ferulic acid production is preserved in a glycerol tube and named as L-7, and then enterococcus faecium L-7 is used as a starting strain to optimize enzyme production conditions.
Four) determination of feruloyl esterase enzyme Activity
1) Preparation of L-7 fermentation broth
(1) Picking single colony of L-7, inoculating the single colony into a 15mL centrifuge tube filled with 5mL LB liquid culture medium for culture overnight, (2) absorbing 1mL of bacterial liquid, inoculating the bacterial liquid into 100mL fermentation culture medium, and after shaking culture for 72 hours at 37 ℃ and 180r/min, ending fermentation and absorbing 1mL of bacterial liquid as fermentation liquid;
2) Preparation of crude enzyme solution: placing 1ml of the fermentation broth obtained in the step 1) into a high-speed centrifuge, centrifuging for 15min at 10000r/min, and taking 250 μl of supernatant as crude enzyme solution;
3) Enzyme activity determination: taking 250 mu L of the crude enzyme solution obtained in the step 2), putting the crude enzyme solution into a 1.5mL centrifuge tube, immediately adding 750 mu L of 0.003mol/L ferulic acid methyl ester solution (0.031 g of ferulic acid methyl ester is accurately weighed and dissolved in 50mL of 0.05mol/L Tris-HCl (pH 8.0) buffer solution), and forming a reaction system by 250 mu L of the crude enzyme solution and 750 mu L of ferulic acid methyl ester solution. Then, the mixture was allowed to react at 50℃for 15 minutes, immediately after the completion of the reaction, the mixture was boiled for 10 minutes to terminate the reaction, and a blank group was set for control. After the reaction system is cooled to room temperature, centrifuging for 10min at 10000r/min, filtering by a 0.22 mu m filter membrane, and filling the sample liquid into a liquid phase vial for high performance liquid chromatography detection analysis. As can be seen from FIG. 3, the peak time of the enzyme reaction solution sample (lower) was approximately 14min and the peak time of the ferulic acid standard sample (upper) were close, indicating that the enzyme reaction solution contained ferulic acid esterase and could decompose ferulic acid methyl ester to ferulic acid.
Definition of enzyme activity: the amount of enzyme required to hydrolyze methyl ferulate to 1. Mu. Mol ferulic acid per minute at 50℃was 1 enzyme activity unit (1U).
Fifth) growth curve of enterococcus faecium L-7
During shake flask culture at 37deg.C and 180r/min, sampling is performed every 2h to measure OD 600 As is clear from FIG. 4, the growth was slow 2 hours before the cultivation, the logarithmic phase was started after 2 hours, the growth period was stable for 16 to 18 hours, and the decay phase was started after 18 hours.
Sixth), detection result of virulence gene and harmful metabolite of enterococcus faecium L-7
The detection results of the virulence genes and the harmful metabolites of enterococcus faecium L-7 are shown in Table 1, and as can be seen from Table 1 and FIG. 5, the detection results of the 6 virulence genes of enterococcus faecium L-7 are all negative, and the 6 virulence genes common to enterococcus faecium are not carried. In addition, the detection of the enterococcus faecium L-7 amino acid decarboxylase, nitrate reductase, indole test and hemolysis test is negative, which shows that enterococcus faecium L-7 has no amino acid decarboxylase and nitrate reductase activity and cannot decompose tryptophan to produce indole substances. As is clear from FIG. 6, enterococcus faecium L-7 did not produce a hemolytic cycle after being cultured on a blood plate for 48 hours, indicating that the strain does not have hemolytic toxicity.
TABLE 1 detection results of enterococcus faecium L-7 virulence genes and deleterious metabolites
Note that: "+" indicates positive and "-" indicates negative.
Seven) morphological identification of enterococcus faecium L-7
As shown in FIG. 7, the bacterial colony of the strain is circular or elliptical, has smooth surface, is opaque, has no spores and no flagella, and is arranged singly, in pairs or in short chain;
the gram staining results were purple, as shown in FIG. 8, and belonged to gram-positive bacteria.
Eighth) pH tolerance
As shown in FIG. 9, enterococcus faecium L-7 has pH tolerance of 4-11, grows well at pH 7-10, and grows most rapidly at pH 8. The minimum pH tolerance of the strain is 4, and the strain can grow under the condition of higher pH (pH 8-10), and belongs to alkali-resistant microorganisms.
Nine) salt tolerance
As shown in FIG. 10, enterococcus faecium L-7 was inhibited from growing when the mass fraction of NaCl exceeded 3%. When the NaCl mass fraction is 8%, the bacterial density of enterococcus faecium is obviously reduced, but OD 600 The value is still higher than 0.6. Thus, the enterococcus faecium has strong salt tolerance.
Ten) ethanol tolerance
As shown in FIG. 11, enterococcus faecium L-7 with the highest ethanol content of 13% can grow normally within the range of 1% -7% ethanol content.
Example 3: enterococcus faecium L-7 enzyme production condition optimization
One) one-factor test
The inoculation amount is 3%, 4%, 5%, 6%, 7%, the rotation speed is 140, 160, 180, 200, 220r/min, the culture temperature is 22, 27, 32, 37, 42 ℃ and the culture time is 48, 72, 96, 120, 144h, etc. 4 factors are selected to be single factor tests, and each group of tests is 3 parallel.
Two) orthogonal test
Design L9 (3) 4 ) Orthogonal test, selecting 4 factors of rotation speed, inoculation amount, culture temperature and culture time for investigation, wherein each investigation factor is 3 in level, and each group of test is 3 in parallel.
On the basis of a single-factor test, the influence of each factor on the fermentation and enzyme production of enterococcus faecium L-7 is examined by an orthogonal test. The orthogonal test design scheme is shown in Table 2, the result is shown in Table 3, and the effect of each factor on the enzyme production of the strain is shown as A > B > C > D in sequence by extremely poor analysis, namely, the culture time is more than the culture temperature is more than the inoculation amount is more than the rotating speed. From the orthogonal test results, the optimal test combination A can be obtained 2 B 1 C 3 D 1 Namely, the culture time is 96 hours, the culture temperature is 32 ℃, the inoculation amount is 6 percent, and the rotating speed is 160 r/mm. As there is no best in the orthogonal table combinationsA preferred combination, so that a verification test is required. When combined as A 2 B 1 C 3 D 1 When the strain produces feruloyl esterase, the enzyme activity reaches 44.68U/L.
TABLE 2 orthogonal test design scheme
TABLE 3 results of orthogonal experiments
Comprehensive analysis, the optimal conditions for producing enzyme by enterococcus faecium L-7 fermentation are as follows: the culture time is 96 hours, the culture temperature is 32 ℃, the inoculation amount is 6 percent, and the rotating speed is 160 r/mm. Under the condition, the enzyme activity of the feruloyl esterase produced by the strain reaches 44.68U/L, which is 68.92 percent higher than that of the feruloyl esterase produced by the strain before the strain is optimized.
Example 4: screening of Burkholderia BK100
First) enrichment
Fermenting in certain winery for 1-30 days, storing Daqu Qu Yang for one month and two months, pulverizing, mixing to obtain yeast powder, placing the yeast powder in a-80 refrigerator, adding yeast powder 10g into 90mL enriched culture medium, and making into 10 -1 Daqu enrichment of (2), and diluting to give 10 -2 -10 -7 Obtaining Daqu diluent;
enrichment medium was measured in g/L: 1L of culture medium comprises 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 20g of glucose;
two) screening of high-yield feruloyl esterase strain BK100
1) And (3) primary screening: take 10 -2 、10 -3 、10 -4 、10 -5 Inoculating four different gradient Daqu dilutions on a primary screening culture medium plate with ethyl ferulate as a carbon source, reversely culturing at 30deg.C for 3d, purifying and culturing single colony on the culture medium, inoculating into the primary screening culture medium based on 30deg.C water-proof constant temperature incubator, culturing for 5d, and obtaining 32 strains by primary screening Bacteria. Colonies with transparent rings were subjected to secondary purification and separation on the primary screening medium to obtain pure colonies, as shown in FIG. 12.
The primary screening medium is calculated in g/L: KCl 0.5g, K was contained in 1L medium 2 HPO 4 ·3H 2 O 1.0g，MgSO 4 ·7H 2 O 0.5g，NaNO 3 2.0g，FeSO 4 ·7H 2 O0.01 g, agar 20g, sucrose 30g, distilled water 1L, pH nature, sterilization at 121 ℃ for 20min. Cooling to 60 ℃ and adding 10mL of ethyl ferulate (10% w/V in N, N-dimethylformamide);
2) And (3) re-screening: the strain which can generate more transparent circles is obtained in the step 1), and the strain is separated and purified for multiple times, wherein the transparent circles of the Burkholderia BK100 are the largest. The BK100 of Burkholderia is streaked on a primary screening culture medium for several times to obtain pure single colonies, and the BK100 of Burkholderia is inoculated in 100mL of LB culture medium and placed at 30 ℃ for culturing for 48 hours; b. the BK100 strain solution of Burkholderia was inoculated into 100ml of a double-sieve medium at an inoculum size of 2% by volume, and after culturing at 30℃for 72 hours at 180r/min, the ferulic acid content in the fermentation broth was measured by high performance liquid chromatography, as shown in FIG. 13. The fermentation liquor has a peak in 13min, and the peak-out time is similar to that of the ferulic acid standard substance, so that the sample liquor can be proved to contain ferulic acid esterase and can metabolize the ferulic acid methyl ester to generate the ferulic acid. According to the high performance liquid chromatography measurement result, a strain producing feruloyl esterase with the enzyme activity of 41.6U/L is obtained, the strain is named as Burkholderia BK100 and is preserved in a glycerol tube, and in the subsequent experiments, the Burkholderia BK100 is taken as a starting strain.
LB medium in g/L: 1L of culture medium contains tryptone 10g, yeast extract 5g, naCl 10g, distilled water 1L, pH is natural, and sterilization is carried out for 20min at 121 ℃;
the rescreening medium is calculated in g/L: the 1L culture medium comprises yeast powder 10g, potassium chloride 0.5g, magnesium sulfate heptahydrate 0.5g, dipotassium hydrogen phosphate 1.0g, agar 20g, distilled water 1L, pH nature, sterilizing at 121deg.C for 20min, cooling to 60deg.C, and adding 10mL (10% W/V solution in N, N-dimethylformamide) of ferulic acid ethyl ester.
Example 5:
first) molecular biological identification
Streaking a target strain, namely Burkholderia BK100, on an LB plate, and observing the shape and color of a colony by referring to a Bojg system bacteriology handbook; and gram staining and strain individual morphology observation were performed. The bacterial strain identification method comprises the steps of (1) primarily judging gram-negative short rod-shaped thalli through morphological identification, inoculating purified burkholderia BK100 into an LB culture medium, culturing overnight, and taking bacterial liquid to send samples to a adult tussilago organism for bacterial strain identification; sequencing data was analyzed by homology search in the national center for biotechnology information (National center of biotechnology information, NCBI) database and phylogenetic tree was constructed using MEGA6.0.26 software as shown in fig. 14. The strain was determined to be burkholderia and designated burkholderia BK100.
Two) Burkholderia BK100 growth curve
Picking single bacterial colony of Burkholderia BK100, inoculating to an LB liquid culture medium, activating for 16 hours at a temperature of 180r/min by a shaking table at 30 ℃, inoculating the activated bacterial liquid to the LB liquid culture medium with an inoculum size of 1%, placing in a constant-temperature shaking table at 30 ℃ at 180r/min for shake culture, and measuring the OD value of the bacterial liquid at 600nm every 2 hours. The blank is LB liquid medium without bacteria, three parallel experiments are carried out, analysis data are recorded, and a growth curve graph is drawn by taking the measured OD value as an ordinate and the time as an abscissa, as shown in FIG. 15. The measurement result shows that Burkholderia BK100 grows slowly in 0-2h and is in a lag phase; 2-16h enter the logarithmic growth phase, and the strain grows fast; after 16h, the stationary phase is entered.
Three) BK100 temperature tolerance of Burkholderia
Picking single bacterial colony of Burkholderia BK100, inoculating to LB liquid medium, shaking at 30 ℃, activating for 16h at 180r/min, inoculating the activated bacterial liquid to LB liquid medium with 1% inoculum size, culturing for 24h at 20 ℃, 25 ℃, 30 ℃, 37 ℃, 42 ℃ and 47 ℃ respectively, measuring OD value at 600nm, taking blank control as the LB liquid medium without bacteria, performing three groups of parallel experiments, recording analysis data, and drawing a graph with the measured OD value as ordinate and temperature as abscissa, as shown in FIG. 16. The measurement result shows that the optimum growth temperature of Burkholderia BK100 is 30 ℃, the Burkholderia BK100 grows well at 30-37 ℃, grows slowly at below 25 ℃ and above 42 ℃ and hardly grows at below 20 ℃.
Four) BK100pH tolerance by Burkholderia
Picking single bacterial colony of Burkholderia BK100, inoculating to an LB liquid culture medium, activating for 16 hours at a temperature of 180r/min by a shaking table of 30 ℃, inoculating the activated bacterial liquid to the LB liquid culture medium with an inoculum size of 1%, inoculating to the LB liquid culture medium with pH values of 5.0, 6.0, 7.0, 8.0 and 9.0 respectively with an inoculum size of 1%, and culturing for 24 hours at a temperature of 30 ℃ and 180 r/min. The OD value was measured at 600nm, the blank was LB liquid medium without bacteria, three parallel experiments were performed, analysis data were recorded, and the graph was drawn with the measured OD value as ordinate and temperature as abscissa, as shown in FIG. 17. The measurement result shows that Burkholderia BK100 grows best at pH 7.0 and grows more stably and well at pH 5.0-8.0.
Five) morphological and physiological biochemical identification of burkholderia BK100
The basic morphology of Burkholderia BK100 on the primary screening medium was round, more regular, pale yellow, opaque, moist and smooth, raised colonies, smooth edges and regular, as shown in FIG. 18. After gram staining, the cells were observed to be red with an optical microscope and were gram-negative, as shown in FIG. 19. The cell was short rod-shaped as shown in FIG. 20.
The physiological and biochemical results of Burkholderia BK100 are shown in Table 1, and the bacterial catalase test is positive, and the methyl red test, the acetyl methyl methanol test, the catalase test and the starch hydrolysis test are negative.
TABLE 4 physiological and biochemical characteristics of strains
Example 6: determination of ferulic acid content and ferulic acid esterase enzyme Activity in fermentation broth of Burkholderia BK100
First) drawing ferulic acid standard curve
Accurately weighing 0.1g of ferulic acid standard substance, and fixing the volume to 10ml by 50% methanol to prepare 0.1g/L mother liquor, and sequentially diluting the mother liquor into 0.001g/L, 0.0015g/L, 0.01g/L and 0.04g/L sample liquor. And measuring the peak area of the sample liquid by using a high performance liquid chromatograph. And a standard curve is drawn by taking the concentration (g/L) of the sample liquid as an abscissa and the peak area as an ordinate, and a linear regression equation is prepared, as shown in FIG. 21. Its regression equation y=314399x+5.94, correlation coefficient R 2 =0.9999, indicating that the two are linearly related well.
Two) determination of ferulic acid content
Selecting single colony of Burkholderia BK100, inoculating to a seed solution culture medium with pH of 7.0, activating for 16h in a shaking table with the temperature of 30 ℃ and the pressure of 180r/min, adding activated seed solution into a fermentation culture medium with the inoculum size of 4%, fully and uniformly stirring, fermenting for 72h in a 30 ℃ incubator with constant temperature, adding PBS buffer solution with the pH of 6.0, continuously leaching for 24h in a shaking table with the temperature of 30 ℃ and the pressure of 180r/min, taking 20mL of fermentation liquor with the pressure of 24h, centrifuging for 5min with 12000r/min, filtering the supernatant with a water-based filter membrane with the pressure of 0.22 mu m, and carrying out high performance liquid chromatography detection analysis, wherein the corresponding peak area is brought into a regression equation of FIG. 21, and the ferulic acid content in the fermentation culture medium is 623.08 +/-0.79 ng/L.
Three) determination of feruloyl esterase enzyme Activity
1) Preparation of Burkholderia BK100 fermentation broth: a. selecting a single colony of Burkholderia BK100, inoculating the single colony to a seed solution culture medium with pH of 7.0, activating the single colony for 16h in a shaking table with the temperature of 30 ℃ and the speed of 180r/min, b, inoculating the activated liquid to 100mL of fermentation culture medium with the inoculum size of 4%, fully and uniformly stirring, placing the mixture in a 30 ℃ incubator for fermentation at constant temperature for 72h, adding PBS buffer solution with the pH of 6.0, and placing the mixture in a shaking table with the speed of 180r/min at 30 ℃ for continuous leaching for 24h, thus obtaining fermentation liquor;
2) Preparation of crude enzyme solution: centrifuging 1mL of the fermentation broth obtained in the step 1) at 10000r/min for 15min, and taking supernatant as crude enzyme liquid;
3) Enzyme reaction assay: taking 250ul and 750ul of 0.003mol/L of ferulic acid methyl ester solution (dissolved in 0.05mol/L of Tris-HCl solution) in the step 2), adding into a test tube, shaking uniformly, reacting the reaction solution with pH of 6.0, placing the test tube in a water bath kettle with the temperature of 50 ℃ for 15min, transferring the test tube to boiling water after the reaction is finished, boiling for 10min, cooling, centrifuging for 5min at 10000r/min, sucking the supernatant, and filtering through a 0.22um water-based filter membrane to obtain a sample solution. A control experiment was performed with an equal amount of Tris-HCl buffer instead of crude enzyme as a blank. The sample solution was subjected to high performance liquid chromatography detection analysis, and the results are shown in fig. 22. The peak time (22 a) of the ferulic acid in the sample liquid and the peak time (22 b) of the ferulic acid standard substance are both about 13min, which proves that the sample liquid contains ferulic acid esterase and can metabolize the ferulic acid methyl ester to generate the ferulic acid.
Definition of enzyme activity: the amount of enzyme required to hydrolyze methyl ferulate at a specific temperature per minute to produce 1. Mu. Mol ferulic acid is 1 enzyme activity unit (1U)
Example 7: enzyme activity determination condition optimization of feruloyl esterase produced by Burkholderia
One) one-factor experiment
1) Effect of reaction temperature on enzyme activity assay: the reaction conditions of other enzymes are unchanged, the reaction temperature is respectively set to 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃ for enzyme activity measurement, and the influence of the reaction temperature on the enzyme activity is examined. Three parallel experiments were performed for each temperature.
2) Effect of reaction time on enzyme activity assay: the reaction temperature is the highest enzyme activity temperature in the single factor test, other enzyme reaction conditions are unchanged, and the enzyme activity is measured by taking reaction solutions with the reaction time of 10min, 15min, 20min, 25min and 30min respectively, so as to examine the influence of the reaction time on the enzyme activity. Three replicates were run at each time.
3) Effect of reaction pH on enzyme activity assay: the reaction temperature and time are respectively the highest temperature and time of the enzyme activity in the single factor test, the reaction conditions of the rest enzymes are unchanged, and the reaction liquids with the reaction pH values of 5.0, 6.0, 7.0, 8.0 and 9.0 are taken for enzyme activity measurement, so that the influence of the reaction pH values on the enzyme activity is examined. Three replicates were run for each pH.
4) Influence of crude enzyme addition on enzyme activity assay: the reaction temperature, time and pH are respectively the highest temperature, time and pH of the enzyme activity in the single factor test, other enzyme reaction conditions are unchanged, and enzyme activity measurement is carried out by taking reaction solutions with the addition amounts of 150 mu L, 200 mu L, 250 mu L, 300 mu L and 350 mu L of crude enzyme solution respectively, so as to examine the influence of the addition amount of the crude enzyme solution on the enzyme activity. Three replicates were run for each additive amount.
As is clear from FIG. 23, the enzyme activity of feruloyl esterase produced by Burkholderia was maximum at a reaction temperature of 40℃but decreased at a temperature exceeding 40℃and very low at a reaction temperature of less than 30℃as measured by enzyme activity, because the rate of enzyme reaction was increased when the temperature was increased but the enzyme was denatured and deactivated because the temperature of the enzyme was too high.
As is clear from FIG. 24, the ferulic acid esterase produced by Burkholderia gradually increased in activity with the increase of the reaction time, and the ferulic acid esterase was maximized at 20 minutes of the reaction time, whereas the ferulic acid esterase was decreased with the continuous increase of the reaction time.
As can be seen from FIG. 25, the enzyme activity of feruloyl esterase produced by Burkholderia is maximum at a reaction pH of 7.0, and the enzyme activity is high in an enzyme reaction system having a pH of 6.0-8.0; the activity of peracid or peralkali feruloyl esterase is low, and the space structure of enzyme is possibly destroyed under the condition of peracid or peralkali, and the dissociation process of groups which maintain the space structure of enzyme molecules is also possibly influenced, so that the conformation of enzyme active sites is influenced, and the activity of enzyme is further influenced.
As can be seen from fig. 26, the enzyme activity increases with an increase in the crude enzyme solution, and the enzyme activity is lower when the amount of the crude enzyme solution added is smaller; the enzyme activity of feruloyl esterase of Burkholderia reaches the maximum when the addition amount of crude enzyme liquid is 300 mu L; the addition of the crude enzyme solution in an amount of more than 300. Mu.L may be due to the fact that the crude enzyme solution contains some substances which affect the measurement of the enzyme activity of ferulic acid esterase, and when the content of the substances exceeds a certain amount, the measurement of the enzyme activity of ferulic acid esterase is affected.
Two) response surface test design
Analyzing the result of the single factor test, applying Design-Expert V8.0.6 software on the basis, designing the test by taking the reaction temperature (A), the reaction time (B), the reaction pH (C) and the crude enzyme liquid addition amount (D) as investigation factors and taking the enzyme activity (E) of ferulic acid esterase as a response value according to the Box-Behnken test Design principle, and optimizing the condition of the enzyme activity measurement of the ferulic acid esterase. The response surface factors and their horizontal settings are shown in table 2. The Box-Behnken experimental design and results are shown in Table 3.
Table 5 response surface factor level encoding table
TABLE 6 regression model analysis of variance results
/>
1) Establishing and analyzing secondary response surface regression model
According to the test results of table 3, design-Expert V8.0.6 software was used to perform a quadratic regression fit on the test data, and the obtained binary regression equation was:
E＝83.49+6.63A+5.95B+1.6C+1.88D+0.41AB+0.072AC+1.05AD+1.42BC+0.013BD+0.99CD-15.07A 2 -9.85B 2 -8.58C 2 -3.93D 2
Analysis of variance of enzyme activity determination condition optimization regression model is shown in Table 4
TABLE 7 regression model analysis of variance results
/>
2) Analysis of the influence of the interaction of factors on the enzyme Activity of feruloyl esterase
The response surface diagram can intuitively reflect the influence of test factors on the enzyme activity of feruloyl esterase, and the steeper the slope of the response surface, the more sensitive the response surface value is to the change of the operation condition. As can be seen from Table 4, A, B appears to be extremely remarkable (P < 0.01), indicating that the reaction temperature and reaction time have a great influence on the enzyme activity of feruloyl esterase, A 2 、B 2 、C 2 、D 2 The four enzyme activity measurement conditions are extremely remarkable (P is less than 0.01), and the influence of the four enzyme activity measurement conditions on the enzyme activity of feruloyl esterase is not a simple linear relation. The influence magnitude sequence of each measurement condition is as follows: the reaction temperature is more than the reaction time is more than the addition amount of the crude enzyme solution is more than the reaction pH.
The enzyme activity measurement conditions after software Design-Expert V8.0.6 optimization are as follows: 42.35 ℃, reaction time: 21.59min, reaction pH of 7.14, crude enzyme adding amount of 314.43 mu L, and theoretical enzyme activity of feruloyl esterase of 85.5953U/L. The results were examined, and the optimal enzyme activity measurement conditions were modified in consideration of actual conditions: reaction temperature: 42 ℃, reaction time: 22min, reaction pH 7.1, crude enzyme solution addition: 320. Mu.L, three parallel experiments were performed under the assay conditions, with an average enzyme activity of feruloyl esterase of 85.08U/L, which is close to the predicted value of the software. Therefore, the response surface method is used for optimizing the enzyme activity measurement conditions of feruloyl esterase.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (13)

1. The enterococcus faecium L-7 for producing feruloyl esterase is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO. M20221555 and a preservation date of 2022 and 10 months and 10 days.
2. Gao Wenba g-resistant Hold's bacillus BK100 is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) No. M20221553, and the preservation date is 2022 and 10 months and 10 days.
3. The enterococcus faecium L-7 producing feruloyl esterase according to claim 1, wherein the enterococcus faecium L-7 has a pH tolerance of between pH 4 and 11, grows well at pH 7 and 10, and grows most rapidly at pH 8.
4. The enterococcus faecium L-7 producing feruloyl esterase according to claim 1, wherein the enterococcus faecium L-7 can normally grow in the range of 1% -3% NaCl concentration and 1% -7% ethanol concentration.
5. The enterococcus faecium L-7 producing feruloyl esterase according to claim 4, wherein the enterococcus faecium L-7 grows well at a NaCl concentration of 2% and an ethanol concentration of 5%.
6. A method for screening the enterococcus faecium L-7 producing feruloyl esterase according to claim 1, comprising the steps of:
and 1) sampling: taking 10g of samples from each corner and the center of the finished Daqu, grinding and mixing uniformly to obtain Daqu samples;
second) enrichment: weighing 2g of the Daqu sample obtained in the step 1), filling the obtained product into a 250mL triangular flask, adding 100mL of sterile physiological saline, shaking uniformly, and performing shake culture at 37 ℃ and 180r/min for 30min to prepare bacterial suspension;
third), screening: serial gradient dilution is carried out on the bacterial suspension prepared in the step 2) by using sterile physiological saline, and 100 mu L of dilution factors are respectively taken to be 10 -3 、10 -4 、10 -5 、10 -6 Coating on a screening culture medium, making three gradients in parallel, and inversely culturing the coated culture dish in a 37 ℃ incubator for 72 hours;
Fourth), purification: observing and screening the colony and the transparent ring size on the culture medium, picking the colony with larger transparent ring, streaking on the LB solid culture medium, culturing for 24 hours in a 37 ℃ incubator, repeating for three times to obtain a single colony of the target strain; the single colonies obtained were inoculated into LB liquid medium and cultured overnight. Inoculating 1ml of bacterial liquid into a fermentation medium, culturing for 72 hours, ending fermentation, centrifuging, taking supernatant, filtering to obtain a liquid phase vial, measuring the ferulic acid content in the fermentation liquid by using a high performance liquid chromatography, and preserving strains producing ferulic acid esterase in a glycerol tube;
wherein, screening culture medium: 2g/L of sodium nitrate, 1g/L of dipotassium hydrogen phosphate, 0.5g/L of potassium chloride, 0.5g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate, 20g/L of agar, 1L of ultrapure water and sterilization at 121 ℃ for 20min; cooling to 70deg.C, adding 1.5% (V/V) ethyl ferulate (10% W/V in N, N-dimethylformamide solution), and sterilizing by filtration through 0.22um sterile filter membrane;
LB solid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L agar, 1L ultrapure water, natural pH, 121 ℃ and sterilization for 20min;
LB liquid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 1L ultrapure water, natural pH, 121 ℃ and sterilization for 20min;
Fermentation medium: wheat bran 10g/L, yeast extract 1g/L, potassium chloride 0.5g/L, magnesium sulfate heptahydrate 0.5g/L, dipotassium hydrogen phosphate 0.1g/L, ultrapure water 1L, pH nature, 121 ℃, sterilization for 20min, and independent sterilization of magnesium sulfate heptahydrate and addition of the magnesium sulfate heptahydrate into a culture medium.
7. Use of enterococcus faecium L-7 according to claim 1 for the fermentative production of feruloyl esterase.
8. The use according to claim 7, wherein the fermentation conditions for producing feruloyl esterase by enterococcus faecium L-7 fermentation are: the inoculation amount is 6%, the rotating speed is 160r/min, the culture temperature is 32 ℃, and the culture time is 96 hours.
9. The Gao Wenba g-resistant burkholderia BK100 of claim 2, wherein the burkholderia BK100 has a temperature tolerance in the range of 20-57 ℃ and grows well at 25-40 ℃ with an optimum growth temperature of 30 ℃.
10. A Gao Wenba gram-resistant halidella BK100 isolation and screening step as claimed in claim 2, comprising the following operative steps:
and 1) enrichment: fermenting in certain winery for 1-30 days, storing Daqu Qu Yang for one month and two months, pulverizing, mixing to obtain yeast powder, placing in-80 refrigerator, adding 10g yeast powder into 90mL enriched culture medium, and making into 10 -1 Daqu enrichment of (2), and diluting to obtain 10 -2 -10 -7 Daqu dilution of (2);
two) screening of strains with high feruloyl esterase yield
1) And (3) primary screening: inoculating four Daqu dilutions with different gradients on a primary screening culture medium plate, inversely culturing for 3d at 30 ℃, purifying and culturing single bacterial colonies on the culture medium, inoculating the culture medium to the primary screening culture medium for 5d based on the culture at 30 ℃, observing whether transparent rings are formed, and inoculating bacterial colonies with the transparent rings to the primary screening culture medium for purifying and separating again to obtain pure bacterial colonies;
2) And (3) re-screening: a. the strain which can generate more transparent circles is obtained in the step 1), and the strain is separated and purified for multiple times, wherein the transparent circles of Burkholderia are the largest. B, inoculating Burkholderia BK100 into 100mL of LB medium, and placing the LB medium at 30 ℃ for culturing for 48 hours; b. inoculating Burkholderia BK100 into 100ml of rescreen culture medium with an inoculum size of 2% by volume, culturing at 30 ℃ for 72 hours at 180r/min, and then measuring the ferulic acid content in the fermentation broth by using high performance liquid chromatography to obtain a strain with the enzyme activity of producing ferulic acid esterase of 41.6U/L, and preserving the strain in a glycerol tube;
wherein the enrichment medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L glucose, 1L distilled water, natural pH, and sterilizing at 121deg.C for 20min;
Primary screening of the culture medium: potassium chloride 0.5g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate heptahydrate 0.5g/L, sodium nitrate 2.0g/L, ferrous sulfate heptahydrate 0.01g/L, agar 20g/L, sucrose 30g/L, distilled water 1L, pH is natural, 121 ℃; sterilizing for 20min, cooling to 60deg.C, and adding 10mL (10% W/V in N, N-dimethylformamide) of ethyl ferulate;
the LB medium is: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, 1L distilled water, natural pH, and sterilizing at 121deg.C for 20min;
re-screening the culture medium: 10g of yeast powder, 0.5g of potassium chloride, 0.5g of magnesium sulfate heptahydrate, 1.0g of dipotassium hydrogen phosphate, 20g of agar, 1L of distilled water, natural pH, sterilization at 121 ℃ for 20min, cooling to 60 ℃ and adding 10mL of ethyl ferulate (10% W/V in N, N-dimethylformamide solution).
11. Use of Gao Wenba g-resistant halidella BK100 as claimed in claim 2 for the fermentative production of feruloyl esterases.
12. The use according to claim 10, wherein the conditions for measuring the enzyme activity of feruloyl esterase produced by fermentation of burkholderia BK100 are that the optimal reaction temperature of the enzyme activity in the crude enzyme solution is 20-60 ℃, the optimal reaction pH of the enzyme activity in the crude enzyme solution is 5.0-9.0, the addition amount of the crude enzyme solution is 250-350 mu L, and the optimal reaction time of the enzyme activity in the crude enzyme solution is 15-30min.
13. The use according to claim 11, wherein the conditions for measuring the enzyme activity of the feruloyl esterase produced by fermentation of burkholderia BK100 are that the optimal reaction temperature of the enzyme activity in the crude enzyme liquid is 42 ℃, the optimal reaction pH of the enzyme activity in the crude enzyme liquid is 7.1, the addition amount of the crude enzyme liquid is 320 μl, and the optimal reaction time of the enzyme activity in the crude enzyme liquid is 22min.
CN202310038026.4A 2023-07-29 2023-07-29 Two strains producing feruloyl esterase Pending CN116731898A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310038026.4A CN116731898A (en) 2023-07-29 2023-07-29 Two strains producing feruloyl esterase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310038026.4A CN116731898A (en) 2023-07-29 2023-07-29 Two strains producing feruloyl esterase

Publications (1)

Publication Number Publication Date
CN116731898A true CN116731898A (en) 2023-09-12

Family

ID=87901797

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310038026.4A Pending CN116731898A (en) 2023-07-29 2023-07-29 Two strains producing feruloyl esterase

Country Status (1)

Country Link
CN (1) CN116731898A (en)

Similar Documents

Publication Publication Date Title
Zhang et al. The two-step biotransformation of monosodium glutamate to GABA by Lactobacillus brevis growing and resting cells
CN113186121B (en) Caproic acid bacteria capable of utilizing various substrates and application thereof
CN111484954A (en) Pseudomonas nigricans for producing alginate lyase
CN111100825B (en) Bacillus and application thereof in industry
CN115322911A (en) Abnormal yeast Weikehan and application thereof
CN110317734B (en) Monascus with high yields of saccharifying enzyme, esterifying enzyme and protease and separation culture method and application thereof
RU2381270C1 (en) STRAIN OF BACTERIA Clostridium acetobutylicum-PRODUCER OF BUTANOL, ACETONE AND ETHANOL
CN113957016A9 (en) Bacillus subtilis and method for preparing milk-flavor cordyceps sinensis fermentation liquor by using same
CN111205987A (en) Monascus, method for preparing strong aromatic Chinese spirit esterification liquid by using same and application thereof
JP5051727B2 (en) Heat-resistant ethanol-producing yeast and ethanol production method using the same
CN116731898A (en) Two strains producing feruloyl esterase
CN112143681B (en) Bacillus belgii capable of producing feruloyl esterase and application thereof
AU2021102656A4 (en) A method and compositions for treatment of alzheimer’s disease
EP3845658B1 (en) Method for preparing vanillin by fermentation with eugenol as substrate
CN113174342A (en) Strain for efficiently degrading ethyl carbamate and application thereof
CN116083307B (en) Lactococcus garvieae for producing esterase and ester compounds and application thereof
CN117143744B (en) Bremia mycorrhizae and application thereof in preparation of yellow water esterified liquid
CN114854625B (en) Wound escherichia and method for preparing carotenoid degrading enzyme by using same
JP2003079363A (en) Culture medium for separating soy sauce lactic bacterium having low turbidity, method for separating soy sauce lactic bacterium having low turbidity by using the medium, and method for producing soy sauce having high clarity by using the lactic bacterium
CN114410546B (en) Ester-producing and aroma-enhancing acinetobacter vinelandii and application thereof in marine fish fermentation products
CN117431189B (en) Lactobacillus paracasei subspecies paracasei strain QH-20029 and application thereof
Easa et al. Biosynthesis of Lovastatin by Gamma Irradiated Aspergillus terreus
CN110607266B (en) Flavobacterium for producing alginate lyase and application thereof
RU2404247C2 (en) Method of obtaining butanol
CN114032185A (en) Hansenula polymorpha culture medium and culture method for grape juice

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination