CN115851670B - Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive - Google Patents
Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive Download PDFInfo
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- CN115851670B CN115851670B CN202211515012.9A CN202211515012A CN115851670B CN 115851670 B CN115851670 B CN 115851670B CN 202211515012 A CN202211515012 A CN 202211515012A CN 115851670 B CN115851670 B CN 115851670B
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a xylanase mutant, a compound preparation of the xylanase mutant and an eucommia ulmoides leaf extract and application of the xylanase mutant and the eucommia ulmoides leaf extract in a feed additive. The xylanase xyn from actinomycetes Actinobacteria bacterium is mutated, xylanase mutants AxynM1, axynM2 and AxynM3 are obtained through screening, compared with the original genes, the tolerance of the xylanase mutants to animal endogenous pepsin is respectively improved from 55% to 61%, 66% and 73%, the tolerance to animal endogenous trypsin is over 95%, and anti-nutritional factors such as xylan in feed ration can be effectively degraded. According to the invention, xylanase mutants and eucommia ulmoides leaf extracts are compounded, so that the feed utilization rate and conversion rate in piglet cultivation can be improved, and the piglet production performance is further improved.
Description
Technical Field
The invention belongs to the field of enzyme engineering, and in particular relates to a xylanase mutant, a compound preparation of the xylanase mutant and an eucommia ulmoides leaf extract and application of the xylanase mutant and the eucommia ulmoides leaf extract in a feed additive.
Background
Xylan is a five-carbon sugar, an important component of hemicellulose, and is also a polysaccharide with a content inferior to that of cellulose in nature. Xylan is widely present in feed materials such as corn, wheat bran, rice bran, straw, soybean meal, etc., and belongs to non-starch polysaccharides in feeds which cannot be effectively degraded in the digestive system of animals. The xylan is difficult to digest by monogastric animals, and simultaneously combines a large amount of water, so that the volume of chyme in the digestive tract of the fed animals is increased, the viscosity is increased, and the actions of nutrients and endogenous enzymes in the digestive tract are reduced, thereby preventing the digestion and absorption of nutrients, particularly fat and protein, and reducing the utilization rate of feed. In the breeding industry, the xylanase is added into the feed to degrade the anti-nutritional factor xylan, so that the feed utilization rate is improved economically and effectively.
Xylanase is a generic term for enzymes capable of degrading xylan into oligosaccharide or xylose, and mainly comprises endo-beta-1, 4 xylanase, xylosidase, arabinosidase and the like, wherein the endo-beta-1, 4 xylanase plays a main role in the enzymes. Most xylanases on the market at present have a suitable temperature of 40-60℃and a pH of 5.0-7.0. Most xylanases have poor tolerance to animal endogenous proteases, and especially after pepsin treatment, the enzyme activity residual rate is only about 30%, so that the enzymolysis effect of the xylanases in animals is greatly affected. The current general approach to solve such problems is to use a coating agent and a carrier to increase the tolerance of the enzyme, which increases the production cost of the enzyme preparation on the one hand and affects the bioavailability of the enzyme preparation on the other hand.
By means of genetic engineering and in-vitro directed evolution means of enzymes, the enzyme characteristics are improved from the genes and structures of the enzymes, and xylanase with excellent heat resistance, acid resistance and endogenous protease resistance is obtained through screening, so that the xylanase has important significance in reducing the production cost of the xylanase for feeding at present and improving the utilization efficiency of the xylanase.
Disclosure of Invention
The invention provides a xylanase mutant, a compound preparation of the xylanase mutant and an eucommia ulmoides leaf extract and application of the xylanase mutant and the eucommia ulmoides leaf extract in a feed additive. According to the invention, xylanase mutants AxynM1, axynM2 and AxynM3 with strong tolerance to animal endogenous pepsin are obtained through screening, and can be mixed with eucommia ulmoides leaf extracts to prepare a feed additive, so that the ratio of feed to weight is effectively reduced, and the growth performance is improved.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides xylanase mutant AxynM, the amino acid sequence of which is shown in SEQ ID NO:3, or as set forth in SEQ ID NO:5, or as set forth in SEQ ID NO: shown at 7.
Further, it specifically includes: consists of a polypeptide with an amino acid sequence of SEQ ID NO:1 to serine from glutamine at position 40 of the xylanase of the invention; consists of a polypeptide with an amino acid sequence of SEQ ID NO:1 from glutamine at position 40 to serine and from alanine at position 54 to serine; consists of a polypeptide with an amino acid sequence of SEQ ID NO:1 from glutamine to serine and from leucine to methionine at position 163.
The invention also provides a coding gene which is the coding gene of the xylanase mutant AxynM, and the nucleotide sequence is shown in SEQ ID NO:4, or SEQ ID NO:6, or SEQ ID NO: shown at 8.
The invention also provides a recombinant engineering bacterium which comprises the coding gene.
The invention also provides a compound preparation which comprises the xylanase mutant AxynM and an eucommia ulmoides leaf extract.
Further, the mass-volume ratio of the powder preparation of the xylanase mutant AxynM to the eucommia ulmoides leaf extract is 2:3-8; the enzyme activity of the xylanase mutant AxynM is not lower than 10 ten thousand U/g.
The invention also provides application of the xylanase mutant in enzymolysis of bran.
Further, the xylanase mutant is used in an amount of 100U/g to 500U/g.
The invention also provides application of the xylanase mutant or the compound preparation in preparing animal feed additives.
Furthermore, the dosage of the xylanase mutant or the compound preparation is 0.01-0.05% of the weight of the animal feed.
Compared with the prior art, the invention has the advantages and technical effects that:
1. the invention uses xylanase gene of actinomycetes Actinobacteria bacterium as a basis for mutation improvement, and the xylanase mutants AxynM1, axynM2 and AxynM3 of which mutation sites are single-point mutation Q40S, double-point mutation Q40S/A54S and Q40S/L163M are obtained by screening. Compared with the original genes, the xylanase mutant obtained by the invention has the advantages that the tolerance to animal endogenous pepsin is respectively improved from 55% to 61%, 66% and 73%, the tolerance to animal endogenous trypsin is over 95%, and the xylanase mutant can effectively degrade anti-nutritional factors such as xylan in feed ration, so that the xylanase mutant has good application prospects in the fields of feeds and the like.
2. The invention utilizes the enzyme preparation prepared by fermenting, filtering and drying the engineering bacteria containing the mutant to compound with the eucommia ulmoides leaf extract, which can improve the feed utilization rate and conversion rate in piglet cultivation, thereby improving the piglet production performance.
Drawings
FIG. 1 shows the amplification results of the xylanase gene.
FIG. 2 is a comparison of the tolerance of the xylanase mutants to endogenous proteases.
FIG. 3 is fermentation data of the xylanase mutants in a 15L fermenter.
FIG. 4 shows the effect of the xylanase mutants on wheat bran degradation.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings and examples, in which the invention is shown, but the scope of the invention is not limited to the specific examples. Reagents and biological materials used in the specific examples are commercially available unless otherwise specified.
The formula of the culture medium used in the invention is as follows:
LB medium: 1% tryptone, 0.5% yeast extract, 1% nacl;
MD medium: 1.34% YNB,0.4mg/L biotin, 2% glucose;
YPD medium: 1% yeast extract, 2% peptone, 2% glucose;
BMGY medium: 1% yeast extract, 2% peptone, 100mmol/L potassium phosphate buffer (pH 6.0), 1.34% YNB,0.4mg/L biotin, 1% glycerol;
BSM medium: 26.7mL of 85% phosphoric acid, 0.93g of calcium sulfate dihydrate, 14.9g of magnesium sulfate dihydrate, 4.13g of potassium hydroxide, 18.2g of potassium sulfate, 40g of glycerol, and 4.0mL of PMT1.
When the culture medium is solid, adding 2% agar powder.
Enzyme activity measurement: reference is made to the determination method in "determination spectrophotometry for xylanase Activity of GBT 23874-2009 feed additive".
Example 1: xylanase mutant gene construction and screening
Referring to the xylanase xyn (GenBank: PZN 45477.1) amino acid sequence of actinomycetes Actinobacteria bacterium, translating the xylanase xyn into a corresponding nucleotide sequence, and artificially synthesizing the nucleotide sequence after gene sequence optimization to obtain the amino acid sequence shown in SEQ ID NO:1, the corresponding nucleotide sequence is shown as SEQ ID NO: 2. The primer was designed as follows, an EcoR I restriction site was designed at the 5 'end, and a Not I restriction site was designed at the 3' end.
xyn-F：CCGGAATTCGATACCACTATTACTCAAAA(SEQ ID NO：9)；
xyn-R：ATAAGCGGCCGCTTAGTTGGCGGTACAGGTAA(SEQ ID NO：10)。
The random mutation is carried out by using a GeneMorph II random mutation PCR kit and using the artificially synthesized gene as a template and the primer sequence, and the PCR amplification result is shown in figure 1. The amplified random mutation PCR product is digested with EcoR I and Not I, purified and recovered, and then connected to a pET-21a (+) vector, and escherichia coli BL21-DE3 is transformed, and ampicillin resistance LB plates are used for screening positive clones to obtain pET-AxynMx. The synthesized original gene is connected to a pET-21a (+) vector and transformed into escherichia coli BL21-DE3 by the same method to obtain pET-xyn0.
The screened single colonies were inoculated into 96-well deep well plates. 2 single colonies expressing xyn0 were inoculated per plate as controls. 300uL of LB liquid medium (containing 100 mug/mL of ampicillin) is filled into each hole, after shaking culture is carried out for 4 hours at 37 ℃ and 200rpm, 50uL of bacterial liquid is transferred to a new 96-hole flat plate for seed preservation, 200uL of LB-Amp medium containing IPTG is added into the remaining bacterial liquid of the flat plate, the final concentration of the IPTG is 1mM, the final concentration of the ampicillin is 100 mug/mL, and shaking culture is carried out for 10 hours at 200rpm at 37 ℃ to induce xylanase expression.
Repeatedly freezing and thawing the induced bacterial liquid for breaking, centrifuging the broken cell liquid to obtain supernatant, and detecting the enzyme activity of xylanase and the enzyme activity after pepsin treatment. Mutant genes with higher enzyme activity residual rates than the control were sequenced.
Sequencing to obtain xylanase mutant AxynM1 with mutation site Q40S and amino acid sequence as shown in SEQ ID NO:3, the nucleotide sequence is shown as SEQ ID NO: 4.
Example 2: random mutation by using xylanase mutant AxynM1 gene as template
The mutant AxynM1 screened in the example 1 is used as a template, the same random mutation method is used for carrying out the second round of mutation and screening, the AxynM1 is used as a control during screening, the enzyme activity of xylanase is detected, and the mutant gene with obviously improved enzyme activity residual rate after endogenous protease treatment is sequenced.
After a large number of screening, two mutants AxynM2 and AxynM3 with obviously improved enzyme activity residual rate after endogenous protease treatment are obtained:
the mutation mode of AxynM2 is Q40S/A54S, and the amino acid sequence is shown in SEQ ID NO:5, the nucleotide sequence is shown as SEQ ID NO:6 is shown in the figure;
the mutation mode of AxynM3 is Q40S/L163M, and the amino acid sequence is shown in SEQ ID NO:7, the nucleotide sequence is shown as SEQ ID NO: shown at 8.
Example 3: transformation of xylanase mutant with Pichia pastoris GS115
The xylanase mutant gene is connected to pPIC9K plasmid by EcoR I and Not I double enzyme cutting sites and transformed into escherichia coli DH5 alpha to obtain an expression vector, and sequencing verification is carried out. The expression vector was then electrotransformed into Pichia pastoris GS115 after being tangential with Sal I enzyme, and the transformants were selected on MD plates and transferred to YPD plates for activation (typically 24-48 transformants were selected). The activated transformant was inoculated in shake flasks for fermentation (20 mL of BMGY medium per flask), shake-cultured at 30℃for 18 hours, then induced by adding 1% methanol, and shake-cultured further, and then 1% methanol was added per 24 hours. After 96h of induction expression, the culture broth was centrifuged to obtain a supernatant, and the average enzyme activity of the broth supernatant was measured.
Endogenous protease tolerance assay: digestion of xyn0, axynM1, axynM2 and AxynM3 in an artificial gastric juice with pH 2.0 in a water bath at 40 ℃ for 2 hours, and measurement of enzyme activity under the conditions of pH 5.5 and 37 ℃, wherein the enzyme activity of untreated enzyme liquid is defined as 100%, and the relative enzyme activity is calculated; and then respectively digesting xyn0, axynM1, axynM2 and AxynM3 in an artificial intestinal juice with the pH of 6.8 in a water bath at the temperature of 40 ℃ for 4 hours, measuring the enzyme activity of the artificial intestinal juice at the pH of 5.5 and the temperature of 37 ℃, and calculating the relative enzyme activity by taking the enzyme activity of untreated enzyme liquid as 100 percent.
The result of the tolerance of the xylanase mutant to endogenous protease is shown in figure 2, and the tolerance of the xylanase mutant to animal endogenous pepsin is respectively improved from 55% to 61%, 66% and 73%, and the tolerance to animal endogenous trypsin is over 95%.
Example 4: xylanase mutant AxynM3 fermentation and preparation in 15L fermentation tank
And (3) respectively streaking the genetic engineering bacteria of the xylanase mutant AxynM3 on a YPD plate, culturing at 30 ℃ for 3 days to grow single colonies, picking the single colonies with good growth vigor, continuing streaking culture on the YPD plate, and inoculating the single colonies of the pichia pastoris obtained by three generations of activation in 20mL of BMGY culture medium, and culturing at 30 ℃ at 200rpm for 24 hours. The seed solution was inoculated into 300mL of BMGY medium at an inoculum size of 2%, and cultured at 30℃and 200rpm until the OD600 was 5, and used as a seed solution inoculation fermenter. The fermentation production process comprises the following steps: BSM culture medium, pH 4.8, temperature 30 ℃, stirring speed 500rpm, ventilation rate 1.5 (v/v), dissolved oxygen control over 20% fermentation process is divided into three stages: (1) a cell culture stage: inoculating seed liquid according to the proportion of 8%, and culturing for 20-24 hours at 30 ℃ to ensure that the glycerol in the fermentation liquid is exhausted; (2) starvation phase: after the carbon source glycerol is exhausted, temporarily adding no carbon source, and ending the starvation stage when the dissolved oxygen rises to 80%; (3) induction expression stage: ammonia water or phosphoric acid is used for regulating the pH value to a required value, methanol is added for induction, dissolved oxygen is maintained to be more than 20%, and the induction time is 160-200 h; after fermentation is finished, the fermentation liquor is processed by a plate-frame filter and then is sprayed into powder preparation by a spray tower for application test.
The fermentation curve is shown in FIG. 3: sampling every 8h, measuring enzyme production level, fermenting for 168h, and reaching the highest enzyme activity level.
Example 5: xylanase mutant with enzymolysis effect on bran
2% bran is used as a substrate, and a buffer solution is used for preparing a suspension. Control group: no enzyme is added; the experimental groups were 4: namely, xyn0 treatment group, axynM1 treatment group, axynM2 treatment group and AxynM3 treatment group, 200U/g xylanase xyn0, axynM1, axynM2 and AxynM3 were added, respectively; each group was repeated 3 times.
The process for simulating in-vitro enzymolysis of bran comprises the following steps: (1) Adjusting the pH to 5.2 with buffer solution, and carrying out constant-temperature shaking digestion for 1h at a temperature of 40 ℃ and at a speed of 120 rpm; (2) Adjusting the pH to 2.5 by using a buffer solution, and carrying out constant-temperature shaking digestion for 1h at a temperature of 40 ℃ and at a speed of 120 rpm; (3) The pH was adjusted to 6.8 with buffer, and the mixture was digested with shaking at a constant temperature of 120rpm at 40℃for 4 hours. After digestion is completed, the chyme reducing sugar content and dry weight before and after digestion are measured, and the dry matter disappearance rate and reducing sugar increase rate are calculated.
As shown in FIG. 4, the disappearance of dry matter of bran after digestion of xyn0 and mutant xynM1, xynM2 and xynM3 was 30.5%, 31.0%, 29.2% and 30.8%, respectively; the growth rates of the reducing sugar are 35.2%, 36.3%, 35.5% and 36.0% respectively. The mutant xylanases xynM1, xynM2 and xynM3 in the invention have equivalent bran raw material degradation efficiency and xyn0 in the simulated digestive tract environment, have no influence on enzymolysis efficiency on the basis of improving xylanase tolerance, have obvious enzymolysis effect on bran, and are beneficial to improving digestion absorption rate and conversion rate of bran.
Example 6: preparation of compound preparation and application experiment in piglet cultivation
The xylanase mutant powder preparation prepared in the example 4 is uniformly mixed with eucommia ulmoides leaf extract according to the mass-volume ratio of 2:5 to prepare a compound preparation. Wherein the enzyme activity of the xylanase mutant in the compound preparation is not lower than 10 ten thousand U/g.
The preparation method of the eucommia ulmoides leaf extract comprises the following steps: extracting folium Eucommiae with water at 55-65deg.C for 2-4 hr, filtering, vacuum concentrating the filtrate at 60deg.C to specific gravity of 1.2, and spray drying in spray drying tower to obtain folium Eucommiae extract.
The 180 healthy piglets close to birth weight at birth are randomly divided into 6 groups, 3 columns, and 10 pigs per column, namely 3 replicates per group. Feeding common basic ration to a control group; feeding folium Eucommiae group with basic ration added with 0.02% folium Eucommiae extract; test groups 1-4 were fed basal diet supplemented with 0.02% xylanase xyn0 and mutants AxynM1, axynM2 and AxynM3, respectively, with no substantial difference in other ingredients of each group.
The test measurement index and the calculation method in the feeding process are as follows: daily gain of piglets = (average weight after test-average weight before test)/test days; daily average consumption = (daily consumption after test-daily consumption before test)/test days; feed to meat ratio = daily average consumption/average daily gain.
After 30 days of cultivation, the test results are shown in the following table 1, and the daily gain of piglets is improved after the eucommia ulmoides leaf extract and xylanase are added; the material-weight ratio is obviously reduced compared with that of a control group, and the xylanase, especially the xylanase mutant of the invention has better effect than that of adding eucommia ulmoides leaf extract. The xylanase mutants AxynM1, axynM2 and AxynM3 are added to improve the feed utilization rate and conversion rate in piglet cultivation, so that the piglet production performance is improved.
Table 1 application of xylanase mutant in piglet cultivation
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (9)
1. The xylanase mutant AxynM is characterized in that the amino acid sequence of the xylanase mutant AxynM is shown as SEQ ID NO:3, or as set forth in SEQ ID NO:5, or as set forth in SEQ ID NO: shown at 7.
2. A coding gene, which is the xylanase mutant AxynM coding gene of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO:4, or SEQ ID NO:6, or SEQ ID NO: shown at 8.
3. A recombinant engineering bacterium comprising the coding gene of claim 2.
4. A combination formulation comprising both the xylanase mutant AxynM of claim 1 and eucommia ulmoides leaf extract.
5. A compound preparation according to claim 4, wherein the mass to volume ratio of the powder preparation of xylanase mutant AxynM to eucommia ulmoides leaf extract is 2:3-8; the enzyme activity of the xylanase mutant AxynM is not lower than 10 ten thousand U/g.
6. The use of a xylanase mutant in enzymatic hydrolysis of bran, characterized in that the xylanase mutant is the xylanase mutant AxynM of claim 1.
7. The use according to claim 6, wherein the xylanase mutant is used in an amount of 100U/g to 500U/g.
8. Use of a xylanase mutant or a complex formulation for the preparation of an animal feed additive, characterized in that the xylanase mutant is a xylanase mutant AxynM according to claim 1; the formulation is the formulation of claim 4.
9. The use according to claim 8, wherein the xylanase mutant or the complex formulation is used in an amount of 0.01% -0.05% by weight of the animal feed.
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| US6277617B1 (en) * | 1996-11-26 | 2001-08-21 | Genencor International, Inc. | Chemically modified enzymes |
| CN1670203A (en) * | 2005-03-10 | 2005-09-21 | 济南高新开发区京鲁生物技术研究开发中心 | Food grade xylanase and its production method |
| CN103131684A (en) * | 2013-01-09 | 2013-06-05 | 中国农业科学院饲料研究所 | Xylanase XynA with C end surplus sequence, gene and application of xylanase XynA with C end surplus sequence, and method for improving xylanase catalytic rate |
| CN103642777A (en) * | 2013-12-10 | 2014-03-19 | 江南大学 | Method of improving thermal stability of aspergillus oryzae xylanase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6277617B1 (en) * | 1996-11-26 | 2001-08-21 | Genencor International, Inc. | Chemically modified enzymes |
| CN1670203A (en) * | 2005-03-10 | 2005-09-21 | 济南高新开发区京鲁生物技术研究开发中心 | Food grade xylanase and its production method |
| CN103131684A (en) * | 2013-01-09 | 2013-06-05 | 中国农业科学院饲料研究所 | Xylanase XynA with C end surplus sequence, gene and application of xylanase XynA with C end surplus sequence, and method for improving xylanase catalytic rate |
| CN103642777A (en) * | 2013-12-10 | 2014-03-19 | 江南大学 | Method of improving thermal stability of aspergillus oryzae xylanase |
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