CN108048350B - Psychrophilic cellulase producing strain - Google Patents

Psychrophilic cellulase producing strain Download PDF

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CN108048350B
CN108048350B CN201711368415.4A CN201711368415A CN108048350B CN 108048350 B CN108048350 B CN 108048350B CN 201711368415 A CN201711368415 A CN 201711368415A CN 108048350 B CN108048350 B CN 108048350B
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cellulase
psychrophilic
producing strain
psychrotrophic
strain
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CN108048350A (en
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林凌
秦娜
杨青
程啸天
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Jiangsu Morig Technology Co ltd
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Anhui Normal University
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12N9/2405—Glucanases
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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)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

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Abstract

The invention discloses a psychrotrophic cellulase producing strain, which has a preservation number of 14938 in China general microbiological culture collection center (CGMCC) and a preservation date of 2017, 11 months and 20 days. The psychrophilic cellulase producing strain has the capability of producing cellulase, the method for preparing the psychrophilic cellulase by using the psychrophilic cellulase producing strain is simple and easy to implement, and the cellulase has the characteristics of psychrophilic property, acid and alkali resistance and wide application prospect.

Description

Psychrophilic cellulase producing strain
Technical Field
The invention relates to a cellulase producing strain, in particular to a psychrotrophic cellulase producing strain, a psychrotrophic cellulase and a preparation method thereof.
Background
Cellulases include three types, endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and β -glucosides (EC 3.2.1.21), which can completely hydrolyze cellulose to glucose through a synergistic effect. Wherein the endoglucanase mainly acts on the amorphous region of the cellulose to randomly cut beta-1, 4-glycosidic bonds and hydrolyze the cellulose. Exoglucanases hydrolyze beta-1, 4-glucosidic bonds in amorphous cellulose to form soluble cellodextrins and cellobiose. The beta-glucosidase hydrolyzes the non-reducing terminal hydrolysis glucose residue, breaking down cellobiose and cellodextrin into glucose.
At present, most of strains for researching cellulase production are usually taken from conventional environments and are places with abundant nutrition and complex ecological structures, and the obtained strains for producing cellulase are not high in tolerance and easy to degrade. Some special extreme ecological environment areas can inoculate strains with special physiological activity, and if strains with special cellulase can be screened in the special extreme ecological environment areas, the special ecological environment can greatly reduce the cost and save resources, and is more suitable for industrial production.
The Qinghai-Tibet plateau area of China belongs to the plateau temperate zone, has high altitude, cold and dry climate, annual average precipitation of 140mm, annual average temperature of 1 ℃ and large day and night temperature difference. The climatic environment of the area is extreme, but various habitats such as mountains, glaciers, lakes and marshes distributed in the area still breed rich and peculiar biological resources, and the area gradually becomes the victory place for scientific investigation and ecological research in recent years. The unique geographical environment and climate conditions of the Qinghai-Tibet plateau bring about that the community structure and physiological characteristics of the microorganism in the region are greatly different from those in other regions, so that the screening of the cellulase-producing strains in the region has important ecological, economic and social benefits, and the obtained novel strains have potential application prospects in the fields of food, feed, pharmacy and the like.
Disclosure of Invention
The invention aims to provide a psychrotrophic cellulase producing strain, a psychrotrophic cellulase produced by the psychrotrophic cellulase producing strain and a preparation method thereof.
In order to achieve the aim, the invention provides a psychrotrophic cellulase producing strain, wherein the preservation number of the psychrotrophic cellulase producing strain in China general microbiological culture Collection center (CGMCC) is 14938, and the preservation date is 2017, 11 and 20 days.
The invention also provides a preparation method of the psychrophilic cellulase, which comprises the following steps: (1) inoculating the psychrophilic cellulase-producing strain according to claim 1 in liquid LB (Luria-Bertani) for culture; (2) transferring the cultured bacterial liquid to a fermentation medium for fermentation; (3) and centrifuging the fermented bacteria liquid, and taking supernatant.
The invention also provides the psychrophilic cellulase prepared by the preparation method.
Through the technical scheme, the psychrophilic cellulase producing strain provided by the invention is classified and named as salt-tolerant Liuzhi Hexuelans (Zhihengliella halotolerans) La12, the 16S rDNA gene sequence of the strain is shown in SEQ ID NO1, the preservation unit is China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of West Luo No.1 of the sunward area of Beijing, institute of microbiology of China academy of sciences), the preservation date is 11 months and 20 days in 2017, and the preservation number is: 14938. the psychrotrophic cellulase producing strain has the capability of producing cellulase, and the cellulase has the characteristics of psychrotrophy, acid and alkali resistance and wide application prospect.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a photograph of a hydrolyzed cellulose of a psychrophilic cellulase producing strain provided by the present invention;
FIG. 2 is a graph showing the effect of temperature on the activity of cellulase according to the present invention in test example 2;
FIG. 3 is a graph showing the effect of pH on cellulase activity provided by the present invention in test example 3;
FIG. 4 is a graph showing the effect of metal ions on the cellulase activity provided by the present invention in test example 4.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a psychrotrophic cellulase producing strain, which has a preservation number of 14938 in China general microbiological culture collection center (CGMCC) and a preservation date of 2017, 11 months and 20 days.
The psychrophilic cellulase producing strain provided by the invention is classified and named as salt-tolerant Liuzhi Hexuelan (Zhihengliella halotolerans) La12, the 16S rDNA gene sequence of the strain is shown as SEQ ID NO1, the preservation unit is China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of Xilu No.1 of Xingyang district in Beijing, institute of microbiology of China academy of sciences), the preservation date is 11 months and 20 days in 2017, and the preservation number is: 14938.
the psychrophilic cellulase producing strain is obtained by screening through the following method: (1) selecting soil 3-20 cm below the vegetation surface at the elevation of 3000-5000 meters of Qinghai-Tibet plateau, dissolving and diluting by water; (2) coating the diluted soil solution on a cellulose enrichment medium for culturing to grow a bacterial colony; (3) selecting a bacterial colony to culture on a cellulose screening culture medium so as to grow a bacterial colony; (4) dyeing, decoloring and checking a transparent ring on the cellulose screening culture medium with the grown bacterial colony; (5) and (4) screening out strains on the cellulose screening culture medium with the hydrolysis ring. As will be described in detail later.
The invention also provides a preparation method of the psychrophilic cellulase, which comprises the following steps: (1) inoculating the psychrophilic cellulase-producing strain according to claim 1 in liquid LB (Luria-Bertani) for culture; (2) transferring the cultured bacterial liquid to a fermentation medium for fermentation; (3) and centrifuging the fermented bacteria liquid, and taking supernatant.
The psychrophilic cellulase producing strain has the capability of producing cellulase, and the cellulase has the characteristics of psychrophilic property and acid and alkali resistance.
In a preferred embodiment of the present invention, in order to increase the yield of the psychrophilic cellulase-producing strain, the liquid LB is preferably formulated as: relative to 1000mL of water, 4-6g of yeast powder, 8-12g of sodium chloride and 8-11g of peptone have pH of 7.2-7.6.
In a preferred embodiment of the present invention, in order to increase the yield of the psychrotrophic cellulase-producing bacteria, it is preferred that the culture conditions in step (1) are: culturing at 24-30 ℃ and at 100-300rpm for 18-36 hours.
In a preferred embodiment of the present invention, in order to increase the yield of the psychrotrophic cellulase-producing strain and increase the fermentation efficiency, the volume ratio of the strain liquid to the fermentation medium in step (2) is preferably 2-3: 100.
In a preferred embodiment of the present invention, in order to increase the yield of the psychrotrophic cellulase-producing bacteria and increase the fermentation efficiency, the fermentation medium preferably has the following formula: relative to 1000mL of water, 4-6g of yeast powder, 8-12g of sodium chloride, 8-11g of peptone and 0.4-0.6g of sodium carboxymethylcellulose, and the pH value is 7.2-7.6.
In a preferred embodiment of the present invention, in order to increase the yield of the psychrotrophic cellulase-producing bacteria and increase the fermentation efficiency, the fermentation conditions in step (2) are preferably: culturing at 24-30 ℃ and at 100-300rpm for 40-50 hours.
In a preferred embodiment of the present invention, in order to increase the extraction rate of the psychrophilic cellulase, it is preferred that the conditions of centrifugation in step (3) comprise: centrifuging at 3000-.
The invention also provides the psychrophilic cellulase prepared by the preparation method.
The psychrophilic cellulase has the characteristics of psychrophilic property, acid and alkali resistance and wide application prospect.
The present invention will be described in detail below by way of examples.
1. Screening of psychrophilic cellulase-producing bacteria
And selecting vegetation surrounding sample points at the altitude of 4000 meters of the Qinghai-Tibet plateau, and collecting soil 5-10 centimeters below the ground surface. Weighing 1g of soil sample, adding the soil sample into a triangular flask containing 99mL of sterile water and glass beads, and fully dissolving and uniformly mixing the soil sample to obtain 10-2Diluting the solution with 10 times of sterile water to 10 times-3、10-4、10-5、10-6The diluent (2). Get 10-4、10-5、10-60.1mL of each of the three dilutions was spread on a cellulose enrichment medium plate, and placed in a 25 ℃ incubator for inverted culture for 2 days. Cellulose enrichment medium: CaCl20.1g，MgSO40.1g，(NH4)2SO42.0g，KH2PO40.5g，K2HPO42.0g, NaCl 6g, agar 1.5%, sodium carboxymethylcellulose 0.5%, and 1000mL of distilled water, adjusting pH to 7.0, and sterilizing at 121 deg.C and 0.11Mpa for 30 min.
The sterilized toothpick is picked to well grow bacteria, spotted on a cellulose screening culture medium plate, and cultured in a constant temperature incubator at 25 ℃ for 3 days. After the bacteria grow out, adding a proper amount of 1mg/mL Congo red dye solution into the flat plate, dyeing for 10 minutes, pouring out the dye solution, adding a proper amount of 1mol/L NaCl solution, soaking for 5 minutes for decolorization, judging and selecting according to the existence and the size of the transparent ring, and obtaining a result shown in figure 1.
2. Molecular identification of psychrotrophic cellulase-producing bacteria
Bacterial genomic DNA was extracted using the TIANMP Bacteria DNA Kit from Tiangen Biochemical technology (Beijing) Ltd, 5mL of the bacterial solution was collected by centrifugation at 10000rpm, and the supernatant was discarded. 200 mu L of buffer solution GA, 20 mu L of LProteinase K solution, 220 mu L of buffer solution GB, 500 mu L of buffer solution GD, 600 mu L of rinsing solution PW and 50 mu L of distilled water are added in turn according to requirements to obtain the strain La1 genome DNA.
The method comprises the steps of performing 16S rDNA amplification (denaturation at 94 ℃ for 3 minutes, unwinding at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extending at 72 ℃ for 1.5 minutes, repeating 2-4 steps for reaction for 30 cycles, and extending at 72 ℃ for 8 minutes) by taking genome DNA of a strain to be detected as a template, performing agarose gel electrophoresis detection after the reaction is finished, recovering a PCR product by using a TIANGEN agarose gel DNA recovery kit, sending the PCR product to Jinsry biotechnology limited for sequencing identification, submitting the obtained 16SrDNA sequence to a BLAST program for sequence comparison in GenBank, and analyzing the molecular biology classification status of the strain.
Example 1
La1 strain (psychrophilic cellulase-producing strain of the present invention) was inoculated into 20mL of liquid LB (yeast powder 5g, sodium chloride 10g, peptone 10g, distilled water 1L, pH adjusted to 7.4) using an inoculating loop, and cultured in a shaker at 28 ℃ and 180rpm for 24 hours. The resulting bacterial solution was inoculated in an amount of 2% into a flask containing a fermentation medium (5 g of yeast powder, 10g of sodium chloride, 10g of peptone, 0.5g of sodium carboxymethylcellulose, 1L of distilled water, pH adjusted to 7.4), and cultured in a shaker at 180rpm at 28 ℃ for 48 hours. After the fermentation is finished, the fermentation liquor is centrifuged for 30 minutes at the rotating speed of 4000rmp at the temperature of 4 ℃, and the supernatant is collected to be used as enzyme liquid for standby.
Example 2
La1 strain (psychrophilic cellulase-producing strain of the present invention) was inoculated into 20mL of liquid LB (yeast powder 4g, sodium chloride 8g, peptone 8g, distilled water 1L, pH adjusted to 7.2) using an inoculating loop, and cultured in a shaker at 30 ℃ and 300rpm for 36 hours. The resulting bacterial solution was transferred to a flask containing a fermentation medium (yeast powder 4g, sodium chloride 8g, peptone 8g, sodium carboxymethylcellulose 0.4g, distilled water 1L, pH adjusted to 7.6) in an inoculum size of 3%, and cultured in a shaker at 30 ℃ and 300rpm for 50 hours. After the fermentation is finished, the fermentation liquor is taken out and centrifuged for 20 minutes at the rotating speed of 5000rmp under the condition of 6 ℃, and the supernatant is collected to be used as enzyme liquid for standby.
Example 3
La1 strain (psychrophilic cellulase-producing strain of the present invention) was inoculated into 20mL of liquid LB (yeast powder 6g, sodium chloride 12g, peptone 11g, distilled water 1L, pH adjusted to 7.6) using an inoculating loop, and cultured in a shaker at 24 ℃ and 100rpm for 18 hours. The resulting bacterial solution was inoculated in an amount of 2% into a flask containing a fermentation medium (6 g of yeast powder, 12g of sodium chloride, 11g of peptone, 0.6g of sodium carboxymethylcellulose, 1L of distilled water, pH adjusted to 7.6), and cultured in a shaker at 24 ℃ and 100rpm for 40 hours. After the fermentation is finished, taking the fermentation liquor, centrifuging for 25 minutes at the rotating speed of 3000rmp at the temperature of 2 ℃, and collecting the supernatant as enzyme liquid for later use.
Detection example 1
Activity assay of psychrophilic cellulase
1. Preparation of glucose concentration standard curve
Respectively taking glucose solutions with different volumes of 1mg/mL, and adding distilled water until the total volume is 1mL to obtain a series of glucose solutions with different concentrations. And (3) subpackaging 1mL of DNS solution into each tube, mixing uniformly, boiling for 5min, placing in cold water for several minutes, and adding distilled water into the reaction liquid of each tube to 10 mL. Absorbance was measured at 540 nm. Using the data obtained, a glucose standard curve was plotted.
2. Enzyme activity assay under standard conditions
0.5ml of a 1.0% sodium carboxymethylcellulose solution having a pH of 5.0 and the enzyme solution obtained in example 1-3 were reacted in a water bath at 30 ℃ for 60min, respectively. After the reaction was complete, 1mL of DNS was added. After boiling water bath for 5min, the reaction mixture was cooled, and 8mL of distilled water was added to dilute the reaction mixture, and the absorbance was measured at 540 nm. The control group was not added with the enzyme solution, and after the reaction was completed, 0.5mL of the enzyme solution and 100mL of the LDNS reagent were added, and then the previous steps were repeated. Taking glucose as a standard solution, taking the enzyme amount required for generating 1 mu mol of glucose per minute as an enzyme activity unit, and calculating the enzyme activity of the cellulase.
The result shows that the enzyme production activity of the strain is 0.3U/mL, and the psychrophilic cellulase has certain activity of hydrolyzing cellulose.
Detection example 2
Referring to the method for measuring enzyme activity in detection example 1, the optimum temperature of the enzyme was measured, and enzyme solutions were added at 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ to measure the enzyme activity, and the relative enzyme activity of each reaction system was calculated. As a result, as shown in FIG. 2, the optimum reaction temperature of the enzyme was 35 ℃.
Detection example 3
The optimum pH of the enzyme was measured by referring to the method for measuring enzyme activity in detection example 1, the enzyme solution was diluted with different buffers having a pH of 2-11 and the activity thereof was measured, and the relative enzyme activity of each reaction system was calculated, and the results are shown in FIG. 3. As can be seen from FIG. 3, the optimum reaction pH of the enzyme was 5.0.
Detection example 4
Referring to detection example 1, the enzyme activity determination method is used for determining the influence of various metal ions on the enzyme activity, and different ions including K are respectively added into an enzyme solution+、Li+、NH4 +、Cu2+、Co2+、Rb2+、Mg2+、Ca2+、Zn2+、Mn2+SDS, T-100 and EDTA, the final concentration is 1mmol/L, meanwhile, the untreated enzyme solution is used as a reference, the influence of each ion on the enzyme activity is measured under the conditions of the optimal temperature and the optimal pH value of the enzyme reaction, and the relative enzyme activity of each reaction system is calculated, and the result is shown in figure 4. As can be seen in FIG. 4, the enzyme is resistant to most metal ions and chemical agents.
The detection result shows that the cellulase produced by the psychrotrophic cellulase producing strain has the characteristics of psychrotrophy and acid and alkali resistance.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Sequence listing
<110> university of teacher's university in Anhui
<120> psychrophilic cellulase-producing bacterium
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agagtttgat cctggctcag gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60
gatgaagccc agcttgctgg gtggattagt ggcgaacggg tgagtaacac gtgagtaacc 120
tgccctcgac tccaggataa gcccgggaaa ctgggtctaa tactggatat tcaatttcta 180
ccgcatggtg gtttttggaa aggattctgg tcgaggaggg actcgcggcc tatcagcttg 240
ttggtgaggt aatggctcac caaggcgacg acgggtagcc ggcctgagag ggtgaccggc 300
cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg gaatattgca 360
caatgggcga aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa 420
acctctttca gtagggaaga agcgaaagtg acggtacctg cagaagaagc gccggctaac 480
tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tatccggaat tattgggcgt 540
aaagagctcg taggcggttt gtcgcgtctg ccgtgaaagt ccggggctta actccggatc 600
tgcggtgggt acgggcagac tagagtgctg taggggagac tggaattcct ggtgtagcgg 660
tgaaatgcgc agatatcagg aggaacaccg atggcgaagg caggtctctg ggcagtaact 720
gacgctgagg agcgaaagca tggggagcga acaggattag ataccctggt agtccatgcc 780
gtaaacgttg ggcactagat gtgggggaca ttccacgttt tccgcgtcgt agctaacgca 840
ttaagtgccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg 900
cccgcacaag cggcggagca tgcggattaa ttcgatgcaa cgcgaagaac cttaccaagg 960
cttgacatgg accggatcgg gctagaaata gtctttccct tcggggctgg ttcacaggtg 1020
gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080
accctcgttc tatgttgcca gcacgtgatg gtggggactc ataggagact gccggggtca 1140
actcggagga aggtggggac gacgtcaaat catcatgccc cttatgtctt gggcttcacg 1200
catgctacaa tggccggtac aatgggttgc gatactgtga ggtggagcta atcccaaaaa 1260
gccggtctca gttcggattg gggtctgcaa ctcgacccca tgaagtcgga gtcgctagta 1320
atcgcagatc agcaacgctg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
agtcacgaaa gttggtaaca cccgaagctg gtggcctaac cccttgtggg aaggagctgt 1440
cgaaggtggg accggcgatt gggactaagt cgtaacaagg tagccgtacc ggaaggtgcg 1500
gctggatcac ct 1512

Claims (1)

1. The psychrotrophic cellulase producing strain is characterized in that the preservation number of the psychrotrophic cellulase producing strain (ZHIHengliuella halotolerans) in China general microbiological culture collection center (CGMCC) is 14938, and the preservation date is 11 months and 20 days in 2017.
CN201711368415.4A 2017-12-18 2017-12-18 Psychrophilic cellulase producing strain Active CN108048350B (en)

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