CN105647904A - Method for screening cellulase producing strains and method for producing cellulase by means of fermentation - Google Patents

Abstract

The invention discloses a method for screening cellulase-producing strains and a method for fermenting and producing cellulase. The strain is selected from decayed wood in Huangshan ecological forest through preliminary screening, protoplast preparation, ultraviolet light mutagenesis, and breeding. A cellulase-producing strain named TP-02 was obtained through re-screening. This strain has the ability to produce high cellulase. It can use soybean meal, ammonium sulfate, urea or ammonium chloride as nitrogen source, and bran juice, lactose, etc. , starch sugar, rice straw or corn stalks are used as carbon sources for liquid fermentation. After 72-84 hours of culture, the enzyme activity of the filter paper can reach 16.98IU/mL, the enzyme activity of the endonuclease can reach 41.33IU/mL, and the activity of the exonuclease can reach 41.33IU/mL. The activity reached 3.65IU/mL, and the enzyme activity of β-glucosidase reached 15.98IU/mL.

Description

A screening method for cellulase-producing strains and a method for fermenting and producing cellulase

technical field

The invention belongs to the field of biotechnology, and in particular relates to a screening method for cellulase-producing strains and a method for fermenting and producing cellulase.

Background technique

Lignocellulosic substances are the most widely distributed and abundantly stored substances on the earth, and they are also the cheapest renewable resources. Cellulase is a general term for a class of multi-component enzymes that can degrade cellulose into glucose. They work synergistically to degrade cellulose to produce oligosaccharides and cellodisaccharides, and finally hydrolyze them into monosaccharides such as glucose and xylose. In turn, alcohol, food, single-cell protein, pharmaceuticals and other chemical and chemical raw materials can be further produced. Therefore, cellulase has good application prospects in fuel ethanol production, papermaking, feed processing and other industries.

Cellulase-producing bacteria include bacteria, fungi, yeast, etc., but currently the strains mainly used for cellulase production are mainly filamentous fungi. The cellulase enzyme system produced by filamentous fungi is relatively complete, and most of the enzymes produced are extracellular enzymes, which are convenient for later separation and extraction. Cellulase is a multi-enzyme complex composed of three different enzymes: exoglucanase, endoglucanase and β-glucosidase (BGL). hydrolysis.

At present, the application of cellulase is still limited to a certain extent by the high production cost. The problem of high production cost of cellulase is mainly manifested in the following two aspects: First, the fermentation enzyme activity of the strain producing cellulase is relatively low, which has always been an urgent problem to be solved in large-scale production, and it is also a long-term restriction on the mass production of cellulase. And the main factor that limits its application; Second, the currently used filamentous fungi have generally long cellulase fermentation time due to factors such as strain characteristics and culture conditions, and the cost is too high. At present, the filamentous fungi used for the production and research of cellulase mainly include Trichoderma, Aspergillus and Penicillium. The fermentation cycle is generally more than 6 days, which has the characteristics of long time consumption and low efficiency.

Contents of the invention

Aiming at the above deficiencies, the present invention provides a method for screening cellulase-producing bacterial strains, which are obtained from rotting wood in Huangshan ecological forest through primary screening, protoplast preparation, ultraviolet light mutagenic breeding, and secondary screening. A cellulase-producing strain named TP-02.

The present invention also provides the application of the bacterial strain screened by the above screening method as the production of vitaminase. This strain has the ability of high cellulase production. It can use soybean meal, ammonium sulfate, urea or ammonium chloride as nitrogen source and bran juice, lactose, starch sugar, rice straw or corn stalk as carbon source for liquid fermentation. After 72 After ~84 hours, the enzyme activity of the filter paper can reach 16.98IU/mL, the enzyme activity of endonuclease can reach 41.33IU/mL, the activity of exonuclease can reach 3.65IU/mL, and the activity of β-glucanase can reach 15.98IU/mL mL.

The present invention also provides a production method of vitrease. Compared with other prior art, the fermentative enzyme production period of this method is greatly shortened.

The technical scheme that the present invention takes is:

A screening method for cellulase-producing strains, the screening method comprising the following steps: primary screening, protoplast preparation, ultraviolet light mutagenic breeding, and secondary screening;

The cellulase-producing strain was preserved on July 16, 2015 by the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Management Committee, and the preservation address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology; the preservation number is CGMCCNo.11119; the classification name is Rhizopus stolonifervar.reflexus.

The screening method specifically includes the following steps:

A. initial screening medium initial screening, the composition of described initial screening medium is: every L medium contains: CMC-Na5-10g; Glucose 3-5g; Agar 10-20g; MandelS inorganic nutrient salt 1000mL;

B. Prepare protoplasts, cultivate the spores of the bacterial strains obtained by primary screening in PDA medium, and vibrate and incubate the mycelia obtained after purification and centrifugation in the wall-breaking liquid, and obtain protoplasts after filtering and centrifugal purification; The composition of wall fluid is helicase: cellulase=3:1;

C. Mutation breeding, the protoplast obtained in step B is irradiated with a 15W ultraviolet lamp for mutagenesis treatment, the treatment solution is coated on a high-osmosis primary screening medium plate, and the colony is transferred to a PDA slant medium for cultivation after cultivation;

The composition of the hypertonic primary screening medium plate is: each L medium contains: CMC-Na5-10g; glucose 3-5g; agar 10-20g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol; The medium plate can maintain proper physiological osmotic pressure and avoid fragmentation of protoplasts.

D. re-screening; the spore suspension of the culture on the PDA slant medium obtained in step C is inoculated in the liquid seed medium for cultivation, and the seed culture solution is inoculated into the liquid fermentation medium for cultivation in a volume ratio of 1:10 , through the determination of the yield of reducing sugar in the enzymatic hydrolysis of fermentation products, the strain with high enzyme activity was screened out, which was the cellulase-producing strain TP-02.

The composition of the liquid seed culture medium is: 10% bran extract juice, and its preparation method is to weigh 100g of bran, add 800mL of distilled water to boil for 30min, filter through 4 layers of gauze, and set the volume to 1000mL.

The composition of the liquid fermentation medium is: each L medium contains: 5-10g of 80-120 mesh corn stalks, rice straw or wheat straw, 80-100g of 5% bran extract, (NH ₄ ) ₂ SO ₄ 20- 30g, KH ₂ PO ₄ 5-10g, MgSO ₄ 7H ₂ O5-10g, CaCl ₂ 10-20g, Tween800.15-0.25g, trace elements: FeSO ₄ 7H ₂ O0.003-0.005g, MnSO ₄ H ₂ O 0.001-0.00156g, ZnSO ₄ ·7H ₂ O 0.001-0.0014g, CoCl ₂ ·6H ₂ O 0.001-0.002g, pH 5.0.

The present invention also provides the application of the bacterial strain screened according to the above screening method as the production of vitaminase.

The present invention also provides a kind of production method of vitaminase, described method comprises the following steps:

(1) use liquid seed culture medium to carry out seed culture to the bacterial strain TP-02 that screens according to the screening method described in claim 1, obtains

to the seed culture solution;

(2) Inoculate the seed culture solution into a liquid fermentation medium for cultivation, and produce cellulase through liquid fermentation.

Described step (1) specifically comprises the following steps: the bacterial strain TP-02 obtained by screening according to the screening method according to claim 1 is successively cultivated through PDA slant culture medium and liquid seed culture medium, and the gained seed liquid is preserved in- 80 ℃ ultra-low temperature glycerin freezing tube; the seed liquid stored in the -80 ℃ ultra-low temperature glycerin freezing tube is successively cultured on PDA slant medium and liquid seed medium to obtain seed culture liquid.

In the step (2), the volume ratio of the seed culture solution to the liquid fermentation medium is 1:9-10.

The culture conditions in the step (2) are 30° C., 200-350 r/min for 84-96 hours.

Further, the culture conditions in the step (2) also include: the stirring speed is 450r/min, the ventilation rate is 4.5vvm, the tank pressure is 0.03MPa, and the pH is in the range of 5.0 to 5.5, and the process is controlled by adding ammonia water The pH is in the range of 5.0 to 5.5.

Compared with the prior art, the present invention has the following advantages:

1. The growth rate of the strain is fast, the rate of enzyme production is high, the fermentation time is short, and the fermentation can be completed within 4 days;

2. The bacteria are wild bacteria, vigorous, not easy to infect bacteria, and easy to pure culture;

3. The strains can use agricultural by-products such as bran juice, rice straw (or wheat straw, corn straw) to ferment to produce cellulase, and the nutrient substrate can be used in a wide range and the ingredients are cheap and easy to obtain;

4. The fermentation performance of repeated shaking flasks is stable, the enzyme activity of the filter paper reaches 13.16IU/mL, the enzyme activity of endonuclease reaches 45.81IU/mL, the activity of exonuclease reaches 0.537IU/mL, and the activity of glucanase reaches 12.45IU /mL;

5. After being cultured in the fermenter for 72 hours, its filter paper enzyme activity can reach 16.98IU/mL, endonuclease activity can reach 41.33IU/mL, exonuclease activity can reach 3.65IU/mL, glucosidase activity can reach Vitality reaches 15.98IU/mL;

6. The fermentation pH is suitable for a wide range, and the process control is simple.

Description of drawings

Fig. 1 is the hydrolysis circle of primary screening bacterial colony on the culture medium;

Fig. 2 is the research on the enzyme production characteristic of TP-02 bacterial strain among the embodiment 5;

Fig. 3 is the study on the enzyme production characteristics of the TP-02 strain in Example 6.

detailed description

Example 1

A screening method for producing cellulase strains, the screening method may further comprise the steps:

Primary screening:

Take 1g of the material containing the target strain (Huangshan ecological forest rotting wood) and pulverize; add 10mL of normal saline and shake for 1h. All the above suspensions were added to 200mL PDA medium, 30°C, 180rpm shaking culture for 12h;

The above cultures were diluted 10 times with normal saline to 10 ^-3 , 10 ^-4 , 10 ^-5 in sequence, and spread on PDA plate medium respectively, and cultured at 30°C until colonies appeared, and a typical single colony was selected for transformation. Connect the PDA slant, and at the same time make slices for purity inspection to obtain pure culture; plant the pure culture on the primary screening medium, the composition of the primary screening medium is: each L medium contains: CMC-Na5-10g ; Glucose 3-5g; Agar 10-20g; MandelS inorganic nutrient salt 1000mL, cultured at 28°C for 48h, stained with 1mg/mL Congo red dye solution for 2h, poured out the dye solution and then washed with 1mol/L NaOH solution Dyeing solution, inhibits enzyme activity, fixes hydrolysis circle size. If the bacteria can secrete cellulase, a clear hydrolysis zone will appear around the colony, and high-viability strains will be selected according to the ratio of the hydrolysis zone to the colony diameter (LD ratio). Select the colony with a large LD ratio and transfer it to the slope of the PDA test tube for culture and storage for future use (see Figure 1).

Protoplast preparation:

The spores of the Rhizopus stoloniferus strain preserved on the slant of the PDA test tube were eluted with physiological saline, mixed and diluted to ¹ ×105/mL; the above-mentioned spore suspension was transferred to a 500mL Erlenmeyer flask containing 100mL of PDA medium, Adjust the spore concentration to 10 ⁷ spores/mL, culture on a shaker with a rotating speed of 150r/min for 12h; centrifuge the obtained culture at 3500r/min, take the precipitate, wash twice with normal saline, centrifuge, and then wash with 0.6mol/L KCl The hypertonic solution was washed once and centrifuged to obtain the precipitate;

Use an electronic balance to accurately weigh 2g of the treated mycelium, add 20mL of 3mg/mL wall-breaking liquid, the composition of the wall-breaking liquid is helicase: cellulase=3:1, mix well, and shake at 30°C and 160rpm Incubate, observe the generation of protoplasts and count them under the microscope every 0.5 hours, determine the enzymatic hydrolysis time, filter the protoplast body fluid with four layers of sterile microscope paper, remove the mycelia fragments and take the filtrate, centrifuge at 3500r/min for 10min, and collect the protoplasts The body was washed twice with an organic sugar alcohol osmotic pressure stabilizer to obtain purified protoplasts.

Mutation breeding:

Take 5mL of the above-mentioned purified protoplasts in a sterile 9cm petri dish, and irradiate them with a 15W ultraviolet lamp at a distance of 30cm for mutagenesis. The ultraviolet lamp is preheated for 30 minutes to stabilize the ultraviolet light. On the hypertonic primary screening medium plate, the composition of the hypertonic primary screening medium plate is: each L medium contains: CMC-Na5-10g; glucose 3-5g; agar 10-20g; MandelS inorganic nutrient salt 1000mL; KCl0 .6mol; culture at a constant temperature of 30°C in the dark and away from light, and transfer to a PDA slant for culture after colonies grow on the plate;

Using the CMC-Congo red plate method, the strains with larger transparent circles and larger colony diameters were initially screened out, and the mutants with high cellulase activity were selected by shake-flask fermentation and re-screening.

Double screening:

Wash the PDA slant culture with physiological saline, mix well and dilute to 1×10 ⁵ cells/mL to prepare a spore suspension; inoculate the spore suspension into a 250mL Erlenmeyer flask containing 50mL liquid seed medium, The composition of the liquid seed medium is 10% bran extract juice (weigh 100g bran, add 800mL distilled water to boil for 30min, filter through 4 layers of gauze, and set the volume to 1000mL), adjust the spore concentration to ¹⁰⁷ /mL, and Cultivate on a shaker at 150r/min for 12h to obtain a seed culture solution;

Add about 200mL of liquid fermentation medium into a 500mL Erlenmeyer flask, insert 20mL of seed culture solution, and cultivate at 30°C and 180rpm for 12h with shaking. The composition of the liquid fermentation medium is: each L medium contains: 5-10g of 80-120 mesh corn stalks, rice straw or wheat straw, 80-100g of 5% bran extract, (NH ₄ ) ₂ SO ₄ 20- 30g, KH ₂ PO ₄ 5-10g, MgSO ₄ 7H ₂ O5-10g, CaCl ₂ 10-20g, Tween800.15-0.25g, trace elements: FeSO ₄ 7H ₂ O0.003-0.005g, MnSO ₄ H ₂ O 0.001-0.00156g, ZnSO ₄ ·7H ₂ O 0.001-0.0014g, CoCl ₂ ·6H ₂ O 0.001-0.002g, pH 5.0.

Through the determination of the yield of reducing sugar by enzymatic hydrolysis of fermentation products, the strains with high enzyme activity are screened out to enter the next round of fermentation and enzyme production conditions.

Table 1 The relationship between the size of the hydrolysis circle and the level of enzyme activity

The screened bacterial strain TP-02 was continuously subcultured for 10 generations, and its enzyme activity was measured by means of liquid fermentation, and its genetic stability was studied as shown in Table 1. It can be seen from Table 1 that the mutant strain TP-02 has been cultured for 10 generations. , the difference in FPA and CMC enzyme activities between any two generations is within 10%, indicating that the strain has stable enzyme production performance and can be used as a starting strain for subsequent experiments.

Table 2 Genetic stability of mutant strain TP-02

algebra 1 2 3 4 5 6 7 8 9 10 FPA (IU/mL) 5.2 5.3 5.1 5.2 5.0 4.8 4.9 5.3 5.2 5.2 CMC (IU/mL) 20.5 21.2 20.8 21.0 20.2 19.8 20.2 22.1 21.8 21.5

Identification of strains:

The ITS-5.8srDNA sequence of this strain was obtained by PCR and compared with the nucleotide sequence on NCBI by sequencing. The recognized standard sequence data of related strains were obtained from NCBI, and ClustalX1.8 was used for multiple analysis and comparison. Using MEGA3.1 and PAUP software, the MP and ML phylogenetic trees were constructed with Trichoderma reesei, a close relative of Rhizopus, as the outgroup. The sequence homology rate between the strains in this study and Rhizopus stoloniferum was as high as 99.8%. It is generally believed that the homology rate greater than 98% can be considered to belong to the same species. Therefore, it was identified as Rhizopus stoloniferus subspecies Rhizopus nodosa according to the molecular level. Combined with the results of morphological identification, the TP-02 strain was preliminarily identified as Zygomycotina, Phycobacteria, Mucorales, Mucoraceae, Rhizopus genus, Rhizopus stoloniferus subspecies Rhizopus nodosa, and its gene sequence table As shown in SEQUENCELISTING1.

Example 2

A screening method for producing cellulase strains, the screening method may further comprise the steps:

Others are the same as in Example 1, except that the composition of the primary screening medium is as follows: every L medium contains: CMC-Na 5g; Glucose 3g; Agar 10g; MandelS inorganic nutrient salt 1000mL;

The composition of the hyperosmotic primary screening medium plate is: each L medium contains: CMC-Na5g; glucose 3g; agar 10g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol;

The composition of the liquid fermentation medium is: Each L medium contains: 5g of 80-120 mesh corn stalks, 80g of 5% bran extract juice, (NH ₄ ) ₂ SO ₄ 20g, KH ₂ PO ₄ 5g, MgSO ₄ 7H ₂ O5g, CaCl ₂ 10g, Tween800.15g, trace elements: FeSO ₄ ·7H ₂ O0.003g, MnSO ₄ ·H ₂ O0.001g, ZnSO ₄ ·7H ₂ O0.001g, CoCl ₂ ·6H ₂ O0.001g, pH5 .0.

Example 3

A screening method for producing cellulase strains, the screening method may further comprise the steps:

Others are the same as in Example 1, except that the composition of the primary screening medium is: each L medium contains: CMC-Na 10g; Glucose 5g; Agar 20g; MandelS inorganic nutrient salt 1000mL;

The composition of the hyperosmotic primary screening medium plate is: each L medium contains: CMC-Na10g; glucose 5g; agar 20g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol;

The composition of the liquid fermentation medium is: each L medium contains: 80-120 mesh rice straw 10g, 5% bran extract juice 100g, (NH ₄ ) ₂ SO ₄ 30g, KH ₂ PO ₄ 10g, MgSO ₄ 7H ₂ O 10g , CaCl ₂ 20g, Tween800.25g, trace elements: FeSO ₄ ·7H ₂ O0.005g, MnSO ₄ ·H ₂ O0.00156g, ZnSO ₄ ·7H ₂ O0.0014g, CoCl ₂ ·6H ₂ O0.002g, pH5. 0.

Example 4

A screening method for producing cellulase strains, the screening method may further comprise the steps:

Others are the same as in Example 1, except that the composition of the primary screening medium is: each L medium contains: CMC-Na8g; glucose 4g; agar 15g; MandelS inorganic nutrient salt 1000mL;

The composition of the hyperosmotic primary screening medium plate is: each L medium contains: CMC-Na8g; glucose 4g; agar 15g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol;

The composition of the liquid fermentation medium is as follows: each L medium contains: 8g of 80-120 mesh wheat straw, 90g of 5% bran extract juice, (NH ₄ ) ₂ SO ₄ 25g, KH ₂ PO ₄ 8g, MgSO ₄ 7H ₂ O8g, CaCl ₂ 15g, Tween800.2g, trace elements: FeSO ₄ ·7H ₂ O0.004g, MnSO ₄ ·H ₂ O0.0014g, ZnSO ₄ ·7H ₂ O0.0012g, CoCl ₂ ·6H ₂ O0.0015g, pH5 .0.

Example 5

A production method of vitaminase, said method comprising the following steps:

The preparation method of seed is with embodiment 2;

Production of cellulase by liquid fermentation:

Take 5mL of seed culture solution and add it to 45mL of liquid fermentation medium. After inoculation, the amount of liquid in the base of the fermentation medium is 250mL of Erlenmeyer flask and 50mL of medium. After inoculation, they were cultured in a shaking constant temperature culture at a temperature of 30°C, a shaker speed of 200 rpm/min, and a total culture time of 4 days. During this period, 1 mL of liquid was drawn from the fermentation broth every 12 hours and its enzyme activity was measured. After 84 hours of fermentation, the cellulase filter paper enzyme activity (FPA), endonuclease activity, exonuclease activity and β-glucosidase enzyme activity in the fermentation broth reached the maximum, respectively 13.16IU/mL, 45.81 IU/mL, 0.537IU/mL, 12.45IU/mL, as shown in Figure 2.

The composition of the liquid fermentation medium is: each L medium contains: 5g of 80-120 mesh wheat straw, 100g of 5% bran extract juice, (NH ₄ ) ₂ SO ₄ 26g, KH ₂ PO ₄ 8g, MgSO ₄ · 7H ₂ O 9g, CaCl ₂ 18g, Tween 800.2g, trace elements: FeSO ₄ ·7H ₂ O0.004g, MnSO ₄ ·H ₂ O0.0015g, ZnSO ₄ ·7H ₂ O0.0013g, CoCl ₂ ·6H ₂ O0.0015g , pH5.0.

Example 6

A production method of vitaminase, said method comprising the following steps:

Seed preparation:

The strain TP-02 was cultured in PDA slant medium for 3 days, and then the spores were washed with sterile water. The number of spores was counted using a hemocytometer and diluted to 1×10 ⁵ /mL. Insert 10% of the inoculum into 200mL (500mL Erlenmeyer flask) liquid seed medium, cultivate it at 30°C and 200rpm for 24 hours, then use it as a seed liquid for later use, and store the obtained seed liquid in -80°C ultra-low temperature glycerol to freeze tube;

Take a -80°C ultra-low temperature glycerol freezing tube at room temperature to dissolve and streak the PDA slant medium, culture at 30°C for 3 days, wash the mycelia or spores with sterile water, wash each plate with 5 mL of sterile water, and pipette 2 mL for washing The mycelia suspension obtained after that is inserted into 48mL liquid seed culture medium, and the liquid volume of seed culture medium after inoculation is 250mL triangular bottle and 50mL culture medium, at 30 ℃, shaker speed is 200rpm, after constant temperature cultivation 24h, namely shaker Bottle fermented seed culture solution for enzyme production.

Production of cellulase by liquid fermentation:

Put 2L of liquid fermentation medium in a 3L fermenter, and insert 200mL of seed culture solution for fermentation. Fermentation conditions: the culture temperature is 30°C, the stirring speed is 450r/min, the ventilation rate is 4.5vvm, the tank pressure is 0.03MPa, and the pH is controlled within the range of 5.0 to 5.5 by adding ammonia water. The specific pH can be 5.0, 5.1, 5.2 , 5.3, 5.4, 5.5 these values. Under the above-mentioned culture conditions, cultured for 88 hours, during which 1 mL of liquid was drawn from the fermentation broth every 4 hours and its enzyme activity was measured. It was found that after culturing for 84 hours, the enzyme activity of the filter paper reached 16.98IU/mL, the enzyme activity of the endonuclease reached 41.33IU/mL, the activity of the exonuclease reached 3.65IU/Ml, and the activity of the glucosidase reached 15.98IU/mL, as shown in the figure 3 shown.

The composition of the liquid fermentation medium is: each L medium contains: 80-120 mesh corn stalks 8g, 5% bran extract juice 90g, (NH ₄ ) ₂ SO ₄ 20g, KH ₂ PO ₄ 5g, MgSO ₄ · 7H ₂ O 7g, CaCl ₂ 15g, Tween 800.15g, trace elements: FeSO ₄ 7H ₂ O 0.003g, MnSO ₄ H ₂ O 0.001g, ZnSO ₄ 7H ₂ O 0.001g, CoCl ₂ 6H ₂ O 0.001g , pH5.0.

Example 7

Detection method of cellulase

Determination of filter paper enzyme activity: Accurately draw 0.5mL enzyme solution diluted 10-50 times, put a 1cm×6cm filter paper in 0.05mol/L citric acid-sodium citrate buffer solution with pH4.8 and 0.5mLpH4.8 0.05mol/L citric acid-sodium citrate buffer solution, keep warm at 50°C for 30min, add 3mL DNS reagent to boiling water bath for 10min, add water to dilute to 25mL after cooling, and measure the absorbance at 520nm.

β-glucosidase: Accurately draw 0.5mL of enzyme solution diluted 10-50 times, add 1mL of 1% salicin (dissolved in 0.05mol/L citric acid-sodium citrate buffer solution with pH 4.8) And 0.5mL pH4.8 0.05mol/L citric acid-sodium citrate buffer solution, keep warm at 50°C for 30min, add 3mLDNS reagent boiling water bath for 10min, add water to dilute to 25mL after cooling, measure the absorbance at 520nm.

Endonuclease enzyme activity assay: Accurately draw 0.5mL of enzyme solution diluted 10-50 times, add 1mL of 1% CMC (dissolved in 0.05mol/L citric acid-sodium citrate buffer solution with pH 4.8) and 0.5mL pH4.8 0.05mol/L citric acid-sodium citrate buffer solution, keep warm at 50°C for 30min, add 3mLDNS reagent to boiling water bath for 10min, add water to dilute to 25mL after cooling, measure the absorbance at 520nm.

Determination of exonuclease enzyme activity: draw 0.5 mL of enzyme solution diluted 10-50 times, add 1 mL of 2% Avicelase (dissolved in 0.05 mol/L citric acid-sodium citrate at pH 4.8) Buffer solution) and 0.5mL pH4.8 0.05mol/L citric acid-sodium citrate buffer solution, incubate at 50°C for 30min, then add 3mLDNS reagent in a boiling water bath for 10min, after cooling, add water to dilute to 25mL, and measure the absorbance at 520nm.

Definition of cellulase activity unit: Under the above reaction conditions, the amount of enzyme required to hydrolyze 1 μmol of glucose per minute is 1 international unit (IU) of enzyme activity.

The above-mentioned detailed description of the screening method of cellulase strains and the method for fermenting and producing cellulase thereof with reference to the above-mentioned examples is illustrative rather than limiting, and several embodiments can be listed according to the limited scope, so Changes and modifications without departing from the general concept of the present invention shall fall within the protection scope of the present invention.

Claims (10)

1. the screening technique of a cellulase producing strain, it is characterised in that described screening technique comprises the following steps: prepared by primary dcreening operation, protoplast, ultraviolet lighting mutagenic and breeding, multiple sieve;

Described cellulase producing strain is now by China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, deposit number is No.11119, and Classification And Nomenclature is that Rhizopus stolonifer is nodded mutation Rhizopusstolonifervar.refiexus.

2. screening technique according to claim 1, it is characterised in that described screening technique specifically includes following steps:

A. primary dcreening operation culture medium primary dcreening operation, the composition of described primary dcreening operation culture medium is: every L culture medium contains: CMC-Na5-10g; Glucose 3-5g; Agar 10-20g;MandelS inorganic nutrient salt 1000mL;

B. preparing protoplast, the spore of the bacterial strain obtained by primary dcreening operation is cultivated in PDA culture medium, the mycelia oscillation incubation in shell-broken liquid obtained after purified, centrifugal, obtains protoplast after filtration, centrifugal purification; The composition of described shell-broken liquid is Snailase: cellulase=3:1;

C. mutagenic and breeding, the protoplast 15W ultra violet lamp obtained by step B carries out mutagenic treatment, and treatment fluid is coated height and oozed on primary dcreening operation culture medium flat plate, and after cultivation, bacterium colony is transferred to the cultivation of PDA slant medium;

Described height oozes the composition of primary dcreening operation culture medium flat plate: every L culture medium contains: CMC-Na5-10g; Glucose 3-5g; Agar 10-20g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol;

D. sieve again; The spore suspension of the culture on PDA slant medium obtained by step C is inoculated in liquid seed culture medium and cultivates, and seed culture fluid is inoculated in liquid fermentation culture medium by 1:10 volume ratio and cultivates, cross the mensuration to tunning enzymolysis reducing sugar yield, filter out the bacterial strain that enzymatic activity is high, be cellulase producing strain TP-02.

3. screening technique according to claim 2, it is characterised in that the composition of described liquid seed culture medium is: 10% wheat bran diffusion juice.

4. screening technique according to claim 2, it is characterised in that the composition of described liquid fermentation culture medium is: every L culture medium contains: the corn straw of 80��120 orders, Caulis et Folium Oryzae or straw 5-10g, 5% wheat bran diffusion juice 80-100g, (NH₄)₂SO₄20-30g, KH₂PO₄5-10g, MgSO₄��7H₂O5-10g, CaCl₂10-20g, Tween800.15-0.25g, trace element: FeSO₄��7H₂O0.003-0.005g, MnSO₄��H₂O0.001-0.00156g, ZnSO₄��7H₂O0.001-0.0014g, CoCl₂��6H₂O0.001-0.002g, pH5.0.

5. the bacterial strain that screening technique according to claim 1 screening obtains is as the application producing dimension element enzyme.

6. the production method tieing up element enzyme, it is characterised in that said method comprising the steps of:

(1) use the bacterial strain TP-02 that screening technique according to claim 1 screening is obtained by liquid seed culture medium to carry out seed culture, obtain seed culture fluid;

(2) seed culture fluid is inoculated in liquid fermentation culture medium and cultivates, liquid fermentation cellulase-producing.

7. production method according to claim 6, it is characterized in that, described step (1) specifically includes following steps: the bacterial strain TP-02 that screening technique according to claim 1 screening is obtained cultivates through PDA slant medium, liquid seed culture medium after first, and gained seed liquor is saved in-80 DEG C of ultralow temperature glycerol freezing pipes;

The seed liquor preserved in-80 DEG C of ultralow temperature glycerol freezing pipes be can be prepared by seed culture fluid through PDA slant medium, liquid seed culture medium after cultivating after first.

8. production method according to claim 6, it is characterised in that in described step (2), the ratio of seed culture fluid and the volume of liquid fermentation culture medium is 1:9��10.

9. production method according to claim 6, it is characterised in that the condition of culture in described step (2) is 30 DEG C, 200��350r/min cultivates 84��96 hours.

10. production method according to claim 9, it is characterised in that the condition of culture in described step (2) also includes: in fermentation tank, ventilation is 4.5vvm, tank pressure is 0.03MPa, pH is 5.0��5.5 scopes.