CN117187099A - Xylanase producing strain HH-22 and application thereof - Google Patents
Xylanase producing strain HH-22 and application thereof Download PDFInfo
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Abstract
The invention relates to a xylanase producing strain HH-22 and application thereof, which can effectively solve the problem that the prior xylanase needs to be cooled in advance before being put into use so as to improve the catalytic activity of the enzyme, and the technical scheme is that the xylanase producing strain HH-22 is classified as microbacterium @Microbacterium sp.) HH-22, deposited in China general microbiological culture Collection center, with accession number: CGMCC No.26621, the preservation date is 2023, 2 and 21 days, and the preservation address is North Chen Silu No.1, 3 in the Chaoyang area of Beijing city; the 16S rDNA gene sequence of the strain is shown as SEQ ID No.1, and the xylanase is effectively used for preparing high temperature resistant xylanase, thus developing a new way for producing xylanase, effectively meeting the industrial requirement for xylanase, and having great economic and social effectsBenefit is provided.
Description
1. Technical field
The invention relates to the technical field of microorganisms, in particular to a xylanase producing strain HH-22 and application thereof.
2. Background art
Xylanase (xylan) is an important industrial enzyme, is widely used in nature, is an important component of hemicellulose, is the second most abundant polysaccharide with the content next to that of cellulose in nature, and almost accounts for one third of renewable organic carbon of the earth. In recent years, the xylanase has an increasing application value in industry, and is widely paid attention to, wherein the alkaline xylanase has higher enzyme activity under alkaline conditions, and the application of the alkaline xylanase in the paper industry comprises the following steps: pulping, assisting bleaching, modifying fiber properties, deinking waste paper and the like. The xylanase can be used as a pulp bleaching auxiliary agent in the paper industry, can improve the whiteness and strength of paper, and reduces the practicability of the traditional chloride bleaching agent; the xylanase is added into the animal feed, so that the xylan can be degraded, and the utilization rate of the animal to the feed is improved; in the food industry, xylanases can also be used to clarify juice beverages, improving bread softness. Xylo-oligosaccharide can be obtained by degrading xylan with xylanase, and can promote proliferation of probiotics in large intestine of human body and improve intestinal function. In nature, the enzyme activity characteristics of xylanases from different sources are greatly different, and the xylanase has respectively specific optimal reaction pH and optimal action temperature.
Furthermore, it can be found by analysis of the overall environment of xylanases currently in use on the market that the xylanase is used in a large number of conditions at high temperatures, such as paper making processes, requiring high temperature operations to remove lignin, etc.; the food production process generally requires high temperature sterilization and other operations. The most suitable reaction temperature of xylanase is 50 ℃, so that the xylanase needs to be cooled in advance before being put into use in the process so as to improve the catalytic activity of the xylanase, and the operation consumes energy and greatly hinders the application and development of xylanase. Therefore, development of xylanase products resistant to high temperatures is imperative.
3. Summary of the invention
Aiming at the situation, the invention aims to solve the defects of the prior art and provide a xylanase producing strain HH-22 and application thereof, which can effectively solve the problem that the prior xylanase needs to be cooled in advance before being put into use so as to improve the catalytic activity of the enzyme.
The xylanase producing strain HH-22 is classified and named as Microbacterium sp HH-22, and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), and the preservation number is: CGMCC No.26621, the preservation date is 2023, 2 and 21 days, and the preservation address is North Chen Silu No.1, 3 in the Chaoyang area of Beijing city; the 16S rDNA gene sequence of the strain is shown as SEQ ID No. 1.
The method for fermenting the high temperature resistant xylanase by utilizing the xylanase-producing strain HH-22 comprises the following steps:
1) Preparing a xylanase fermentation broth: inoculating xylanase producing strain HH-22 into a fermentation enzyme producing culture medium according to an inoculum size of 5% by mass, and shake-flask culturing at 30-37 ℃ and 150-2000 rpm for 48-72 hours to obtain xylanase fermentation liquor;
2) Preparation of xylanase supernatant: centrifuging the xylanase fermentation broth obtained in the step 1) at the temperature of 4 ℃ and at the speed of 12000rpm for 10-15 min to obtain xylanase supernatant;
3) Fermentation supernatant: slowly adding ammonium sulfate into the xylanase supernatant obtained in the step 1) to enable the mass content of the ammonium sulfate to reach 70%, and salting out at 4 ℃ overnight;
4) Preparing a high temperature resistant xylanase crude enzyme solution: salting out the fermentation supernatant fluid after overnight salting out in the step 3) at 4 ℃, 12000rpm for 10-15 min, collecting precipitate, dissolving the precipitate with Tris-HCl buffer solution with pH of 7.2 to obtain high temperature resistant xylanase crude enzyme liquid for standby;
5) Octyl FF hydrophobic interaction chromatography: adjusting the mass concentration of ammonium sulfate in the high temperature resistant xylanase crude enzyme solution obtained in the step 4) to 40%, performing hydrophobic interaction chromatography, and performing column chromatography with the concentration of 2.6X18 cm, wherein the concentration is the balance solution: pH8.0, 0.02mol/L ammonium sulfate with 40% saturation ammonium sulfate, flow rate: 1mL/min, loading, linearly eluting with 1M ammonium sulfate, and collecting active components;
6) DAE FF gel filtration chromatography: column 1.6X50 cm, equilibration solution: 0.02mol/L Tris-HCl buffer, pH8.0, flow rate: collecting active components at 0.5 mL/min;
7) Ff strong cation exchange chromatography: column type 2.6X10 cm, equilibrium liquid: 0.02mol/L Tris-HCl buffer, pH8.0, flow rate: 1mL/min, performing linear elution with an equilibrium buffer solution containing 1.0M NaCl after sample loading, and collecting active components (after column passing, determining components through protein electrophoresis strips and protein sizes) to obtain a high temperature resistant xylanase sample;
8) Preparing xylanase dry powder: and 7) putting the high temperature resistant xylanase sample obtained in the step 7) into a freeze dryer, and freeze-drying at-55 ℃ under 7Pa to obtain xylanase dry powder.
In the step 1), the fermentation enzyme production culture medium comprises the following components: KH (KH) 2 PO 4 1～1.5g，(NH4) 2 SO 4 2～5g，MgSO 4 ·7H 2 0.4-0.5 g of O, 5-5.5 g of yeast extract, 0.5-1.0 g of xylan and pH 7.0.
The xylanase is a novel Microbacterium (Microbacterium) HH-22, has the effect of producing xylanase, is effectively used for preparing high-temperature resistant xylanase, develops a novel xylanase production path, can effectively meet the industrial requirement on xylanase, and has huge economic and social benefits.
4. Description of the drawings
FIG. 1 is a schematic representation of the xylanase of the invention on a plate of inorganic salt.
FIG. 2 is a phylogenetic tree of the construction of 18 species with the xylanase of the invention closest to the 16S rRNA sequence.
FIG. 3 is a schematic diagram showing the relative enzyme activities and stabilities of xylanase of the invention at different temperatures
FIG. 4 is a schematic representation of the relative enzyme activities and stabilities of the xylanases of the invention at different pHs.
FIG. 5 is a schematic representation of the isolation and purification route of the xylanase of the invention.
5. Detailed description of the preferred embodiments
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples.
Example 1
In the specific implementation, the method for fermenting the high-temperature resistant xylanase by the xylanase-producing strain HH-22 comprises the following steps of:
1) Preparing a xylanase fermentation broth: inoculating xylanase producing strain HH-22 into a fermentation enzyme producing culture medium according to an inoculum size of 5% by mass, and shake-culturing at 30 ℃ and 180rpm for 48 hours to obtain xylanase fermentation liquor;
2) Preparation of xylanase supernatant: centrifuging the xylanase fermentation broth obtained in the step 1) at the temperature of 4 ℃ and at the speed of 12000rpm for 10min to obtain xylanase supernatant;
3) Fermentation supernatant: slowly adding ammonium sulfate into the xylanase supernatant obtained in the step 1) to enable the mass content of the ammonium sulfate to reach 80%, and salting out at 4 ℃ overnight;
4) Preparing a high temperature resistant xylanase crude enzyme solution: salting out the fermentation supernatant fluid after overnight salting out in the step 3) at 4 ℃, 12000rpm for 10min, collecting precipitate, dissolving the precipitate with Tris-HCl buffer solution with pH of 7.2 to obtain high temperature resistant xylanase crude enzyme liquid for standby;
5) Octyl FF hydrophobic interaction chromatography: regulating the mass concentration of ammonium sulfate in the high temperature resistant xylanase crude enzyme solution obtained in the step 4) to 40%, performing hydrophobic interaction chromatography, and performing column chromatographyBalancing solution: pH8.0, 0.02mol/L ammonium sulfate with 40% saturation ammonium sulfate, flow rate: 1mL/min, loading, linearly eluting with 1M ammonium sulfate, and collecting active components;
6) DAE FF gel filtration chromatography: column typeBalancing solution: 0.02mol/L Tris-HCl buffer, pH8.0, flow rate: collecting active components at 0.5 mL/min;
7) Ff strong cation exchange chromatography: column typeBalancing solution: 0.02mol/L Tris-HCl buffer, pH8.0, flow rate: 1mL/min, performing linear elution by using a balance buffer solution containing 1.0M NaCl after sample loading, and collecting active components to obtain a high-temperature-resistant xylanase sample;
8) Preparing xylanase dry powder: and 7) putting the high temperature resistant xylanase sample obtained in the step 7) into a freeze dryer, and freeze-drying at-55 ℃ under 7Pa to obtain xylanase dry powder.
The strain for producing the high-temperature resistant xylanase is a new strain separated from soil samples collected by a Yunnan Kunming phosphorite factory (altitude: 1950m; longitude and latitude: 102.593489, 24.666158) in 10 months in 2018, 13 known strains closest to the 16srRNA sequence are selected according to the sequence comparison, a phylogenetic tree (shown in figure 2) is constructed, the strain with the highest xylanase enzyme activity is identified as a small bacterial genus (Microbacterium) HH-22, and the strain is preserved in China general microbiological culture Collection center, with the preservation number: CGMCC No.26621.
The xylanase producing strain HH-22 provided by the invention is effectively applied to producing high temperature resistant xylanase, and the optimal reaction temperature and the optimal pH are respectively 60 ℃ and 7.0; the applicable temperature range is 30-70 ℃, the applicable reaction pH value is 5.0-10.0, and the related test data are as follows:
1. molecular biological Properties
According to the 16srRNA sequence comparison, 13 known strains with similar 16srDNA sequence comparison result sequences are selected, and a phylogenetic tree is constructed, so that the evolution position of the phylogenetic tree is further determined.
16S rDNA sequence:
AGGGCGGGGCGGGTGCTTACCATGCAAGTCGAACGGTGAAGCAGGAGCTTGCTCTTGTGGATCAGTGGCGAACGGGTGAGTAACACGTGAGCAACCTGCCCCGAACTCTGGGATAAGCGCTGGAAACGGCGTCTAATACTGGATATGCACCAGGAAGGCATCTTCTCTGGTGGGAAAGATTTTTTGGTTCGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGGAAGCCTGATGCAGCAACGCCGCGTGAGGGACGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAAAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTCTGTCGCGTCTGCTGTGAAAACCCGAGGCTCAACCTCGGGCCTGCAGTGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGGAGCAAACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGGCCTTTCCACGGTCTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATATTCGAGAACGCTGCAGAAATGCAGAACTCTTTGGACACTCGTATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCAATACCGCGAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAAGTCATGAAAGTCGGTAACACCTGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAAGGTGATCCAATTG
the classification was designated as Microbacterium sp. HH-22, accession number: CGMCC No.26621. 2. Culture and culture conditions (the following culture medium solutions were all water)
Enrichment medium: 1g of yeast powder, 2g of peptone, 2g of xylan and 7.0 pH value;
plate screening medium: xylan 10g KNO 3 1g、MgSO 4 0.2g、NaCl 0.5g、K 2 HPO 4 0.5g, 20g of agar and pH value of 7.0;
slope preservation medium: yeast powder 0.5g, peptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g, soluble starch 0.5g, KH 2 PO 4 0.3g，，MgSO 4 0.024g, 0.3g of sodium pyruvate, 15.0g of agar and pH value of 7.2+/-0.2;
seed culture medium: yeast powder 0.5g, peptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g, soluble starch 0.5g, KH 2 PO 4 0.3g，，MgSO 4 0.024g, 0.3g of sodium pyruvate, 15.0g of agar and pH value of 7.2+/-0.2;
basal enzyme-producing medium: KH (KH) 2 PO 4 1～1.5g，(NH 4 ) 2 SO 4 2～5g，MgSO 4 ·7H 2 0.4-0.5 g of O, 5-5.5 g of yeast extract, 0.5-1.0 g of xylan and pH 7.0;
shake flask culture conditions: the seed age is 24 hours, the inoculation amount is 5 percent, the shaking bottle liquid filling amount is 50mL, the temperature is 30-37 ℃, and the shaking bottle culture is carried out at 150-20 rpm for 48-72 hours.
3. The route for isolation and purification of the thermostable xylanase is shown in FIG. 5.
4. Identification of enzymatic Properties
The most suitable action temperature of the high temperature resistant xylanase is 60 ℃, the temperature is kept for 180min at 40-60 ℃, the residual enzyme activity is more than 85.5%, the temperature is kept for 60min at 70 ℃ and 80 ℃, the residual enzyme activity is 75.3% and 38.2%, respectively, and the enzyme can keep higher activity under the high temperature condition, thus the high temperature resistant xylanase is a novel high temperature resistant xylanase.
The optimum action pH of the enzyme is 7.0, the pH is 5.0-10.0, the enzyme is placed for 60min, and the residual enzyme activity is 53.3-71.6%.
The specific test procedure is as follows (example 1):
1. screening and identification of xylanase-producing strains
1) Enrichment culture
Adding 0.1g of ground phosphate rock into 10mL of sterile water, shaking and mixing uniformly, then sucking 1mL of bacterial suspension, inoculating into 5mL of enrichment medium, and performing shaking culture in a water bath at 30 ℃ for 48h;
2) Transparent ring method primary screen
Gradually diluting the enriched culture by 10 times, and respectively taking dilution factors of 10 -3 、10 -5 And 10 -7 0.5mL of each bacterial suspension is coated on a plate screening culture medium, cultured for 24 hours at 37 ℃, single bacterial colony with a relatively large transparent ring is selected for separation and purification, and the single bacterial colony is connected to a slant preservation culture medium for preserving an original bacterial strain;
3) Shaking, fermenting and re-screening:
inoculating the primary strain to 100mL basic enzyme production medium, culturing at 30deg.C and 180r/min for 2-3d, centrifuging fermentation broth to obtain supernatant, and respectively measuring enzyme activity of xylanase in fermentation broth by DNS;
2. enzyme activity assay for xylanases
Xylanase enzyme activity assay: 180. Mu.L of a substrate containing 1% xylan is added with 20. Mu.L of an appropriately diluted enzyme solution, the reaction is stopped by adding 200. Mu.L of 3, 5-dinitrosalicylic acid (dinitrosalicylic acid, DNS) reagent solution after accurate reaction for 10min at 50 ℃, the reaction is boiled in boiling water for 5min to develop color, the reaction solution is rapidly cooled to room temperature, 2.1mL of water is added, and then the OD value is measured at a wavelength of 540 nm. And calculating the sugar content of the reaction system according to a regression equation of the xylose standard curve.
The xylanase activity units in the assay are defined as: the amount of enzyme required to hydrolyze 1. Mu. Mol of reducing sugar (in xylose) per minute of xylanase at 50℃and pH7.0 was defined as 1 enzyme activity unit (1U).
IU＝D×R/10min×0.1mL
Wherein: d is the dilution factor of the enzyme solution; r is the xylose content calculated by a regression equation.
And performing SDS-PAGE analysis on the xylanase to obtain a single band, and determining that the xylanase has xylanase activity through zymogram analysis to reach electrophoretic purity.
3. Enzymatic Properties of high temperature resistant xylanases
1) Determination of the relative molecular weight of the high temperature resistant xylanase
SDS-PAGE is carried out on crude enzyme liquid obtained by fermentation and xylanase obtained by purification respectively, and relative molecular weight is about 37.8kDa.
2) Influence of temperature on enzyme Activity
The enzyme activities of the purified xylanase were measured at 20, 30, 40, 50, 60, 70, 80, 90, 100, pH7.0, repeated 3 times, averaged, and the relative enzyme activities at different temperatures were calculated.
The results are shown in FIG. 4, and the optimum operating temperature is 50 ℃.
3) Temperature stability assay of the xylanase: the purified xylanase enzyme solutions are respectively placed under the conditions of different temperatures (30 ℃, 40 ℃,50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃) and pH values of 7.0, the heat is preserved for 2 hours, the enzyme activities are sampled and measured every 30 minutes, the respective residual enzyme activities are calculated, and the enzyme activities of the initial enzyme solution are defined as 100 percent. As a result, as shown in FIG. 3, the temperature stability of the enzyme was good in the range below 70℃and the change in relative xylanase activity was small, whereas when the temperature exceeded 70℃the temperature stability of the enzyme was poor and the relative enzyme activity was rapidly decreased.
4. Influence of pH on enzyme Activity
1) Determination of the optimum action pH of the xylanase: the enzyme activities of the purified xylanases were measured at different pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0) at 50℃and the relative enzyme activities at different pH values were calculated with the highest enzyme activity being 100%.
As a result, the optimum pH was 7.0 as shown in FIG. 4.
2) Determination of the pH stability of the xylanase: the purified xylanase enzyme solution was adjusted to different pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0) and incubated at 50℃for 2 hours, and then the residual enzyme activities were measured, wherein the highest enzyme activity was defined as 100%. The results show that the enzyme has better stability within the pH range of 5.0-10.0.
Meanwhile, the xylanase produced by other embodiments of the invention obtains the same or similar results in experiments, is not listed one by one, prevents tiredness, has practical production significance in finding out the strain of the invention, and is suitable for large-scale popularization and application.
5. Conclusion(s)
From the above, it can be seen that the present invention provides a xylanase producing strain, named microbacterium HH-22 after strain identification, for producing xylanase, and the xylanase after purification is subjected to enzymatic property test measurement, the relative molecular weight is 37.8kDa, and the optimum action temperature and pH are 60 ℃ and 7.0, respectively; the enzyme activity is kept relatively stable within the temperature range of 30-70 ℃ and the pH range of 5.0-10.0, the quality is good, the enzyme can be effectively used in a plurality of industrial fields such as paper making, food and feed, the application range is wide, and the enzyme is innovative in xylanase production and has huge economic and social benefits.
It is noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and the present invention is not limited thereto, and that any person skilled in the art can make modifications and equivalents within the scope of the present invention without departing from the scope of the present invention.
Claims (3)
1. A xylanase-producing strain HH-22, characterized in that it is classified as MicrobacteriumMicrobacterium sp.) HH-22, deposited in China general microbiological culture Collection center, with accession number: CGMCC No.26621, the 16S rDNA gene sequence of the strain is shown as SEQ ID No. 1.
2. The method for fermenting a thermostable xylanase of xylanase-producing strain HH-22 according to claim 1, comprising the steps of:
1) Preparing a xylanase fermentation broth: inoculating xylanase producing strain HH-22 into a fermentation enzyme producing culture medium according to an inoculum size of 5% -10% by mass, and shake-flask culturing at 28-30 ℃ and 180rpm for 60-72 hours to obtain xylanase fermentation liquor;
2) Preparation of xylanase supernatant: centrifuging the xylanase fermentation broth obtained in the step 1) at a temperature of 4 ℃ and a speed of 12000rpm for 10-15 min to obtain xylanase supernatant;
3) Fermentation supernatant: slowly adding ammonium sulfate into the xylanase supernatant obtained in the step 1) to enable the mass content of the ammonium sulfate to reach 80%, and salting out at 4 ℃ overnight;
4) Preparing a high temperature resistant xylanase crude enzyme solution: collecting the fermentation supernatant which is salted out overnight in the step 3) at the temperature of 4 ℃, at the speed of 12000rpm for 10-15 min, centrifuging, collecting the precipitate, and dissolving the precipitate with Tris-HCl buffer solution with the pH value of 7.2 to obtain high-temperature-resistant xylanase crude enzyme solution for later use;
5) Octyl FF hydrophobic interaction chromatography: adjusting the mass concentration of ammonium sulfate in the high temperature resistant xylanase crude enzyme solution obtained in the step 4) to 40%, performing hydrophobic interaction chromatography, and performing column chromatography with the concentration of 2.6X18 cm, wherein the concentration is the balance solution: pH8.0, 0.02mol/L ammonium sulfate with 40% saturation ammonium sulfate, flow rate: 1mL/min, loading, linearly eluting with 1M ammonium sulfate, and collecting active components;
6) DAE FF gel filtration chromatography: column 1.6X50 cm, equilibration solution: 0.02mol/L Tris-HCl buffer pH7.2, flow rate: collecting active components at 0.5 mL/min;
7) Ff strong cation exchange chromatography: column type 2.6X10 cm, equilibrium liquid: 0.02mol/L Tris-HCl buffer pH7.2, flow rate: 1mL/min, performing linear elution by using a balance buffer solution containing 1.0M NaCl after sample loading, and collecting active components to obtain a high-temperature-resistant xylanase sample;
8) Preparing xylanase dry powder: and 7) putting the high temperature resistant xylanase sample obtained in the step 7) into a freeze dryer, and freeze-drying at-55 ℃ under 7Pa to obtain xylanase dry powder.
3. The method for fermenting thermostable xylanase with xylanase-producing strain HH-22 according to claim 2, wherein in said step 1), the fermentation enzyme-producing medium consists of: KH (KH) 2 PO 4 1～1.5g， (NH4) 2 SO 4 2～5g，MgSO 4 •7H 2 O0.4-0.5 g, yeast extract 5% to the upper5.5g, xylan 0.5 g-1.0 g, pH 7.0.
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