CN107475145B - Strain for producing medium-high temperature resistant cellulase and screening method thereof - Google Patents
Strain for producing medium-high temperature resistant cellulase and screening method thereof Download PDFInfo
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Abstract
The invention provides a screening method of a strain for producing medium-high temperature resistant cellulase, which is characterized in that a selenium-rich hot spring is used as a raw material to separate and enrich thermophilic bacteria, cellulose decomposition bacteria are obtained through enrichment culture and separation purification, and a cellulase strain N1214 is obtained through Congo red dyeing identification and liquid fermentation, wherein the strain is preserved in China center for type culture Collection of Wuhan university with the preservation number of CCTCC M2017142, is the strain N1214 for producing medium-high temperature resistant cellulase and is named in classification as follows: geobacillusGeobacillus sp.(ii) a The 16s rDNA sequence of the strain is shown as SEQ ID NO.1 in a sequence table; NCBI serial number is KY 433297; the screened cellulase producing strain can produce medium-high temperature resistant and alkali resistant cellulase, and can be widely applied to multiple industries such as biofuel, medicine, daily chemical industry, food fermentation, pharmacy and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of microorganisms, and particularly relates to a strain for producing medium-high temperature resistant cellulase and a screening method thereof.
Background art:
cellulosic materials are the most abundant renewable resources in nature. The modern biotechnology is utilized to convert the cellulose materials into liquid fuels such as ethanol and the like, so that the cellulose materials can be used as new resources and new energy to benefit mankind, and the problem of environmental pollution caused by fossil energy is relieved or solved, and meanwhile, the high-efficiency utilization of agricultural residual resources can be realized, and the cellulose materials are generally regarded by countries in the world. Therefore, screening of microbial strains with high-activity cellulase and related research are hot and difficult points of current research. The most commonly used cellulase-producing microorganisms currently under study are normal-temperature and medium-temperature filamentous fungi which are capable of producing complete cellulase systems and completely degrading crystalline cellulose into glucose, such as Trichoderma (Trichoderma)Trichoderma sp.) "Penicillium" (a)Penicillium sp.) Humicola insolens (A) and (B)Humicola sp.) And so on. The cellulase produced by the strain is required to exert better efficiency below 50 ℃ and is easy to inactivate at higher temperature. The research on bacteria is rarely reported, and particularly, the cellulase production by the bacteria resistant to medium and high temperature is less reported; due to thinnessThe optimum pH value of cellulase produced by bacteria is neutral to slightly alkaline, and bacterial cellulase preparations have shown good application prospects along with the successful application of neutral cellulase and alkaline cellulase in cotton washing finishing processes and detergent industries in recent 20 years. Thermophilic enzymes are often produced by thermophilic bacteria. Thermophilic bacteria generally refer to microorganisms that grow well above 55 ℃ but do not grow at 19 ℃.
The microorganisms capable of producing the alkaline cellulase are mostly bacterial strains, including some strains of clostridium, cellulomonas, vibrio, mixed fiber vibrio, bacillus and the like, and strains of bovine bezoar rumen clostridium, rumen albus, fibrinolytic bacteria, clostridium thermocellum, cellulolytic clostridium and faecal fiber monad and the like, and the industrial production of the enzymes is mainly performed by the strains of bacillus (such as bacillus strains) abroadBacillus sp.KSM-635) is a fermentation strain. Domestic research is limited by high-yield strains lacking in alkaline cellulase, large-scale fermentation production is fresh, and the development of new strains producing cellulase by microorganisms is still necessary.
The high-temperature cellulase has a good application prospect in the field of biological energy. How to provide a cellulase strain resistant to medium-high temperatures, e.g.Geobacillus sp.The method has great application value in degrading and utilizing hydrocarbon cellulose and displaying in microbial oil recovery and high temperature resistant biological enzyme. Has wide market prospect.
Disclosure of Invention
The invention provides a strain for producing medium-high temperature resistant cellulase and a screening method thereof, which uses a selenium-rich hot spring as a raw material to separate and enrich to obtain thermophilic bacteria, obtains a plurality of cellulose decomposition bacteria through enrichment culture and separation purification, and obtains a thermophilic bacteria bacillus terreus N1214 with higher cellulase secretion amount through Congo red dyeing identification and DNS method liquid fermentation. The screened cellulase producing strain can produce medium-high temperature resistant and alkali resistant cellulase, and can be widely applied to multiple industries such as biofuel, medicine, daily chemical industry, food fermentation, pharmacy and the like.
The screened bacillus amyloliquefaciens N1214 is an ideal material for cloning cellulase genes, has potential theoretical and practical significance for constructing cellulose decomposition engineering strains and exploring the action mechanism of the cellulase, is simple and quick, aims to simplify the complicated steps in screening and strain identification by the traditional physiological biochemical method, and is easy to implement. The invention has potential theoretical and application value for researching the production of the cellulase preparation.
The invention relates to a strain for producing medium-high temperature resistant cellulase, which is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university with the preservation number of CCTCC M2017142 and the preservation date of the strain is 3 months and 24 days in 2017.
The strain is a strain N1214 for producing medium-high temperature resistant cellulase, and is classified and named as: bacillus soil sampleGeobacillus sp.。
The 16s rDNA sequence of the strain is shown as SEQ ID NO.1 in a sequence table; NCBI serial number is KY 433297; the growth temperature is controlled to be 50-60 ℃, the pH value is 7.0-8.5, and the enzyme activity is 30.2U/mL.
The invention also aims to provide a screening method of the strain for producing the medium-high temperature resistant cellulase, wherein the strain for producing the medium-high temperature resistant cellulase is obtained by separating and enriching thermophilic bacteria which are obtained by taking selenium-enriched hot springs as raw materials, obtaining cellulose decomposition bacteria through enrichment culture, separation and purification, and obtaining the cellulase strain N1214 through Congo red dyeing identification and liquid fermentation.
The screening method of the strain for producing the medium-high temperature resistant cellulase comprises the following steps:
(1) obtaining a sample, separating and enriching the selenium-rich hot spring to obtain a thermophilic bacteria sample, collecting and then putting into a sterile centrifuge tube, preserving and transporting at 2-6 ℃ within 4-6h, and preserving at low temperature to obtain a hot spring water strain sample;
(2) enrichment culture, adding 5ml of the step (1) thermal spring water strain sample into 50ml TYE culture medium, culturing for 24-36 h at 55-60 ℃ in a 250 ml conical flask at the rotating speed of 160 rpm to obtain a culture solution, properly diluting the culture solution, coating the culture solution on a TYE solid plate, culturing for 36-48 h at 55-60 ℃, performing streak separation, and repeating for a plurality of times to obtain a single colony; culturing the single colony in a CMC-Congo red plate screening culture medium; is a cellulase decomposing bacterium;
(3) separating and purifying, namely separating and screening a plurality of cellulose decomposing bacteria by using a CMC-Congo red plate screening culture medium, and separating and purifying by using a DNS method to obtain the medium-high temperature resistant cellulase strain N1214.
Further, the enrichment culture is carried out in the step (2), the single colony is cultured in a CMC-Congo red plate screening culture medium, the culture comprises seed culture and fermentation culture, the seed culture is to inoculate the screened single colony to a seed culture medium, and the seed culture is carried out for 24-30h at 55-60 ℃ to obtain seed liquid; the CMC-Congo red plate screening medium comprises the following components: each 1L of culture medium contains 10g of sodium carboxymethylcellulose, 10g of peptone, 5g of yeast powder and KH2PO 41 g, MgSO40.2 g, NaCl 10g, glucose 2g, agar powder 20 g; pH 7.0, 1000ml water.
Dyeing with 1 mg/ml Congo red solution for 30 min, washing with distilled water, and decolorizing with 1 mol/L NaCl solution for 30 min. And (4) selecting the strains with larger transparent circles, transferring the strains to a new screening plate, and separating and purifying until single colonies are obtained.
The seed culture medium comprises the following components: 10g of CMC-Na, 3.0 g of peptone and KH 2 PO 4 4.0 g、MgSO 4·7H 20.03 g of O, 5g of NaCl, the initial pH value of 7.0 and 1000ml of water;
the fermentation culture: inoculating seed liquid cultured by seeds to a fermentation culture medium by an inoculation amount with the volume concentration of 1-2%, and culturing for 25-30h at 55 ℃ under the condition of 180r/min to obtain cellulase decomposing bacteria; the components of the fermentation control medium are as follows: each liter of fermentation medium contains: 10g of CMC-Na, 2.5g of peptone, 1g of yeast extract and KH2PO4 1 g，(NH4)2SO422g ，MgSO 4·7H 2 O 0.2 - 0. 5g，NaCl 1-10g ，CaCl 2 ·2H 2 O 0.3，FeSO4 ·7H 2 O 0.005，MnSO4 0.002， ZnCl 2 0.002，CoC1 20.002, and controlling pH to 7.0-7.2.
A strain N1214 for producing medium-high temperature resistant cellulase is applied to a material for cloning cellulase genes.
The strain for producing the medium-high temperature resistant cellulase has the beneficial effects that the screening method has the following advantages:
1) the screening method of the invention obtains the cellulase strain suitable for the pH neutrality, while the cellulase produced generally at home is suitable for the acidic condition, and the neutral and alkaline cellulases are obviously superior to the acidic cellulase in the aspect of application;
2) the screened strain is Bacillus licheniformis which is different from the strain produced by adopting trichoderma as cellulase at home at present. The bacillus gasseri prepared by the method is a strain which is easy to store, can be amplified and propagated quickly and is suitable for large-scale production, and the production period can be shortened;
3) the cellulase prepared by the screening method has high activity, wide application range of pH and good stability;
4) the bacillus gasseri has high enzyme production capacity, can be widely applied to multiple industries such as biofuel, medicine, daily chemical industry, food fermentation, pharmacy and the like, and has wide application prospect.
Morphological identification of the selected strain N1214 of the invention: the gram staining method is used for staining the strain N1214, and the result shows that the strain is gram-positive bacteria, and the thallus is rod-shaped, has spores, is oval and is positioned at the center or near the center. Its propagules are thermolabile and can be killed after 20min sterilization at 120 deg.C. The pH fermentation condition of the screened strain is 7.0-8.5, and compared with other bacillus, the strain has the characteristic of producing medium-high temperature resistant cellulase.
Description of the drawings:
FIG. 1 shows a strain Bacillus licheniformis for producing medium-high temperature resistant cellulase of the inventionGeobacillus spN1214 colonial and gram stain maps on TYE medium;
FIG. 2 shows a strain Bacillus licheniformis for producing medium-high temperature resistant cellulase of the inventionGeobacillus spThe 16S rRNA gene sequence of N1214;
FIG. 3 shows the medium-high temperature resistant cellulase producing bacteria of the present inventionThe strain utilizes an adjacent phase linkage method to draw a phylogenetic tree based on a 16S rRNA gene sequence of bacteria, and shows that the screened strain for producing the medium-high temperature resistant cellulase, namely the bacillus amyloliquefaciens N1214 and a databaseGeobacillus spRepresenting the genetic relationship of the strains; the sequences are compared by a ClustalX program, and the phylogenetic tree is generated by MEGA6.0 software after 1000 times of repeated operation;
FIG. 4 shows the effect of different time on the activity of the bacillus amyloliquefaciens Geobacillus sp for producing the medium-high temperature resistant cellulase.
The specific implementation mode is as follows:
the present invention will be described in further detail with reference to specific embodiments,
the raw material used in the invention is thermophilic bacteria obtained by separating and enriching selenium-rich hot springs from hot water town in Yichun city of Jiangxi province, the hot water selenium-rich hot springs are positioned at 115 degrees of east longitude and 28 degrees of north latitude and are positioned in Mingyue mountain scenic spots of Jiangxi province and China AAAA grade scenic spots. Collecting hot spring water sample, placing into sterile 50ml centrifuge tube, storing at 4 deg.C within 4h, transporting back to laboratory, and preserving at low temperature for use.
The invention relates to screening of a strain N1214 for producing medium-high temperature resistant cellulase, wherein the strain N1214 is mainly applied to producing the medium-high temperature resistant cellulase.
Culturing the separated selenium-rich hot spring water sample by using a TYE culture medium, then separating and screening by using a CMC-Congo red plate screening culture medium to obtain a plurality of cellulose decomposing bacteria, and obtaining a bacterium N1214 with high cellulase secretion by using a DNS method (3, 5-dinitrosalicylic acid).
The specific screening of the strain N1214 producing the medium-high temperature resistant cellulase comprises the following steps:
(1) obtaining a sample, separating and enriching the selenium-rich hot spring to obtain a thermophilic bacteria sample, collecting and then putting into a sterile centrifuge tube, preserving and transporting at 2-6 ℃ within 4-6h, and preserving at low temperature to obtain a hot spring water strain sample;
(2) enrichment culture, adding 5ml of the step (1) thermal spring water strain sample into 50ml of TYE culture medium, placing in a 250 ml conical flask, rotating at 160 rpm and 55-Culturing at 60 deg.C for 24-36 h to obtain culture solution, diluting the culture solution, spreading on TYE solid plate, culturing at 55-60 deg.C for 36-48 h, streaking, and repeating for several times to obtain single colony. Preferably, the TYE culture medium comprises the following components: KH (Perkin Elmer)2PO4 0.28g/L，MgSO4·7H2O 0.25g/L，NH 1.3g/L，CoCl2 0.01mg/L，MnCl2·4H2O 1.8mg/L，CaCl2·2H2O 0.07g/L，Na2B4O7·10H2O 4.5mg/L，ZnSO4·7H2O 0.22mg/L，CuSO4·2H2O 0.05mg/L，Na2MoO4·2H2O0.03 mg/L, Yeast extract 5.0g/L, Tryptone 4.0g/L, 1L pure water, 20g agar powder, controlling pH to 7.0-7.5;
culturing the single colony in a CMC-Congo red plate screening culture medium; is a cellulase decomposing bacterium;
(3) separating and purifying, namely separating and screening a plurality of cellulose decomposing bacteria by using a CMC-Congo red plate screening culture medium, and separating and purifying by using a DNS method to obtain the medium-high temperature resistant cellulase strain N1214.
And further (2) carrying out enrichment culture, wherein the single colony is cultured in a CMC-Congo red plate screening culture medium, the culture comprises seed culture and fermentation culture, the seed culture is to inoculate the screened single colony to a seed culture medium, and the seed culture is carried out for 24-30h at 55-60 ℃ to obtain a seed solution. The CMC-Congo red plate screening medium comprises the following components: each 1L of culture medium contains 10g of sodium carboxymethylcellulose, 10g of peptone, 5g of yeast powder and KH2PO 41 g, MgSO40.2 g, NaCl 10g, glucose 2g, agar powder 20 g; pH 7.0, 1000ml water.
Dyeing with 1 mg/ml Congo red solution for 30 min, washing with distilled water, and decolorizing with 1 mol/L NaCl solution for 30 min. And (4) selecting the strains with larger transparent circles, transferring the strains to a new screening plate, and separating and purifying until single colonies are obtained.
Seed culture: inoculating the screened single colony to a seed culture medium, and culturing at 55 ℃ for 24h to obtain a seed solution; the above-mentionedThe seed culture medium comprises the following components in parts by mass: 10g of CMC-Na, 3.0 g of peptone and KH 2 PO 4 4.0 g、MgSO 4·7H 2O0.03 g. NaCl 5g, initial pH 7.0, 1000ml of water.
The fermentation cellulase production culture is fermentation culture: inoculating seed liquid cultured by seeds to a fermentation culture medium by an inoculation amount with the volume concentration of 1-2%, and culturing for 25-30h at 55 ℃ under the condition of 180r/min to obtain cellulase decomposing bacteria; the components of the fermentation control medium are as follows: each liter of fermentation medium contains: 10g of CMC-Na, 2.5g of peptone, 1g of yeast extract and KH2PO4 1 g，(NH4)2SO42 2g ，MgSO 4·7H 2 O 0.2 - 0. 5g，NaCl 1-10g ，CaCl 2 ·2H 2 O 0.3，FeSO4 ·7H 2 O 0.005，MnSO4 0.002， ZnCl 2 0.002，CoC1 20.002, and controlling pH to 7.0-7.2.
Centrifuging the fermentation liquor at 10000r/min and 15 ℃ for 5min, and taking supernatant fluid to obtain the enzyme source capable of measuring the activity of the cellulase.
Table 1 below shows the reaction temperature and pH for Bacillus terreus of the present inventionGeobacillus sp.N1214 cellulase production activity.
TABLE 1 Effect of different reaction temperatures and pH on the cellulase production activity (U/ml) of Bacillus terreus N1214
| 50℃ | 55 |
60℃ | |
| pH=6 | 23.7 | 28.7 | 20.9 |
| pH=7 | 29.9 | 30.2 | 28.3 |
| pH=8 | 23.3 | 26.5 | 22.5 |
Description of the drawings: the invention provides a strain for producing medium-high temperature resistant cellulase, namely bacillus amyloliquefaciensGeobacillus sp.The optimal reaction pH and the optimal reaction temperature for CMCase activity of N1214; the change of the activity of the bacterial CMCase was measured at 55 ℃ and different reaction pH by the above method under different reaction pH (pH 5.0-7.0) conditions of the enzyme solution with the optimum culture time.
Example 1
The invention relates to separation, purification and identification of a strain N1214 producing medium-high temperature resistant cellulase.
First, 0g of agar powder for producing cellulase thermophilic bacteria N1214.
The color of the colonies on the TYE culture medium is white, the surfaces of the colonies are wet and glossy, and the edges of the colonies are wavy; inoculating the single colony to a CMC-Congo red plate screening culture medium, and screening to obtain multiple strains of cellulose decomposing bacteria;
culturing the single colony in a CMC-Congo red plate screening culture medium; is a cellulase decomposing bacterium;
the CMC-Congo red plate screening medium comprises the following components: each 1L of culture medium contains 10g of sodium carboxymethylcellulose, 10g of peptone, 5g of yeast powder, 41 g of KH2PO, 40.2 g of MgSO40, 10g of NaCl, 2g of glucose and 20g of agar powder; pH 7.0, 1000ml water.
Dyeing with 1 mg/ml Congo red solution for 30 min, washing with distilled water, and decolorizing with 1 mol/L NaCl solution for 30 min. Selecting a strain with a larger transparent circle, transferring the strain to a new screening flat plate, separating and purifying until single screening, namely screening of the medium-high temperature resistant cellulase strain N1214 is obtained;
obtaining a sample: thermophilic bacteria obtained by separating and enriching selenium-enriched hot spring from Wen soup town in Yichun city of Jiangxi province are collected and put into a sterile 50ml centrifugal tube, and are stored and transported back at 4 ℃ within 4h and are stored at low temperature for standby.
The hot spring water sample is cultured by a TYE culture medium, then a plurality of cellulose decomposing bacteria are obtained by separating and screening a CMC-Congo red plate screening culture medium, and a bacterium N1214 with high cellulase secretion is obtained by a DNS method (3, 5-dinitrosalicylic acid).
The samples were isolated from hot spring water, 5mL of the sample was added to 50mL of TYE medium, incubated at 55 ℃ for 24h at 180 rpm in a 250 mL Erlenmeyer flask, the medium was diluted appropriately and spread on TYE solid plates, streaked after 3 days of incubation at 55 ℃ and repeated several times until a single colony was obtained, the single colony in the plate was picked, the colony morphology was numbered and recorded, and the colony was incubated in fresh solid medium.
Thermophilic bacteria isolation medium TYE (ph 7.2): KH (Perkin Elmer)2PO4 0.28g/L，MgSO4·7H2O 0.25g/L，NH 1.3g/L，CoCl2 0.01mg/L，MnCl2·4H2O 1.8mg/L，CaCl2·2H2O 0.07g/L，Na2B4O7·10H2O 4.5mg/L，ZnSO4·7H2O 0.22mg/L，CuSO4·2H2O 0.05mg/L，Na2MoO4·2H2O0.03 mg/L, Yeast extract 5.0g/L, Tryptone 4.0g/L, 1L ultrapure water, 2 colonies.
And (3) performing seed culture and fermentation culture on the single colony, wherein the seed culture comprises the following steps: inoculating the screened single colony to a seed culture medium, and culturing at 55 ℃ for 24h to obtain a seed solution; the final concentration of the seed culture medium is as follows: 10g of CMC-Na, 3.0 g of peptone and KH 2 PO 4 4.0 g、MgSO 4·7H 2O0.03 g. NaCl 5g, initial pH 7.0, 1000ml of water.
And (3) fermentation and cellulase production culture: inoculating the seed liquid to a fermentation medium by an inoculation amount with a volume concentration of 1%, wherein the fermentation medium comprises the following components: each L of the culture medium contains: 10g of CMC-Na, 2.5g of peptone, 1g of yeast extract and KH2PO4 1 g，(NH4)2SO42 2g ，MgSO 4·7H 2 O 0. 5g，NaCl 1g ，CaCl 2 ·2H 2 O 0.3, FeSO4 ·7H 2 O 0.005，MnSO4 0.002 ZnCl 2 0.002, CoC1 20.002, pH 7.0-7.2, culturing at 55 deg.C and 180r/min for 25h to obtain fermentation liquid, centrifuging the fermentation liquid at 10000r/min and 15 deg.C for 5min, and collecting supernatant as enzyme source. The method is used for determining the activity of the cellulase.
Secondly, identifying and analyzing the strain,
(1) gram stain
Inoculating bacteria on the glass slide; primary dyeing: dripping crystal violet to cover the mycoderm, dyeing for 1-2 min, and washing with water; mordant dyeing: washing with iodine solution to remove residual water, covering with iodine solution for about 1 min, and washing with water; and (3) decoloring: sucking residual water on the slide by using filter paper, inclining the slide, adding 95% ethanol in a dropping tube for decoloring in a white background, and immediately washing with water (the decoloring time is generally 20-30 s) when the flowing ethanol is not purple; counterdyeing: re-dyeing with safranine solution for about 2 min, and washing with water; microscopic examination: after drying, the observation was performed with an oil mirror. Gram-positive bacteria are stained bluish purple. The strain is a gram-positive bacterium, the thallus is rod-shaped, has spores, is oval and is terminal (see figure 1).
(2) Molecular identification of medium-high temperature resistant cellulase strain
The invention relates to extraction of medium-high temperature resistant cellulase strain DNA: LB liquid medium was inoculated at 1%, centrifuged, and extracted with a bacterial genome DNA extraction kit, Ezup column type bacterial genome DNA extraction kit from Shanghai, a certain company.
Analyzing the sequence of the medium-high temperature resistant cellulase strain 16S r RNA, and analyzing the strain 16S r RNAGene cloning and sequence analysis; the universal upstream primer sequences for cloning the bacterial 16S rRNA genes were: AGAGTTTGATCCTGGCTCAG, and the sequence of the universal downstream primer is as follows: GGTTACCTTGTTA CGACTT are provided. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 60 s; annealing at 56 ℃ for 45 s; extension at 72 ℃ for 90 s; repeating the steps 2-4, and reacting for 30 cycles; extension at 72 ℃ for 10 min. Connecting the 16S rRNA of the strain to a T vector, screening positive clones, and sequencing by Shanghai biological engineering technology service company Limited. The obtained 16S rRNA sequences were submitted to the NCBI GenBank database (see FIG. 2). Searching homologous sequences of the determined sequences by Blast, screening and downloading strain sequences with higher similarity to the sequences and taking the strain sequences as reference sequences, carrying out sequence analysis by using Mega6.0 software and calculating evolutionary distance, constructing a phylogenetic tree by using a Neighbour-Joining method, marking self-development test Bootstrap values on branches as shown in figure 3, and obtaining the phylogenetic tree repeatedly for 1000 times so as to evaluate the confidence coefficient of the phylogenetic tree; strain N1214 andGeobacillus spsimilarity up to 99%.
Example 2
Screening a strain N1214 with the highest cellulase activity and researching the enzymological properties of the strain.
The medium components for determining the activity of the medium-high temperature resistant cellulase strain N1214 cellulase provided by the invention are as follows: each 1L of the culture medium contains: 10g of CMC-Na, 2.5g of peptone, 1g of yeast extract and KH2PO4 1 g，(NH4)2SO42 2g ，MgSO 4·7H 2 O 0. 5g，NaCl 1g ，CaCl 2 ·2H 2 O 0.3, FeSO4 ·7H 2 O 0.005，MnSO4 0.002 ZnCl 2 0.002, CoC1 20.002, pH 7.0-7.2, culturing at 55 deg.C and 180r/min for 25h to obtain fermentation broth, centrifuging the fermentation broth at 10000r/min and 15 deg.C for 5min, and collecting supernatant as enzyme source; the method is used for determining the activity of the cellulase.
Preparing a crude enzyme solution; the previous step of culturing the strain N1214 is carried out by picking out, fermenting, producing enzyme, this time using liquid culture medium, and culturing time, pH, temperature, and each two groups by controlled variable method. The pH values are respectively 50,55 and 60 ℃ and 6.0,7.0 and 8.0. Shaking the flask for 24h, 25h and 26h, taking out 5ml of each group, pouring into a centrifuge tube, centrifuging at 4 deg.C at 5000 rpm for 10min, collecting the supernatant as extracellular enzyme solution, and determining enzyme activity at different culture time and temperature.
The enzyme activity is measured by a DNS (3, 5-dinitrosalicylic acid) method: and (4) drawing a glucose standard curve. Accurately weighing 104.8 mg of glucose with constant weight, dissolving the glucose in distilled water, dissolving the solution in a 100 mL volumetric flask, and preparing a 1 mg/mL standard glucose solution as a standard glucose mother solution for later use. And (3) finishing the addition and reaction of each reagent, cooling, carrying out color comparison at 540 nm respectively, and recording the light absorption value. And drawing a glucose standard curve.
And (5) measuring the activity of the CMC enzyme of the strain. Adding 6.0 mL of 1.0% sodium carboxymethyl cellulose solution (pH 7.0, 6.0 and 8.0) into a 10.0mL test tube, adding 2.0 mL of crude enzyme solution with different culture times, performing enzymolysis in a water bath at 50 ℃ for 30 min, adding 2.0 mL of DNS solution, performing boiling water bath for 8 min, taking out, cooling in ice water to room temperature, fixing the volume to 10.0mL, measuring the light absorption value at 540 nm, and calculating the enzyme activity according to a glucose standard curve.
0.5 ml of the crude enzyme solution diluted n times was taken out and put into a test tube, and 1.5 ml of disodium hydrogenphosphate-citric acid buffer (0.05 mol/L, pH 7.0) was added to prepare a substrate solution containing 1% CMC-Na. Keeping the temperature at 50 ℃ for 50min, adding 1.5 ml DNS reagent, boiling for 10min for color development, immediately cooling to room temperature in ice bath, fixing the volume to 10.0ml, measuring the light absorption value at 540 nm, and calculating the enzyme activity according to a glucose standard curve.
The enzyme activities mentioned in the invention are uniformly defined as: 1ml of enzyme solution, at 50. + -. 0.1 ℃ and pH7, hydrolyses the substrate per minute, yielding an amount of reducing sugar equivalent to 1. mu.g of glucose, expressed as 1 enzyme activity unit in IU/ml.
The enzyme activity calculation formula is as follows: x = (B × n × 1000)/(0.5 × 50)
In the formula: b: calculating the concentration of glucose (mg/ml) from the standard curve; n: dilution times; 1000: mg converted to μ g; 0.5: the milliliter number of the enzyme solution is absorbed during the determination; 50: glucose standard curve for determining cellulase activity by 50min DNS method: y=0.7734X-0.0197 R2=0.9953
the influence of different culture times on the activity of the cellulase is used, preliminary experiments in the early period of the experiment explore that the enzyme activity is small before 24 hours in 0-48 hours, and the enzyme activity changes greatly in 24 hours-26 hours, so that the detection of the activity of the cellulase in 24-26 hours is designed; the results show that the enzyme activity of 23.5h is 13U/ml, the enzyme activity of 24h is 16.3U/ml, the enzyme activity of 24.5h is 22.7U/ml, the enzyme activity of 25h is 28.6U/ml, the enzyme activity of 25.5h is 30.1U/ml, the enzyme activity of 26h is 29.0U/ml and the enzyme activity of 26.5h is 26.2U/ml. Therefore, the enzyme activity of the N1214 cellulase reaches the maximum value in the experiment of 25.5h, and the later experiment takes 25h as a detection value.
The most suitable reaction temperature influences the activity of the cellulase, and the more suitable temperature range for preliminary experiment exploration in the early stage of the experiment is 50-60; therefore, the detection of the activity of the cellulase at the temperature of 50,55 and 60 ℃ respectively is designed; as shown in FIG. 4, 55 ℃ was the optimum temperature.
The most suitable reaction pH influences the activity of the cellulase, and preliminary experiments in the early stage of the experiment explore a more suitable temperature range pH range of 6.0-8.0, so that the detection of the activity of the cellulase under the pH values of 6.0,7.0 and 8.0 is designed; the results are shown in FIG. 4 for N1214 Geobacillus sp .The enzyme activity determination of (1) shows that 55 ℃ is the optimal temperature for growth, pH =7 is the pH value most suitable for growth, when the OD value is maximum after the culture for 25h, the enzyme activity of the cellulase reaches the maximum value of 30.2U/ml, the enzyme activity value is higher, and the number N1214 of the producing strain is higher Geobacillus sp .Can produce medium-high temperature resistant and alkali resistant cellulase, has simple and quick method and easy implementation, and can be widely applied to multiple industries such as biofuel, medicine, daily chemical industry, food fermentation, pharmacy and the like. Bacillus soil sampleGeobacillus sp. N1214 is an ideal material for cloning cellulase genes, and has potential theoretical and practical significance for constructing cellulose decomposition engineering strains and exploring cellulase action mechanisms.
Example 3
The medium-high temperature resistant cellulase strain N1214 of the inventionGeobacillus spExploration of the ability to produce amylase and lipase.
Detection of Amylase-producing ability
Culturing the strain N1214 in a TYE culture medium, taking out, placing in an opened sterile operating platform for observation, generating obvious bacterial colonies, preparing iodine solution, pouring 5ml of iodine solution into each culture medium, standing for two minutes, observing the phenomenon, finding that the iodine solution near the bacterial colonies fades, generating obvious bacteriolysis rings with the diameter of 3cm, and displaying the strain N1214 according to the experimental resultGeobacillus sp.Can produce amylase.
Detection of Lipase-producing ability
The strain N1214 was cultured in M2 medium and removed (M2 medium components: ammonium sulfate 1.3g, potassium dihydrogen phosphate 0.28g, magnesium sulfate 0.112g, calcium chloride 0.074g, trace elements (mg/L): iron chloride 1.9, manganese chloride 0.18, zinc chloride 0.022, copper chloride 0.005, sodium borate 0.44, sodium molybdate 0.003; pH =7.0,22g agar powder, sterilized at 121 ℃ for 20 min). Carrying out lipase identification by using tween80 culture medium; the preparation work is the same as before, but the characteristic of producing lipase by the flora is tested by adding tween80 instead of starch into the fat identification culture medium, the culture medium is prepared at first, then the container and the culture medium are sterilized and inoculated, and the container and the culture medium are placed into an incubator at 55 ℃ for culture for two days, so that the obvious phenomenon appears, an obvious transparent ring appears around the colony, and the diameter of the halo is 1.3 cm; the experimental results show that the strain N1214 Geobacillus sp.Has strong capability of producing lipase.
Experiments show that the strain N1214Geobacillus sp not only produces cellulase and has high enzyme activity, but also can produce amylase and lipase, so that the strain is difficult to obtain, has practical and multifunctional functions, and is expected to be widely applied to papermaking, washing and petroleum industry.
N1214（KY433297）.seq
1 gagcttgctc tggtttggtc agcggcggac gggtgagtaa cacgtgggca acctgcccgc
61 aagaccggga taactccggg aaaccggagc taataccgga taacaccgaa gaccgcatgg
121 tctttggttg aaaggcggcc tttggctgtc acttgcggat gggcccgcgg cgcattagct
181 agttggtgag gtaacggctc accaaggcga cgatgcgtag ccggcctgag agggtgaccg
241 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc
301 cgcaatgggc gaaagcctga cggagcgacg ccgcgtgagc gaagaaggcc ttcgggtcgt
361 aaagctctgt tgtgagggac gaaggagcgc cgttcgaaga gggcggcgcg gtgacggtac
421 ctcacgagga agccccggct aactacgtgc cagcagccgc ggtaatacgt agggggcgag
481 cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg ttccttaagt ctgatgtgaa
541 agcccacggc tcaaccgtgg agggtcattg gaaactgggg gacttgagtg caggagagga
601 gagcggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga
661 aggcggctct ctggcctgca actgacgctg aggcgcgaaa gcgtggggag caaacaggat
721 tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttagag gggtcacacc
781 ctttagtgct gcagctaacg cgataagcac tccgcctggg gagtacggcc gcaaggctga
841 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc
901 aacgcgaaga accttaccag gtcttgacat cccctgacaa cccaagagat tgggcgttcc
961 cccttcgggg ggacagggtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat
1021 gttgggttaa gtcccgcaac gagcgcaacc ctcgcctcta gttgccagca cgaaggtggg
1081 cactctagag ggactgccgg cgacaagtcg gaggaaggtg gggatgacgt caaatcatca
1141 tgccccttat gacctgggct acacacgtgc tacaatgggc ggtacaaagg gctgcgaacc
1201 cgcgaggggg agcgaatccc aaaaagccgc tctcagttcg gattgcaggc tgcaactcgc
1261 ctgcatgaag ccggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc
1321 gggccttgta cacaccgccc gtcacaccac gagagcttgc aacacccgaa gtcggtgcgg
1381 taacccttac gggagccagc cgccgaaggt ggggcaagtg
Claims (2)
1. A strain for producing medium-high temperature resistant cellulase is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university with the preservation number of CCTCC M2017142;
the strain is a strain N1214 for producing medium-high temperature resistant cellulase, and is classified and named as: geobacillusGeobacillus sp.；
The sequence of the strain 16s rDNA is shown as SEQ ID NO.1 in a sequence table; NCBI serial number is KY 433297; the growth temperature is controlled to be 50-60 ℃, the pH value is 7.0-8.5, and the enzyme activity is 30.2U/mL.
2. The use of the strain for producing the medium-high temperature resistant cellulase according to claim 1 in a cellulase gene cloning material.
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| US7319029B2 (en) * | 2004-08-02 | 2008-01-15 | Universiti Purta Malaysia | Lipase from Geobacillus sp. strain T1 |
| CN102057040A (en) * | 2008-06-06 | 2011-05-11 | 丹尼斯科美国公司 | Geobacillus stearothermophilus alpha-amylase (AMYS) variants with improved properties |
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| US7319029B2 (en) * | 2004-08-02 | 2008-01-15 | Universiti Purta Malaysia | Lipase from Geobacillus sp. strain T1 |
| CN102057040A (en) * | 2008-06-06 | 2011-05-11 | 丹尼斯科美国公司 | Geobacillus stearothermophilus alpha-amylase (AMYS) variants with improved properties |
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| Title |
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| 嗜热溶胞菌SY14的基本特性研究;宋玉栋等;《环境科学》;20070930;第28卷(第9期);2106-2111 * |
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