CN101792717B - Thermophilic acid-resisting beta-glucosaccharase producing strain and preparation method thereof - Google Patents

Abstract

The invention discloses a thermophilic acid-resistant β-glucosidase-producing bacterium and a preparation method thereof. The strain (CCTCC M 209312) is isolated from spoiled corn stalks. The screening method is to collect suitable spoiled corn stalks, cut After being diluted with sterile water, it undergoes cellobiose induction and enrichment culture, gradient concentration is diluted and coated on a screening plate, and the colony with a large transparent circle is selected and saved as a primary screening, and after multiple primary screenings; the steam-exploded corn stalk powder induction medium is used After cultivation, the enzyme activity is measured for re-screening, and the supernatant of the screened strain fermentation liquid can be used as a liquid enzyme preparation after centrifugation, or can be spray-dried to obtain a solid enzyme preparation, which can adapt to various acidic and high-heat industrial conditions. The present invention screens a new bacterial strain from rotten corn stalks through biotechnology, and uses cheap medium such as steam-exploded straw powder to produce thermophilic and acid-resistant β-glucosidase, which is applied to cellulose degradation systems, food, Textile, paper and other related industries.

Description

A thermophilic acid-resistant β-glucosidase-producing bacterium and its preparation method

Technical field:

The present invention relates to a thermophilic acid-resistant β-glucosidase producing bacterium and a preparation method thereof, and specifically provides a method for screening and fermenting a thermophilic acid-resistant β-glucosidase producing bacterium and utilizing the produced bacterium The practical application of enzymes in industrial processes belongs to the related fields of microbial fermentation and its product utilization.

Background technique:

β-glucosidase (EC3.2.1.21), also known as β-D-glucoside hydrolase, can hydrolyze the non-reducing β-D-glucosidic bond bound to the terminal and release β-D-glucose at the same time and the corresponding ligands. It widely exists in many plants, insects, yeasts, Aspergillus, Trichoderma and bacteria in nature. It participates in the glucose metabolism of organisms and plays an important role in maintaining the normal physiological functions of organisms. β-glucosidase is also very important in biotechnology applications and biotransformation. In the process of hydrolyzing cellulose into reducing sugars, relying on the synergy of endo-cellulase, exo-cellulase and β-glucosidase However, β-glucosidase is the rate-limiting enzyme in this process, and the low activity of β-glucosidase will cause a large accumulation of cellobiose. Strong inhibitory effect, so the nature and enzyme activity of β-glucosidase determine the overall enzyme activity in the process of cellulose hydrolysis. Most of the reported β-glucosidase pIs are in the acidic range, and the optimum pH value is generally between 3.5 and 5.5, with pH 4.5 being the majority. Rarely below pH 3.5. However, many biological applications need to be carried out in an acidic environment, such as steam-exploded straw powder after acid treatment, textile and paper industry, and industrial and agricultural waste and residue treatment. The requirements on the environment are very high, and the reaction needs to be carried out under the conditions of acid resistance and high temperature. Simultaneously, due to the restriction of production strain and production conditions, the price of commercialized β-D-glucoside hydrolase is very high, there is no way to realize large-scale industrialized use, and the task of the present invention is to find a kind of β-D-glucoside hydrolase that can be produced with cheaper raw materials An excellent strain of thermophilic acid-resistant β-glucosidase.

Invention content:

The purpose of the present invention is to provide a strain for producing thermophilic acid-resistant β-glucosidase, which is a new bacterial strain.

The invention discloses a preparation method of the above-mentioned bacteria and a culture medium used therefor,

The invention discloses a fermentation method of the above bacteria,

A thermophilic acid-resistant β-glucosidase-producing bacterium disclosed by the present invention has a preservation number of CCTCC M 209312 and is named Tolypocladium cylindrosporum syzx4. According to the taxonomy of fungi and the phylogenetic tree identified, the strain belongs to Ascomycota (Ascomycota), Pezizomycotina, Hypocreales, Tolypocladium cylindrosporum.

The preparation method of the thermophilic acid-resistant β-glucosidase producing bacteria of the present invention is characterized in that:

Cut the rotten part of corn stalks into small pieces and pulverize them in sterilized water, and insert the obtained bacterial solution into a medium containing cellobiose as a carbon source for enrichment culture. The culture conditions are: 50°C, 120rpm, 3-5d; After the enrichment culture, the bacterial solution was diluted with sterilized water in equal proportions and then coarsely sieved for culture. After 24 hours of culture at 30°C, a single colony with a black transparent circle was selected for purification and stored; the strain was fermented with a double-sieve medium, 30°C, 120rpm , cultured for 5-8 days, centrifuged to take the supernatant, and selected the strain with higher β-glucosidase activity and stored it on a PDA slant.

In the above preparation method, the enrichment medium used is composed of peptone 2.5g/L, cellobiose 2.5g/L, (NH ₄ ) ₂ SO ₄ 1g/L, KH ₂ PO ₄ 0.5g/L, MgSO ₄ 0.2g /L composition.

In the above preparation method, the coarse sieving medium used is composed of yeast powder 2.5g/L, peptone 2.5g/L, cellobiose 0.25g/L, (NH ₄ ) ₂ SO ₄ 1g/L, KH ₂ PO ₄ 0.5g /L, MgSO ₄ 0.2g/L, agar 15-20g/L, 1g/L aescin, 2.5g/L ferric ammonium citrate.

In the above preparation method, the double sieving medium used is as follows: 20 g of steam-exploded corn stalk powder and 5 g of bean cake powder are added to 1 L of Mandels nutrient salt solution.

The fermentation method of the thermophilic and acid-resistant β-glucosidase-producing bacteria of the present invention comprises culturing the bacteria of the present invention in a fermentation medium, and the culture conditions are: 120 rpm, 30° C., and culture for 8 days. The fermentation medium consists of steam-exploded straw powder 25.72g/L, soybean meal 6.82g/L, KH ₂ PO ₄ 1.90g/L, (NH ₄ ) ₂ SO ₄ 3.21g/L, (H ₂ N) ₂ CO 0.3g/L, MgSO ₄ 7H ₂ O 0.3g/L, CaCl ₂ 0.3g/L, FeSO ₄ 7H ₂ O 0.005g/L, MnSO ₄ H ₂ O 0.0016g/L, ZnSO ₄ 7H ₂ Composed of O 0.0014g/L and CoCl ₂ 0.002g/L.

The statistical method disclosed by the present invention is to the analysis and the result of the optimization of bacterial strain enzyme production condition:

Firstly, a single factor design was used to optimize the appropriate industrial and agricultural wastes including: carbon sources (wheat bran, steam-exploded straw, untreated straw, and wheat straw) and nitrogen sources (corn steep liquor, distiller's grains, soybean meal, and soybean meal) respectively, and the above components After drying, the fine powder obtained by passing through a 40-mesh sieve was used as the medium component. The results showed that the carbon source with high enzyme activity was steam-exploded straw, and the nitrogen source was soybean meal; on this basis, the Plackett-Burman design method was used to determine the The factors with greater enzyme production activity were studied, and the results showed that the factors with greater influence and their order were: steam-exploded straw powder > KH ₂ PO ₄ > soybean meal = (NH ₄ ) ₂ SO ₄ ; The composition was further optimized. Through SAS software analysis and Matlab7.0 drawing, it can be known that the influence of medium composition on enzyme production is not a simple linear relationship, and the square term and interaction term are relatively strong. The effects of steam-exploded straw powder and (NH ₄ ) ₂ SO ₄ on enzyme production were significant, and the square terms of steam-exploded straw powder, KH ₂ PO ₄ , soybean meal and (NH ₄ ) ₂ SO ₄ also had significant effects on enzyme activity , the synergistic interaction between steam-exploded straw powder and (NH ₄ ) ₂ SO ₄ also had a significant effect on enzyme activity. After statistical analysis, the best enzyme production conditions are: steam-exploded straw powder 25.72g/L, bean curd 6.82g/L, KH ₂ PO ₄ 1.90g/L, (NH ₄ ) ₂ SO ₄ 3.21g/L , other medium components remained unchanged, the culture conditions were: 120rpm, 30°C, cultured for 8 days, and the enzyme activity obtained at this time was 2.21U/mL.

The invention discloses a preparation method of a β-glucosidase preparation as follows:

After the bacterial strain Tolypocladium cylindrosporum syzx4 (CCTCC M 209312) obtained by screening is fermented, the fermentation liquid is centrifuged at 6000rpm for 10min, and the supernatant and the bacteria are separated. The clarified supernatant obtained can be used as a β-glucosidase enzyme preparation or can be sprayed Dried to make powder enzyme preparation. The β-glucosidase enzyme preparation can be applied to β-glucosidase activity supplementation in cellulose degradation systems, food, textile, papermaking and other related industrial processes.

The positive effects of the present invention are: screening a new bacterial strain from rotten corn stalks through biotechnology, using cheap medium such as steam-exploded straw powder to produce β-glucosidase with thermophilic and acid resistance, and applying it to cellulose Degradation system, food, textile, paper and other related industrial fields.

Description of drawings

Fig. 1 is the black transparent circle of screening bacterial strain of the present invention and control bacterial strain picture;

(1) strain syzx 1; (2) strain syzx 2; (3) strain syzx 3; (4) strain syzx4; (5) control strain, Aspergullus niger (CICC 40613).

Fig. 2 is the response surface and contour plot of the effects of steam-exploded straw powder (xl) concentration and (NH ₄ ) ₂ SO ₄ (x4) concentration on β-glucosidase activity (Y).

Figure 3 is a table showing the influence of the pH value on the activity of β-glucosidase in the range of 2.0 to 5.8.

Take pH 4.4 as 100% enzyme activity.

Figure 4 is a table showing the influence of reaction temperature on the activity of β-glucosidase at 30-90°C.

Enzyme activity at 60°C is 100%

Detailed ways:

Example 1

Strain screening method

Collect the rotten parts of corn stalks that have accumulated for many years, cut them into small pieces with scissors, break them with glass beads in 100mL sterilized water, and insert the obtained bacterial liquid into the medium containing only cellobiose as carbon source for 50 days. ℃ enrichment culture, the enrichment medium is: peptone 2.5g/L, cellobiose 2.5g/L, (NH ₄ ) ₂ SO ₄ 1g/L, KH ₂ PO ₄ 0.5g/L, MgSO ₄ 0.2g/L L. The culture conditions are: 50°C, 120rpm, enrichment culture for 3~5 days; dilute the obtained bacterial solution by ^10-1 ~ ^10-6 times, take 100μL and spread it on the coarse sieve plate, the coarse sieve medium is: Yeast Powder 2.5g/L, peptone 2.5g/L, cellobiose 0.25g/L, (NH ₄ ) ₂ SO ₄ 1g/L, KH ₂ PO ₄ 0.5g/L, MgSO ₄ 0.2g/L, agar 15~ 20g/L, add filtered 1g escin and 2.5g ferric ammonium citrate solution after sterilization, incubate at 30°C for 24 hours, put the plate in the refrigerator for 1 hour to develop color, observe the formation of transparent circles, and select larger black ones The single colony in the transparent circle (Fig. 1) was purified and preserved; the second screening was carried out by measuring the activity of glucosidase produced by the strain, and the original medium for the second screening was: steam-exploded corn stalk powder 20g/L, bean cake powder 5g/L plus Mandels Nutrients ((NH ₄ ) ₂ SO ₄ 3.5g/L, KH ₂ PO ₄ 2g/L, (H ₂ N) ₂ CO 0.3g/L, MgSO ₄ 7H ₂ O 0.3g/L, CaCl ₂ 0.3g /L, FeSO ₄ ·7H ₂ O 0.005g/L, MnSO ₄ ·H ₂ O 0.0016g/L, ZnSO ₄ ·7H ₂ O 0.0014g/L, CoCl ₂ 0.002g/L), 30℃, 120rpm, culture After 5-8 days, centrifuge to take the supernatant and measure its β-glucosidase activity with the standard method pNPG method, and select the strain with higher β-glucosidase activity for preservation, which can be compared and screened repeatedly. Get suitable strains and use PDA slant for preservation.

Test example 1

Fermentation of strains to produce β-glucosidase Fermentation

Apply statistical methods to optimize the fermentation conditions of the strains identified in Example 1, and perform statistical analysis on the model fitted by RSM, and use Matlab7.0 to make a graph. Figure 2 shows the steam-exploded straw powder and (NH ₄ ) ₂ SO ₄ The effect of interaction on enzyme activity, from the figure, it can be seen that the concentration of steam-exploded straw powder and (NH ₄ ) ₂ SO ₄ has a significant impact on the activity of β-glucosidase during fermentation, and the interaction is significant. The optimal enzyme production conditions obtained by the statistical analysis method are: the fermentation medium is composed of steam-exploded straw powder 25.72g/L, soybean meal 6.82g/L, KH ₂ PO ₄ 1.90g/L, (NH ₄ ) ₂ SO ₄ 3.21g/L, (H ₂ N) ₂ CO 0.3g/L, MgSO ₄ 7H ₂ O 0.3g/L, CaCl ₂ 0.3g/L, FeSO ₄ 7H ₂ O 0.005g/L, MnSO ₄ H ₂ O 0.0016g/L, ZnSO ₄ ·7H ₂ O 0.0014g/L, CoCl ₂ 0.002g/L. The culture conditions are: 120rpm, 30°C, cultured for 8 days, and the enzyme activity obtained at this time is 2.21U/mL.

Test example 2

Determination of β-glucosidase activity in fermentation broth of strain Tolypocladium cylindrosporum syzx4 (CCTCC M 209312)

After culturing with the medium and culture conditions obtained in Test Example 1, centrifuge at 6000 rpm for 10 min to obtain a clarified fermentation supernatant as a crude enzyme liquid. Use p-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate for the determination of β-glucosidase activity. The reaction system was 1ml of enzyme solution containing 0.9ml of 1mM pNPG and 0.1ml of enzyme solution appropriately diluted with citrate buffer (100mM, pH 4.5), and reacted at 60°C for 30min. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol p-nitrophenol from the substrate per minute.

It was determined that the enzyme activity of the fermented broth after centrifugation reached 2.21U/mL. The fermentation liquid of the strain has strong β-glucosidase activity.

Test example 3

Study on the properties of β-glucosidase produced by the strain

The properties of β-glucosidase obtained by fermentation and centrifugation under the optimal fermentation conditions were analyzed, and the changes in enzyme activity were explored in the range of pH value from 2.0 to 5.8 (Figure 3) and temperature from 30 to 90 °C (Figure 4).

It can be seen from Figure 3 that the activity of this enzyme in the acidic pH range is higher than that in the neutral region, and the activity is the highest at pH 2.6, which is 124% of the standard method, an increase of 24%. However, high activity was retained. Figure 4 shows that the activity of this enzyme reaches the highest at 60°C, and retains 89.88% of its activity at 70°C, and retains about 90% of its activity in the range of 30°C to 70°C. The results show that the separation is obtained The β-glucosidase produced by the strain has strong thermophilic and acid resistance properties, and can be applied to a variety of treatment methods, especially the blasted straw powder after acid treatment, the textile and paper industry, and the treatment of industrial and agricultural waste and residues, etc. application in industrial production.

Claims (2)

1. A bacterial strain producing thermophilic and acid-resistant β-glucosidase, which is classified as Tolypocladium cylindrosporum syzx4 and has been preserved in the China Center for Type Culture Collection with a registration number of CCTCC No: M 209312. 2. the fermentation method that bacterial strain described in claim 1 prepares β-glucosidase is characterized in that: Cultivate the bacterial strain of claim 1 in a fermentation medium under the following conditions: 120rpm, 30°C, and cultivate for 8 days; The fermentation medium is composed of 25.72g/L steam-exploded straw powder, 6.82g/L bean curd, 1.90g/L KH ₂ PO ₄ , 3.21g/L (NH ₄ ) ₂ SO ₄ , (H ₂ N) ₂ CO 0.3g/L, MgSO ₄ 7H ₂ O 0.3g/L, CaCl ₂ 0.3g/L, FeSO ₄ 7H ₂ O 0.005g/L, MnSO ₄ H ₂ O 0.0016g/L, ZnSO ₄ 7H ₂ O 0.0014g/L, CoCl ₂ 0.002g/L.

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