CN110564631B - Yeast for producing cellulase at low temperature and screening method thereof - Google Patents
Yeast for producing cellulase at low temperature and screening method thereof Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- 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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
Abstract
The invention relates to a yeast for producing cellulase at low temperature and a screening method thereof, wherein the yeast comprises yeast, the preservation organization is China general microbiological culture Collection center, the preservation number is CGMCC No.18002, and the preservation date is 2019, 6 months and 19 days. The invention separates and screens high-efficiency low-temperature cellulase yeast from soil in different low-temperature areas, and the yeast is characterized in that the yeast is a milky round bacterial colony on a potato glucose culture medium, the surface of the yeast is slightly dry, the bacterial colony is thick, the yeast colony is easy to pick up, has wine aroma and presents a typical yeast bacterial colony; and the influence of the conditions on temperature, carbon source, nitrogen source, pH and the like on the cellulase is studied. And the influence of straw burning on the environment in the northeast is reduced by applying the method to returning the straws to the field in the northeast low-temperature cold region, so that a solid foundation is laid for agricultural sustainable development.
Description
Technical Field
The invention relates to saccharomycetes for producing cellulase at low temperature and a screening method thereof, belonging to the field of microbiology.
Background
The low-temperature microorganisms refer to microorganism groups having growth and reproduction ability in a low-temperature environment, and they grow and reproduce in various low-temperature environments. According to the selection of the growth temperature of the microorganism, the low-temperature microorganism is divided into two types, namely a psychrophilic microorganism and a cold-resistant microorganism, wherein the psychrophilic microorganism can grow and propagate, the optimal temperature is about 15 ℃, and the survivable temperature is about 20 ℃; the cold-resistant microorganism has a survival temperature of about 0 ℃ and an optimal growth and propagation temperature of about 20 ℃. The growth and propagation temperature of the low-temperature microorganisms can be generally carried out at normal temperature, other energy sources are not needed, the low-temperature microorganisms are extremely easy to obtain and are widely distributed, and therefore people are beginning to focus on researching the low-temperature microorganisms and are also concerned by some experts and scholars. So far, the development and utilization of low temperature microorganisms are relatively few, and from the research progress at home and abroad, the research of high temperature microorganisms is still abundant, and the research of soil low temperature microorganism degrading bacteria is few.
So far, most of microbial strains capable of secreting cellulase reported at home and abroad are fungal flora, such as: trichoderma, aspergillus, etc., and yeast producing cellulase at low temperature are rarely reported.
And the varieties of enzyme-producing strains researched at the present stage are limited, and the defects of high production cost, insufficient enzyme-producing capability and enzyme-producing quantity, high efficiency, stability, narrow pH adaptation range and the like exist mostly, so that a new strain with high activity and wider action range needs to be continuously searched and developed, and the source idea of cellulase is further expanded. But scholars at home and abroad have high attention to the high-temperature cellulase and have little concern about bacteria producing the cellulase under the low-temperature condition. The low-temperature cellulase has more advantages and potentials in the aspects of energy conservation and cost reduction, and has great development prospects.
In a word, a yeast strain for producing cellulase at low temperature and a screening method thereof are not reported.
Disclosure of Invention
The invention aims to solve the problems and provides a screening method and application of yeast for producing cellulase at low temperature.
In order to achieve the above purpose of the present invention, the present invention adopts the following technical scheme:
the preservation organization of the microzyme H4 is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number of the microzyme H4 is CGMCC No.18002, and the preservation date is 2019, 6 months and 19 days.
The 26S rDNA sequence of the strain is published in GenBank, the accession number is DPYBCB8U014, and the 26S rDNA sequence is as follows:
Tatgtcacatcctaagcacgtacgtggacgaatcccggccataaaggcgtgctgcgttcctcagtcccaaccactgtatgtgacaggaggctataagtttacccgagggtactacgttcctccagcctttatccagcgatcaaaactgatgttgaccccactcaaaggaagtacaccggcagaaccggctgaatccaaagagcacgactgacttcaatcgtttccctttcaacaatttcacatactgtttaactctctttccaaagtgcttttcatctttccctcacggtacttgttcgctatcggtctctcgccaatatttagctttagatggaatttaccacccattttgagctgcattcccaaacaactcgactcgttgaaaatgtatcacagagcactggtagttcatgtcaaggacgggattctcaccctctatgacgccctgttccaagggacttgtacatgagccagcacggaaaacacttctatagattacaactcggatgacctgaggccaccagatttcaaatttgagctcttcccgcttcactcgccgttactaggggaatccttgttagtttcttttcctccgcttccgggtatatgca。
the yeast is characterized in that the yeast is milky round colony on a potato glucose culture medium, the surface of the colony is slightly dry, the colony is thick, the colony is easy to pick up, and the colony has wine fragrance and presents typical yeast colony.
The separation method of the strain comprises the following steps:
inoculating the collected suspension of the forest soil in the cold area of the northeast region into an inorganic salt culture medium containing 100mg/L straw powder according to the inoculation amount of 10%, shaking at 10 ℃ for 150r/min, culturing for 3 days, transferring the suspension into the inorganic salt culture medium with the straw concentration of 300mg/L according to the inoculation amount of 10% at 5 ℃, and continuing culturing; and continuously circulating for 3 times according to the mode, sequentially setting the straw concentration in the inorganic salt culture medium used for three times to be 300mg/L, 600mg/L and 1000mg/L, then carrying out plate coating, putting the culture dish in a constant-temperature culture box at 5 ℃ for culturing for 72h, picking out a single bacterial colony, and transferring the single bacterial colony into the inorganic salt culture medium with the straw concentration of 1000mg/L to obtain the straw degrading bacteria bacterial suspension.
The inorganic salt culture medium comprises (NH) 4 ) 2 SO 4 2g，KH 2 PO 4 2g，Na 2 HPO 4 1.3g, straw 100-1000m g, naCl 5g and the balance of water with constant volume of 1L; PH =4.8 E.C. of culture medium7.2。
Optimizing the fermentation conditions of the strains:
a. influence of carbon source on growth of yeast H4 and cellulase;
selecting one carbon source of glucose, maltose, sucrose, corn flour, glycerol, lactose, fructose, mannitol, citric acid and soluble starch as a carbon source in a culture medium, and preparing a liquid culture solution; inoculating 1mL of yeast H4 seed culture solution into the culture solution, inoculating 1000mg/L of inorganic salt liquid straw powder fermentation culture medium, carrying out shaking culture at 20 ℃ for 2d at 120rmp, centrifuging at 13000rmp for 15min, measuring the cellulase content in a supernatant, measuring the bacterial suspension concentration in the culture solution, and screening a carbon source;
b. the influence of different nitrogen sources on the growth of saccharomycetes H4 and cellulase;
selecting one of peptone, yeast extract, soybean meal, casein, urea, ammonium sulfate, potassium nitrate and beef extract as a nitrogen source in a culture medium, and preparing a nitrogen source liquid culture solution; the other methods are the same as the above, the cellulase content in the supernatant is measured, meanwhile, the bacterial suspension concentration in the culture solution is measured, and a nitrogen source is screened;
c. the influence of different inorganic salts on the growth of saccharomycete H4 and cellulase;
MgSO4, naCl, KH2PO4 and inorganic salts in the culture medium are used as liquid culture solutions of different inorganic salts. The other methods are the same as the above, the cellulase content in the supernatant is measured, meanwhile, the bacterial suspension concentration in the culture solution is measured, and inorganic salt is screened.
d. Effect of different initial pH on Yeast H4 growth and cellulase
Preparing a liquid culture medium with pH values of 2,3,4,5,6,7,8,9, 10 and 11. And (4) measuring the cellulase content in the supernatant, measuring the bacterial suspension concentration in the culture solution and screening the initial pH value by the same method as the above method.
e. Effect of different temperatures on H4 Strain growth and cellulase
Preparing a liquid culture medium; inoculating 1mL of yeast H4 seed culture solution into the liquid culture solution, placing in a shaking incubator at 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C and 40 deg.C respectively, measuring cellulase content in the supernatant, measuring the bacterial suspension concentration in the culture solution, and screening temperature.
A screening method of yeasts for producing cellulase at low temperature comprises the following screening steps:
a. primary screening: adding 5ml of 1000mg/L inorganic salt culture medium into a test tube with the diameter of 15mm multiplied by 150mm, sterilizing at 121 ℃ for 30min, cooling to room temperature, adding 1ml of straw degrading bacteria suspension into the test tube, repeating 2 tubes for each strain, culturing at low temperature of 5 ℃ in a dark place for 5 days, and observing and recording the growth condition for 1 time every 24 hours; the purified strain is dibbled on a cellulose Congo red culture medium, cultured for 4 days at 5 ℃, and initially selected according to the ratio D/D of the diameter D of the hydrolysis ring to the diameter D of the bacterial colony, wherein the larger the D/D value is, the better the D/D value is;
b. re-screening: adding 50ml of 1000mg/L inorganic salt culture medium into 250ml triangular bottles, sterilizing at 121 ℃ for 30min, cooling to room temperature, adding 1ml of straw degrading bacteria suspension obtained by primary screening into each triangular bottle, culturing at 5 ℃ in a dark place for 10 days, detecting the residual quantity of straws, simultaneously establishing a blank control group, replacing the bacteria suspension with sterile water in the blank control group, and calculating the degradation rate; and respectively inoculating 1mL of the bacterial suspension of the straw decomposing strain obtained by primary screening into 1000mg/L inorganic salt liquid fermentation culture medium, culturing for 3 days at 5 ℃, measuring the cellulase activity, and checking.
The invention has the advantages that:
1. according to the invention, the low-temperature forest soil in the northeast region is collected, the enrichment decomposition straw is cultured at low temperature, and the microbial strains capable of efficiently degrading the straw at low temperature are screened, so that the high-efficiency cellulase can be produced at low temperature (5 ℃) to decompose the straw, and the decomposition microbial inoculum can be used for returning the straw to the field in the northeast low-temperature cold region. Through the microbial inoculum low-temperature fermentation degradation corn stalks, the soil granular structure is improved after the corn stalks are returned to the field in different positions, beneficial microbial colony advantages are created, the formation of the soil granular structure is promoted, the micro-ecosystem of the soil is greatly improved, the soil air permeability is enhanced, and the water and fertilizer retention capacity of the soil is improved.
2. The invention makes the soil have the development potential of sustainable production increase. And nutrients in the straws are effectively converted, micromolecule nutrients and active factors are released, the soil fertility is improved, and the soil is fertilized.
3. The fertilizer product produced by the invention has the unique characteristic of decomposing straws at low temperature, is low in cost, and can effectively improve the physical and chemical properties of soil, improve the emergence rate of seeds and enhance the drought resistance, cold resistance and pest resistance of seedlings after being applied.
Drawings
FIG. 1 is a graph showing the variation of H4 cellulase under different carbon source conditions according to the present invention.
FIG. 2 is a graph showing the change of H4 cellulase under different nitrogen source conditions according to the present invention.
FIG. 3 is a graph of the change in H4 cellulase under different inorganic salt conditions of the present invention.
FIG. 4 is a graph showing the change of H4 cellulase under different pH conditions according to the present invention.
FIG. 5 is a graph showing the change of H4 cellulase under different temperature conditions according to the present invention.
FIG. 6 is a graph showing the change of enzymatic kinetics of H4 cellulase under the condition of liquid culture at 5 ℃ in the present invention.
FIG. 7 is a graph showing the effect of low-temperature cellulase-producing yeast of the present invention on the composting temperature of corn stover.
FIG. 8 is a diagram showing the effect of the yeast for producing cellulase at low temperature in the invention on lignin in corn straw compost.
FIG. 9 is a graph showing the effect of low temperature cellulase producing yeast of the present invention on cellulose in corn stover compost.
Detailed Description
The invention is described in further detail below with reference to the accompanying figures 1-9 and examples.
The preservation organization of the microzyme H4 is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number of the microzyme Saccharomyces sp is CGMCC No.18002, and the preservation date is 2019, 6 months and 19 days.
The 26S rDNA sequence of the strain is published in GenBank, the accession number is DPYBCB8U014, and the 26S rDNA sequence is as follows:
Tatgtcacatcctaagcacgtacgtggacgaatcccggccataaaggcgtgctgcgttcctcagtcccaaccactgtatgtgacaggaggctataagtttacccgagggtactacgttcctccagcctttatccagcgatcaaaactgatgttgaccccactcaaaggaagtacaccggcagaaccggctgaatccaaagagcacgactgacttcaatcgtttccctttcaacaatttcacatactgtttaactctctttccaaagtgcttttcatctttccctcacggtacttgttcgctatcggtctctcgccaatatttagctttagatggaatttaccacccattttgagctgcattcccaaacaactcgactcgttgaaaatgtatcacagagcactggtagttcatgtcaaggacgggattctcaccctctatgacgccctgttccaagggacttgtacatgagccagcacggaaaacacttctatagattacaactcggatgacctgaggccaccagatttcaaatttgagctcttcccgcttcactcgccgttactaggggaatccttgttagtttcttttcctccgcttccgggtatatgca。
the yeast is characterized in that the yeast is milky round colony on a potato glucose culture medium, the surface of the colony is slightly dry, the colony is thick, the colony is easy to pick up, and the colony has wine fragrance and presents typical yeast colony.
The separation method of the strain comprises the following steps:
inoculating the collected cold region forest soil suspension in the northeast region into an inorganic salt culture medium containing 100mg/L straw powder according to the inoculation amount of 10%, shaking the mixture for 150r/min at 10 ℃, culturing the mixture for 3 days, transferring the mixture into the inorganic salt culture medium with the straw concentration of 300mg/L according to the inoculation amount of 10% at 5 ℃, and continuing to culture the mixture; and continuously circulating for 3 times according to the mode, sequentially controlling the straw concentration in the inorganic salt culture medium used for three times to be 300mg/L, 600mg/L and 1000mg/L, then carrying out plate coating, putting the culture dish into a constant-temperature incubator at 5 ℃ for culturing for 72 hours, picking out single bacterial colonies, and transferring the single bacterial colonies into the inorganic salt culture medium with the straw concentration of 1000mg/L to obtain the straw degrading bacteria bacterial suspension.
The inorganic salt culture medium comprises (NH) 4 ) 2 SO 4 2g，KH 2 PO 4 2g，Na 2 HPO 4 1.3g, straw 100-1000m g, naCl 5g and the balance of water with constant volume of 1L; the PH of the medium = 4.8-7.2.
Optimizing the fermentation conditions of the strains:
a. influence of carbon source on growth of yeast H4 and cellulase;
selecting one carbon source of glucose, maltose, sucrose, corn flour, glycerol, lactose, fructose, mannitol, citric acid and soluble starch as a carbon source in a culture medium, and preparing a liquid culture solution; inoculating 1mL of yeast H4 seed culture solution into the culture solution, inoculating 1000mg/L of inorganic salt liquid straw powder fermentation culture medium, carrying out shaking culture at 20 ℃ for 2d at 120rmp, centrifuging at 13000rmp for 15min, measuring the cellulase content in a supernatant, measuring the bacterial suspension concentration in the culture solution, and screening a carbon source;
b. the influence of different nitrogen sources on the growth of saccharomycete H4 and cellulase;
selecting one of peptone, yeast extract, soybean meal, casein, urea, ammonium sulfate, potassium nitrate and beef extract as a nitrogen source in a culture medium, and preparing a nitrogen source liquid culture solution; the other methods are the same as the above, the cellulase content in the supernatant is measured, meanwhile, the bacterial suspension concentration in the culture solution is measured, and a nitrogen source is screened;
c. the influence of different inorganic salts on the growth of saccharomycete H4 and cellulase;
MgSO4, naCl, KH2PO4 and inorganic salts in the culture medium are used as liquid culture solutions of different inorganic salts. The other methods are the same as the above, the cellulase content in the supernatant is measured, meanwhile, the bacterial suspension concentration in the culture solution is measured, and inorganic salt is screened.
d. Effect of different initial pH on Yeast H4 growth and cellulase
Preparing liquid culture medium with pH value of 2,3,4,5,6,7,8,9, 10, 11. The other methods are the same as the above, the cellulase content in the supernatant is measured by measuring the supernatant, the bacterial suspension concentration in the culture solution is measured at the same time, and the initial pH value is screened.
e. Effect of different temperatures on H4 Strain growth and cellulase
Preparing a liquid culture medium; inoculating 1mL of yeast H4 seed culture solution into the liquid culture solution, placing in a shaking incubator at 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C and 40 deg.C respectively, measuring cellulase content in the supernatant, measuring the bacterial suspension concentration in the culture solution, and screening temperature.
A screening method of yeasts for producing cellulase at low temperature comprises the following screening steps:
a. primary screening: adding 5ml of 1000mg/L inorganic salt culture medium into a test tube with the diameter of 15mm multiplied by 150mm, sterilizing at 121 ℃ for 30min, cooling to room temperature, adding 1ml of straw degrading bacteria suspension into the test tube, repeating 2 tubes for each strain, culturing at low temperature of 5 ℃ in a dark place for 5 days, and observing and recording the growth condition for 1 time every 24 hours; the purified strain is dibbled on a cellulose Congo red culture medium, cultured for 4 days at 5 ℃, and initially selected according to the ratio D/D of the diameter D of the hydrolysis ring to the diameter D of the bacterial colony, wherein the larger the D/D value is, the better the D/D value is;
b. re-screening: adding 50ml of 1000mg/L inorganic salt culture medium into 250ml triangular flasks, sterilizing at 121 ℃ for 30min, cooling to room temperature, adding 1ml of straw degrading bacteria suspension obtained by primary screening into each triangular flask, culturing at 5 ℃ in a dark place for 10 days, detecting the residual quantity of straws, simultaneously establishing a blank control group, replacing the bacteria suspension with sterile water in the blank control group, and calculating the degradation rate; and respectively inoculating 1mL of bacterial suspension of the straw decomposing strain obtained by primary screening into 1000mg/L inorganic salt liquid fermentation culture medium, culturing for 3 days at 5 ℃, measuring the cellulase activity of the straw decomposing strain, and checking.
According to the yeast strain disclosed by the invention, the cellulase peak value is reached in 72 hours on the aspect of enzymology kinetics, the most suitable carbon source is glucose, the most suitable nitrogen source is peptone, the most suitable inorganic salt is potassium dihydrogen phosphate, the most suitable pH is 4, and the most suitable fermentation temperature is 25 ℃.
Example of the embodiment
CK: the water content of 1 ton of corn straw is regulated to 50%.
H4: on the basis of CK, 4kg of microbial inoculum H is added.
Composting time: 2019.3.13-2019.4.25.
A composting site: shenyang.
The specific process comprises the steps of composting 1000kg per pile, neatly stacking the corn straws parallel to the ground, respectively scattering a little inoculation microbial inoculum on the surface when each liter is 20cm high, simultaneously treading tightly, keeping the pile height at 1.5, covering the outer surface with an agricultural plastic film, and stacking for 45 days.
Indexes are as follows: and (3) measuring the straw compost temperature and the lignin and cellulose content of the straw in different periods.
The results show that: under the condition that the average temperature in spring is 6 ℃, the low-temperature cellulase-producing yeast is added, so that the average temperature of the straw compost reaches 46.7 ℃, the straw compost is increased by 48.6% compared with a control, the lignin is reduced by 24.2% compared with the control, and the cellulose content is reduced by 32.9% compared with the control. The low-temperature production of the cellulose enzyme saccharomycetes can improve the composting temperature and accelerate the decomposition of the lignin and cellulose content in the straw.
Sequence listing
<110> Liaoning province academy of agricultural sciences
<120> saccharomycete for producing cellulase at low temperature and screening method thereof
<141> 2019-08-09
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 611
<212> DNA
<213> Saccharomyces
<400> 2
tatgtcacat cctaagcacg tacgtggacg aatcccggcc ataaaggcgt gctgcgttcc 60
tcagtcccaa ccactgtatg tgacaggagg ctataagttt acccgagggt actacgttcc 120
tccagccttt atccagcgat caaaactgat gttgacccca ctcaaaggaa gtacaccggc 180
agaaccggct gaatccaaag agcacgactg acttcaatcg tttccctttc aacaatttca 240
catactgttt aactctcttt ccaaagtgct tttcatcttt ccctcacggt acttgttcgc 300
tatcggtctc tcgccaatat ttagctttag atggaattta ccacccattt tgagctgcat 360
tcccaaacaa ctcgactcgt tgaaaatgta tcacagagca ctggtagttc atgtcaagga 420
cgggattctc accctctatg acgccctgtt ccaagggact tgtacatgag ccagcacgga 480
aaacacttct atagattaca actcggatga cctgaggcca ccagatttca aatttgagct 540
cttcccgctt cactcgccgt tactagggga atccttgtta gtttcttttc ctccgcttcc 600
gggtatatgc a 611
Claims (1)
1. A yeast for producing cellulase at low temperature, which is characterized in that,
the yeast is classified under the nameSaccharomyces sp.The preservation organization of the microzyme is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.18002, and the preservation date is 2019, 6 months and 19 days.
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| CN113088459B (en) * | 2021-04-25 | 2023-03-17 | 天津科技大学 | Heat-resistant high-yield candida tropicalis as well as preparation method and application thereof |
Citations (4)
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