CN111925944A - Penicillium brevicompactum and application thereof - Google Patents
Penicillium brevicompactum and application thereof Download PDFInfo
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- 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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/80—Penicillium
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- 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
- C12N1/04—Preserving or maintaining viable microorganisms
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- 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)
- C12N9/24—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)
Abstract
The invention discloses a mould and application thereof, and relates to the technical field of microorganisms. The strain of the present invention is named as W-B23, and the mold strain is Penicillium brevicompactum having cellulose degrading ability. The morphological characteristics are as follows: the bacterial colony is cultured on a PDA culture medium, the shape of the bacterial colony is nearly circular, dark green, the periphery is white, the surface is villous, a large number of spore-forming structures are formed, the back of the bacterial colony is dark black, and no water-soluble pigment exists. The invention can efficiently degrade cellulose.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to penicillium brevicompactum and application thereof in cellulose degradation.
Background
Cellulose is the most abundant and important renewable resource in the world, and the cellulose is degraded into monosaccharide by using cellulase produced by microorganisms and then converted into energy, food, chemical raw materials and the like, so that the problems of environmental pollution, resource shortage and the like can be solved for human beings.
Cellulases are produced by a wide variety of organisms in nature, including bacteria, molds, actinomycetes, and the like, as well as plant and animal sources.
Compared with other microorganisms producing cellulase, the mold has the following advantages: firstly, the produced cellulase is extracellular enzyme, and is convenient for separation and extraction; secondly, the enzyme production efficiency is high, and the structure of the produced cellulase system is more reasonable. From the perspective of industrial preparation and application of cellulase, it is of great significance to research and use mold to produce enzyme.
Disclosure of Invention
The present invention aims to provide a novel mold belonging to the genus Penicillium brevicaulis.
The penicillium brevicompactum has the morphological characteristics that: the bacterial colony is cultured on a PDA culture medium, the shape of the bacterial colony is nearly circular, dark green, the periphery is white, the surface is villous, a large number of spore-forming structures are formed, the back of the bacterial colony is dark black, and no water-soluble pigment exists.
The strain is preserved in China general microbiological culture Collection center (address: China academy of sciences, No. 3, West Lu No. 1, Beijing, Chaoyang, North-China), at 6.4.2020, with the preservation number: CGMCC NO: 19908, name: penicillium brevicompactum W-B23.
The invention relates to a method for separating Penicillium brevicompactum W-B23, which comprises the following steps:
separation of Penicillium brevicompactum:
weighing 5g of the mildew fruit scraps, placing the mildew fruit scraps in a triangular flask containing 90mL of sterile physiological saline and glass beads, and shaking at 28 ℃ for 60min at 180 r/min. Taking the supernatant as 10-1Sequentially diluting to 10-2，10-3，10-4， 10-5. Suction 100mu.L of diluted bacterial solution was spread on a primary screen medium plate. And placing the coated plate in a constant temperature incubator at 28 ℃ for culturing for 4-5 d. And picking the separated single colony by using an inoculating loop, coating and purifying the single colony on a PDA culture medium plate, and culturing at 28 ℃ until the single colony grows out. Picking single colony to dibble on the slant of PDA culture medium, culturing at 28 deg.C, and storing at 4 deg.C. After the seed-protecting strain plate is coated and activated, selecting single colonies, dibbling the single colonies on an enzyme-producing screening culture medium plate, and culturing the single colonies for 3-5 days at a separation temperature. After the culture is finished, 1-2mL of 1mg/mL Congo red dye solution is dripped on the flat plate to dye for 30min (dripping on bacterial colonies is avoided as much as possible), after 1mol/L NaCl solution is used for decoloring for 30min, the diameter (D) of the bacterial colonies and the diameter (D) of a hydrolysis ring are measured, and the ratio of the D/D is calculated so as to judge the decomposition capacity of the separated bacterial strains on cellulose. Selecting the strain with higher D/D ratio, and numbering.
(II) culture medium:
primary screening of culture medium: potato (peeled) 200g, glucose 20g, agar 20g, deionized water 1000mL, natural pH.
Production of cellulase screening culture medium: CMC-Na (sodium carboxymethylcellulose) 6g, peptone 0.75 g, Na2HPO40.75g，KH2PO40.45g agar 6g deionized water 300mL, natural pH.
Producing cellulase fermentation liquor: liquid enzyme-producing culture medium: 10g of CMC-Na, 3.0g of peptone, 0.2g of yeast extract and KH2PO44.0 g，MgSO4·7H20.3g of O and 1000mL of deionized water.
And (III) preserving and passaging strains:
1. and (3) strain preservation culture medium:
PDA culture medium: potato (peeled) 200g, glucose 20g, agar 20g, deionized water 1000mL, natural pH.
2. The storage method comprises the following steps:
(1) and (3) bevel low-temperature preservation: inoculating the separated and purified strain to a slant PDA culture medium, culturing at 28 deg.C in a constant temperature incubator for 7d, allowing the colony to grow over the slant of the test tube, observing to confirm no contamination, and storing in a 4 deg.C refrigerator. The preservation method is suitable for preservation of various strains under experimental conditions, the preservation time is generally three months to six months, and strain mutation and degeneration are easily caused when the subculture frequency is excessive.
(2) Preservation method of glycerol: and (3) selecting spores of the purified hyphae by an inoculation needle, respectively inoculating the spores into 250mL triangular flasks filled with 100mL of newly prepared PDB, placing the flasks on a constant temperature shaker with the rotating speed of 180rpm, culturing at 28 ℃ for 7d, transferring 500 mu L of bacterial liquid into a 1.5mL centrifuge tube, adding 300 mu L of 50% glycerol, attaching a strain number label to the tube wall of the 1.5mL centrifuge tube, and then storing in a refrigerator at-20 ℃. The method can preserve the isolated strain for 1-2 years longer than the tube slant preservation method.
Ecological characteristics of the strain:
the colony formed by the bacillus subtilis is cultured on a PDA culture medium at 28 ℃ for 7 days, the shape of the colony is nearly circular and dark green, the periphery is white, the surface is villous, a large number of spore-forming structures are formed, and the back of the colony is dark black and has no water-soluble pigment.
Culture characteristics of the strain:
(1) the culture temperature is 20-30 ℃, and the optimal temperature is 28-30 ℃;
(2) the culture pH is 6-7.5, and the optimal range is 6.5-7.2.
According to the colony morphological characteristics, the thallus morphological characteristics and the related molecular biology identification experiments of the strains, the mould strains are identified as Penicillium brevicornum according to the fungal identification handbook.
The invention relates to application of penicillium brevicompactum in degrading cellulose.
Compared with the prior art, the invention discovers the bacterial strain with stronger degradation capability on cellulose. The enzyme activity of the cellulase produced by the strain through liquid fermentation is higher than that of the existing industrial enzyme-producing strain.
Drawings
FIG. 1 is a filter paper enzyme activity curve of Penicillium brevicompactum W-B23 and Trichoderma koningii 40108;
FIG. 2 shows the activity curves of Penicillium brevicompactum W-B23 and Trichoderma koningii 40108 with carboxymethyl cellulose (CMC).
Detailed Description
The invention relates to a method for separating Penicillium brevicompactum W-B23, which comprises the following steps:
separation of Penicillium brevicompactum:
weighing 5g of the mildew fruit scraps, placing the mildew fruit scraps in a triangular flask containing 90mL of sterile physiological saline and glass beads, and shaking at 28 ℃ for 60min at 180 r/min. Taking the supernatant as 10-1Sequentially diluting to 10-2，10-3，10-4， 10-5. Draw 100 mul of diluted bacterial liquid and coat the primary screen culture medium plate. And placing the coated plate in a constant temperature incubator at 28 ℃ for culturing for 4-5 d. And (3) picking the separated single colony by using an inoculating loop, coating the single colony on a PDA culture medium plate for purification, and culturing at 28 ℃ until the single colony grows out. Picking single colony to dibble on the slant of PDA culture medium, culturing at 28 deg.C, and storing at 4 deg.C. After the seed-protecting strain plate is coated and activated, selecting a single colony to dibble on a enzyme-producing screening culture medium plate, and culturing for 3-5 days at a separation temperature. After the culture is finished, 1-2mL of 1mg/mL Congo red dye solution is dripped on the flat plate to dye for 30min (dripping on bacterial colonies is avoided as much as possible), after 1mol/L NaCl solution is used for decoloring for 30min, the diameter (D) of the bacterial colonies and the diameter (D) of a hydrolysis ring are measured, and the ratio of the D/D is calculated so as to judge the decomposition capacity of the separated bacterial strains on cellulose. Selecting the strain with higher D/D ratio, and numbering.
(II) culture medium:
primary screening of culture medium: potato (peeled) 200g, glucose 20g, agar 20g, deionized water 1000mL, natural pH.
Production of cellulase screening culture medium: CMC-Na (sodium carboxymethylcellulose) 6g, peptone 0.75 g, Na2HPO40.75g，KH2PO40.45g agar 6g deionized water 300mL, natural pH.
Producing cellulase fermentation liquor: liquid enzyme-producing culture medium: 10g of CMC-Na, 3.0g of peptone, 0.2g of yeast extract and KH2PO44.0 g，MgSO4·7H20.3g of O and 1000mL of deionized water.
And (III) preserving and passaging strains:
1. and (3) strain preservation culture medium:
PDA culture medium: potato (peeled) 200g, glucose 20g, agar 20g, deionized water 1000mL, natural pH.
2. The storage method comprises the following steps:
(1) and (3) bevel low-temperature preservation: inoculating the separated and purified strain to a slant PDA culture medium, culturing at 28 deg.C in a constant temperature incubator for 7d, allowing the colony to grow over the slant of the test tube, observing to confirm no contamination, and storing in a 4 deg.C refrigerator. The preservation method is suitable for preservation of various strains under experimental conditions, the preservation time is generally three months to six months, and strain mutation and degeneration are easily caused when the subculture frequency is excessive.
(2) Preservation method of glycerol: and (3) selecting spores of the purified hyphae by an inoculation needle, respectively inoculating the spores into 250mL triangular flasks filled with 100mL of newly prepared PDB, placing the flasks on a constant temperature shaker with the rotating speed of 180rpm, culturing at 28 ℃ for 7d, transferring 500 mu L of bacterial liquid into a 1.5mL centrifuge tube, adding 300 mu L of 50% glycerol, attaching a strain number label to the tube wall of the 1.5mL centrifuge tube, and then storing in a refrigerator at-20 ℃. The method can preserve the isolated strain for 1-2 years longer than the tube slant preservation method.
Ecological characteristics of the strain:
the colony formed by the bacillus subtilis is cultured on a PDA culture medium at 28 ℃ for 7 days, the shape of the colony is nearly circular and dark green, the periphery is white, the surface is villous, a large number of spore-forming structures are formed, and the back of the colony is dark black and has no water-soluble pigment.
Culture characteristics of the strain:
(1) the culture temperature is 20-30 ℃, and the optimal temperature is 28-30 ℃;
(2) the culture pH is 6-7.5, and the optimal range is 6.5-7.2.
According to the colony morphological characteristics, the thallus morphological characteristics and the related molecular biology identification experiments of the strain, the strain is identified as the penicillium brevicornum according to the fungal identification manual.
Test example 1: determination of cellulose decomposing ability
The method adopts a DNS method to respectively use sodium carboxymethylcellulose (CMC-Na) and filter paper as substrates to detect the cellulase enzyme activities of the crude enzyme solution of the strain Penicillium brevicaulis W-B23 and the industrial strain Trichoderma koningii 40108.
The method comprises the following specific steps:
preparation of a DNS reagent: 6.3g of 3, 5-dinitrosalicylic acid, 21g of NaOH, 182.0g of sodium potassium tartrate, 5.0g of phenol and 5.0g of sodium sulfite are respectively dissolved by deionized water and then mixed, and the mixed solution is cooled to room temperature and the volume is up to 1000 mL. Stored in a brown bottle , and can be used after being placed at room temperature for 7 days.
(182 g of sodium potassium tartrate is dissolved in 500ml of distilled water, the solution is heated, 6.3g of 3, 5-dinitrosalicylic acid, 21g of NaOH and 5g of phenol are sequentially added into the hot solution, the solution is stirred until the solution is dissolved, the solution is cooled and then the volume is fixed to 1000ml by using distilled water, and the solution is stored in a brown bottle and is stored for 7 days at room temperature for use.
However, it should be noted that the 3, 5-dinitrosalicylic acid and NaOH must be added in a relatively close time, or the NaOH must be added first. Otherwise, insoluble precipitates can be formed, leading to failure of the solution formulation. In addition, the heating temperature of the solution is not suitable to exceed 50 ℃ in the preparation process. )
Glucose standard curve: taking 10 25mL colorimetric tubes, respectively adding 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8mL of 1mg/mL glucose standard solution, supplementing deionized water until the volume is 2.0mL, adding 2.0mL deionized water into a control tube, adding 3.0mL of DNS reagent into each tube along the wall, boiling for 5min, immediately cooling by running water, fixing the volume of the deionized water to 25mL, covering and shaking uniformly, measuring the absorbance A540 of each sample at the wavelength of 540nm, and using the glucose content in each tube and the A540 value regression curve. After a glucose standard curve is made, the glucose concentration can be calculated according to the A540 value of the sample to be measured.
And (3) enzyme activity determination of carboxymethyl cellulose (CMC): taking 0.5mL of crude enzyme solution, adding 1mL of CMC-Na citric acid buffer solution, uniformly mixing, carrying out water bath at 50 ℃ for 30min, then adding 3mL of DNS color developing solution, carrying out boiling water bath for 10min, cooling, and measuring the absorbance at 540 nm.
Meanwhile, the inactivated enzyme solution after boiling for 10min at 100 ℃ is used as a reference, and other steps are the same.
And (3) filter paper enzyme activity determination: taking 0.5mL of crude enzyme solution, adding 1mL of citric acid-sodium citrate buffer solution with the pH value of 4.80.1mol/mL, adding a piece of filter paper (1cm x 6cm) into a test tube with a plug, carrying out water bath at 50 ℃ for 1h, then adding 3mL of DNS color development solution, carrying out water bath for 10min for development, cooling to room temperature, adding distilled water to fix the volume to 10mL, uniformly mixing, standing for 20min, and measuring the absorbance at 540 nm.
Meanwhile, the inactivated enzyme solution after boiling for 10min at 100 ℃ is used as a reference, and other steps are the same.
And obtaining the corresponding reducing sugar concentration according to the obtained absorbance value and a glucose standard curve, thereby calculating the enzyme activity of the cellulase.
The enzyme activity calculation formula is as follows:
u is the enzyme activity of cellulase;
n: dilution factor
180.16: molecular weight of glucose
t: reaction time, min
v: volume of enzyme solution, mL
The result shows that the filter paper enzyme activity of the separated penicillium brevicompactum W-B23 reaches the highest value at 8 days of fermentation, and is 3.15U/ml; the CMC enzyme activity reaches the highest at 8 th day and is 2.23U/ml. And the highest filter paper enzyme activity and the highest CMC enzyme activity of the industrial cellulase-producing strain 40108 (trichoderma koningii) are respectively 2.16U/ml and 0.85U/ml.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the present invention without departing from the technical spirit of the present invention.
Claims (5)
1. A Penicillium brevicompactum characterized by: the mould strain has a preservation number of CCC NO: 19908, name: penicillium brevicompactum W-B23.
2. The Penicillium brevicompactum strain according to claim 1, wherein: the mold colony is nearly circular and dark green, the periphery is white, the surface is villous, a large number of spore-forming structures are formed, the back of the mold colony is dark black, and no water-soluble pigment exists.
3. The Penicillium brevicompactum strain according to claim 1, wherein: the culture characteristics of the mould strains are as follows:
(1) the culture temperature is 20-30 ℃, and the optimal temperature is 28-30 ℃;
(2) the culture pH is 6-7.5, and the optimal range is 6.5-7.2.
4. The Penicillium brevicompactum strain according to claim 1, wherein: the method for preserving the mould strains comprises the following steps:
(1) and (3) bevel low-temperature preservation: inoculating the separated and purified strains to a slant PDA culture medium, culturing for 7d at 28 ℃ in a constant-temperature incubator until the slant of the test tube is full of bacterial colonies, observing and confirming that no pollution exists, placing the strains in a refrigerator at 4 ℃ for storage, wherein the storage time is three months to six months, and the strains are easy to mutate and degenerate when the subculture times are excessive;
or (2) glycerol preservation: inoculating the spores of the purified hyphae into an inoculating needle, respectively inoculating into bottles filled with newly prepared PDB, placing on a constant temperature shaking table with the rotating speed set to 180rpm, culturing at 28 ℃ for 7d, taking the bacterial liquid, transferring into a centrifuge tube, adding 50% glycerol with the volume ratio of 3:5, and then placing into a refrigerator at-20 ℃ for preservation for 1-2 years.
5. Use of penicillium brevicompactum according to claim 1 for the degradation of cellulose.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU734261A1 (en) * | 1977-04-11 | 1980-05-15 | Институт Биохимии И Физиологии Микроорганизмов Ан Ссср | Method of ribonuclease isolation |
| CN1796538A (en) * | 2004-12-28 | 2006-07-05 | 杭州华东医药集团生物工程研究所有限公司 | Short dense Penicillium and application |
| US20120100525A1 (en) * | 2008-08-26 | 2012-04-26 | Intelligentnano Inc. | Ultrasound enhanced growth of microorganisms |
| CN104334738A (en) * | 2012-03-30 | 2015-02-04 | 诺维信北美公司 | Processes of producing fermentation products |
| CN110637082A (en) * | 2017-01-04 | 2019-12-31 | 诺维信生物农业公司 | Bacillus isolate and uses thereof |
-
2020
- 2020-07-07 CN CN202010645582.4A patent/CN111925944A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU734261A1 (en) * | 1977-04-11 | 1980-05-15 | Институт Биохимии И Физиологии Микроорганизмов Ан Ссср | Method of ribonuclease isolation |
| CN1796538A (en) * | 2004-12-28 | 2006-07-05 | 杭州华东医药集团生物工程研究所有限公司 | Short dense Penicillium and application |
| US20120100525A1 (en) * | 2008-08-26 | 2012-04-26 | Intelligentnano Inc. | Ultrasound enhanced growth of microorganisms |
| CN104334738A (en) * | 2012-03-30 | 2015-02-04 | 诺维信北美公司 | Processes of producing fermentation products |
| CN110637082A (en) * | 2017-01-04 | 2019-12-31 | 诺维信生物农业公司 | Bacillus isolate and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| MOHAMED A.ABDEL-SATER ET AL.: "Immobilization of Cellulases Produced by Penicillium brevicompactum AUMC 10987, using Cross-Linkage, Chitosan-Coating and Encapsulation", 《CATRINA》 * |
| 刘清锋等: "纤维素降解菌青霉T24-2的分离及产酶特性", 《工业微生物》 * |
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