CN104498365A - Bacterial strain capable of producing chitin deacetylase and application of bacterial strain in production of chitin deacetylase through fermentation - Google Patents

Abstract

The invention provides a chitin deacetylase-producing strain and its application in the production of chitin deacetylase by fermentation. The strain is Aspergillus versicolor X, which has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2014459 , the date of deposit is October 8, 2014. At the same time, it also provides a method for fermenting and producing enzyme by using the bacterium, and the enzyme activity of the prepared chitin deacetylase can reach 1.41U/mL. The invention not only expands the range of chitin deacetylase-producing microorganisms, but also reduces the production cost, and has relatively broad application prospects.

Description

A chitin deacetylase-producing strain and its application in the production of chitin deacetylase by fermentation

technical field

The invention discloses a strain producing chitin deacetylase and a method for producing chitin deacetylase by fermenting the strain, belonging to the field of biotechnology.

Background technique

Chitin is the second largest biological polysaccharide next to cellulose on the earth, and it is also the only natural alkaline polysaccharide that exists in large quantities on the earth. It is widely found in the shells of insects, crustaceans, shrimps and crabs, and fungi ( Yeast, mold) and plant cell walls. The annual biosynthesis of chitin is about 10 billion tons.

Chitin is a linear polymer connected by N-acetylamino-D-glucose through β-(1,4) glycosidic bonds. There are a large number of hydrogen bonds in the molecule, and its solubility is very poor. Chitosan is the product of partial or complete removal of the acetylamino group in chitin molecules. Due to the presence of a large number of free amino groups in its molecule, the molecule is positively charged, its chemical properties are active, and its solubility has been greatly improved. Therefore, it is widely used in China. The application outside is very extensive. Such as: used as a sewage treatment agent, food thickener and preservative, degradable packaging materials, ultrafiltration membranes, etc.; can also be used as immobilized carriers of enzymes and cells, slow-release materials, etc. In addition, chitosan has received more and more attention in the field of medicine. It can be used to make artificial skin and added to health products as dietary fiber, which can reduce blood fat and promote bone growth. At present, the production of chitosan mainly adopts the high-temperature concentrated alkali method, but this method has great environmental pollution, and at the same time, there are problems such as uneven deacetylation degree of the product, large molecular weight variation, and high production cost.

Chitin deacetylase (chitin deacetylase, E.C.3.5.1.41, referred to as CDA) can catalyze the hydrolysis of the acetylamino group in the chitin molecule, and convert chitin into chitosan. Using it to replace the existing concentrated alkali method to produce chitosan can not only solve the environmental problems existing in chitosan production, but also obtain high-quality chitosan with narrow molecular weight distribution range and uniform acetylation. Since Araki et al. first discovered chitin deacetylase from Mucor rouxii in 1974, researchers have successively discovered the existence of the enzyme from various bacteria, molds and Saccharomyces cerevisiae. But the chitin deacetylase produced by most of the strains is an intracellular enzyme, and the enzyme activity and yield are low, which will affect the application of the enzyme in the chitin biotransformation process.

Contents of the invention

The purpose of the present invention is to provide a chitin deacetylase-producing bacterial strain Aspergillus versicolor X in order to solve the deficiencies in the prior art, and also provide a method for producing enzyme by fermentation of Aspergillus versicolor X.

The technical scheme that the present invention realizes above-mentioned purpose is:

Aspergillus versicolor X is obtained from the chitin production waste soil of a biological product factory in Shandong as raw material, and is screened and separated by serial dilution coating plate method, in which 4-nitroacetanilide is used as the color reagent, and then obtained by separation and purification. It was deposited in the China Center for Type Culture Collection, referred to as CCTCC, on October 8, 2014, with the preservation number CCTCC NO: M2014459, and the preservation address is Wuhan University, Wuhan, China (zip code 430072). The specific screening steps are as follows:

(1) Primary screening and purification of strains

The raw material (chitin production waste soil) was added to sterile physiological saline in an amount of 10% (w/v), shaken for 20 minutes, left to stand for 15 minutes, and the supernatant was serially diluted and coated on a solid screening medium. The medium composition is colloidal chitin 20g/L, dipotassium hydrogen phosphate 0.7g/L, potassium dihydrogen phosphate 0.3g/L, magnesium sulfate 0.5g/L, sodium chloride 0.1g/L, 4-nitroacetanilide (PN) 0.4g/L, agar 20g/L. Incubate at a constant temperature of 30°C for 60h.

In the solid screening medium, yellow single colonies appearing around the colonies were picked, and they were separated by streaking in the solid screening medium, and the characteristics of the single colonies were consistent after continuous isolation.

(2) Re-screening of strains

Inoculate the strains obtained from the preliminary screening into the liquid medium respectively, the composition of the medium is the same as that of the primary screening medium (without adding PN, agar), culture at 30°C and 160rpm for 72 hours at a constant temperature, separate and collect the supernatant (crude enzyme) to test the enzyme live. Duplicate 2 bottles of each bacteria. Select the strain with the highest enzyme-producing activity.

Determination of CDA activity: determined by MBTH method. Add 100μl 50mM pH7.0 PBS buffer solution to the test tube, then add 100μl aqueous solution containing 100μg N-acetyl chitosan and 50μl centrifuged fermentation supernatant, react at 30°C for 30min, add 250μl 5% KHSO ₄ to terminate reaction. Add 250 μl 5% NaNO ₂ to the reaction solution, shake intermittently for 15 minutes, then add 250 μl 12.5% ammonium sulfamate, add 250 μl 0.5% MBTH after 5 minutes, heat in boiling water for 3 minutes, add 250 μl 0.5% FeCl ₃ after cooling , Measure the absorbance at 650 nm after 30 min. The standard curve was made with standard glucosamine hydrochloride. One unit of enzyme activity is defined as: the amount of enzyme required to produce 1 μg of glucosamine per minute with N-acetylchitosamine as a substrate is one unit.

(3) Identification of strains

Molecular biological identification was entrusted to Guangdong Microbial Analysis and Testing Center. The DNA sequence is shown in Seq ID No:1.

The obtained 18S rDNA sequences were compared by BLAST and a phylogenetic tree was constructed, as shown in Figure 1. It can be seen from the figure that the 18S rDNA nucleotide sequence of Aspergillus sp.X has a high homology (99%) with Aspergillus versicolor HDJZ-ZWM-16. It can be considered that Aspergillus sp.X belongs to Aspergillus versicolor.

The identification of morphological characteristics found that the strains grew slowly on Chapei's medium, and the diameter of the colony was 12-17mm at 25°C for 7 days. green. After growing on the wort medium for 7 days at 25°C, the diameter is 16-22mm, relatively flat, with many conidia structures, olive green, and reddish brown on the back. The conidia are spherical at first, and then radial; the molecular spore stem is short, colorless, thick-walled; the apical capsule is hemispherical, 12-15 μm in diameter; the sporulation structure is double-layered.

According to the results of molecular identification, morphological characteristics, and cultural characteristics, the strain X was Aspergillus versicolor or its subspecies.

The application of the above-mentioned strains in the production of chitin deacetylase by fermentation, specifically, after the activation of Aspergillus versicolor (Aspergillus versicolor) X, its spores are made into a spore suspension and inoculated into the fermentation medium to make it in the initial stage of the medium. The pH is 5.0-9.0, the temperature is 25-35° C., and the rotation speed is 120-200 rpm on a shaker for 72-100 hours of fermentation. After separation, the supernatant is taken to obtain the crude chitin deacetylase enzyme liquid.

The initial pH value of the fermentation medium is 6.0-7.0, the culture temperature is 28-30° C., and the rotation speed is 160-200 rpm. The culture time is 90-96h.

The spore suspension is 10 ⁶ spores/mL, and is inoculated into the fermentation medium with an inoculation amount of 2-6%.

The fermentation medium comprises the following components:

The carbon source is one or more of glucose, sucrose, xylose, lactose, maltose and soluble starch; preferably sucrose with a concentration of 6-12g/L.

The nitrogen source is one or more of yeast extract, tryptone, soybean peptone, bacteriological peptone, urea, ammonium nitrate, ammonium chloride and ammonium sulfate; preferably yeast extract and urea, the concentration is 8-16g/ L, 1-4g/L.

The inorganic salt is one or more of magnesium sulfate, manganese sulfate, calcium chloride, ferrous chloride, zinc sulfate and copper sulfate. It is preferably manganese sulfate with a concentration of 0.3-0.7g/L.

The components of the fermentation medium include 6-12g/L of sucrose, 0.3-0.7g/L of manganese sulfate, 2.0-12.5g/L of yeast extract and 1-3g/L of urea.

The fermentation medium also includes 0.5-3 g/L dipotassium hydrogen phosphate.

The composition of the fermentation medium is 10 g/L of sucrose, 10 g/L of yeast extract, 2 g/L of urea, 0.5 g/L of manganese sulfate, and 1 g/L of dipotassium hydrogen phosphate.

The present invention has following beneficial effect:

(1) The invention provides a chitin deacetylase-producing strain Aspergillus versicolor, and it is first discovered that Aspergillus versicolor can produce chitin deacetylase through fermentation, and the enzyme activity in the culture supernatant can reach 1.41U/mL. It not only expands the scope of chitin deacetylase-producing microorganisms (it has been reported that chitin deacetylase-producing microorganisms are mainly concentrated in Rhizopus sp., Colletotrichum sp.), but also increases the types of recalcitrant biomass that Aspergillus versicolor can hydrolyze (removing lignin In addition, chitin was added).

(2) The present invention provides a culture medium that can be fermented by Aspergillus versicolor X CCTCC M2014459 to produce enzymes through culture medium optimization, which further improves the production of extracellular enzymes. The culture medium has low cost and is the industrialization of Aspergillus versicolor X. Application provides favorable conditions.

Description of drawings

Figure 1 is a phylogenetic tree of Aspergillus versicolor X and selected strains.

Figure 2 is a microscopic view (400X) of Aspergillus versicolor (Aspergillus versicolor) X spore peduncle.

Figure 3 is the morphology of Aspergillus versicolor (Aspergillus versicolor) X growing on Chapei's medium.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto.

Example 1

Screening of enzyme-producing strain Aspergillus versicolor X

Primary screening of enzyme-producing strains: Weigh 10g of soil collected from chitin production waste, add 90mL of sterile water containing glass beads, shake at 200rpm for 30min, and after standing for 15min, take 0.5ml of the supernatant and add it to 4.5ml of sterile water, followed by Gradually dilute to 10 ^-7 , take 200 μL respectively, and apply to solid screening medium. The solid screening medium was incubated at a constant temperature of 30°C for 4 days, and a single colony that turned yellow around the colony in the medium was picked, and the method of streak separation was used to continue purification, and repeated 3 times until the characteristics of the colonies were consistent. 27 strains producing chitin deacetylase were screened out, including 1 strain of yeast, 6 strains of bacteria and 20 strains of mold. Inoculate the completely purified single colony on the slant medium to grow for a suitable time, and store it at 4°C for later use.

Re-screening of enzyme-producing strains: Shake flask re-screening of the strains that passed the primary screening on the plate. YPD medium was used to prepare seed solution for yeast, LB medium was used for bacteria to prepare seed solution, and sterile saline was used to prepare spore suspension for mold. The seeds were inoculated into 250mL Erlenmeyer flasks (containing 50mL liquid fermentation medium, composed of colloidal chitin 20g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1g/L, magnesium sulfate 0.3 g/L, sodium chloride 0.1g/L), 30°C, 160rpm constant temperature culture for 72h, and isolate the bacteria. The activities of extracellular enzymes and intracellular enzymes were measured respectively. Among them, the extracellular enzyme activity produced by Aspergillus versicolor X was the highest, up to 0.69U/mL.

Example 2

Carbon source optimization experiment of Aspergillus versicolor (Aspergillus versicolor) X-producing chitin deacetylase medium. Glucose, sucrose, xylose, lactose, maltose and soluble starch were respectively selected as the carbon source of the medium, the concentration was 8g/L, and the other components were 5g/L of bactochemical peptone, 1g/L of dipotassium hydrogen phosphate, and 0.5 g of magnesium sulfate. g/L, the initial pH of the medium was 6.0. The spore suspension of the strain was inoculated into the fermentation medium at an inoculation ratio of 6%, cultured at a constant temperature of 30° C. and 160 rpm for 96 hours, and the mycelium was separated by filtration, and the supernatant was the extracellular enzyme. The mycelium was crushed with a mortar, dissolved in 50mM PBS buffer, centrifuged at 12000rpm at 4°C, the supernatant was taken as intracellular enzymes, and the enzyme activities of intracellular enzymes and extracellular enzymes were measured respectively. The wet cells were dried at a constant temperature of 105°C to constant weight, and the biomass was measured. The results are shown in Table 1 below.

Table 1

Among them, when the carbon source is sucrose, the activity of extracellular enzyme is the highest, which is 0.62U/mL, and the activity of intracellular enzyme is 0.34U/mL, and the biomass can reach 6.67g/L.

Example 3

Nitrogen source optimization experiment of Aspergillus versicolor (Aspergillus versicolor) X chitin deacetylase production medium. Yeast extract, tryptone, soybean peptone, bacteriological peptone, urea, ammonium nitrate, ammonium chloride and ammonium sulfate were respectively selected as the nitrogen source of the medium, the nitrogen content was 1g/L, and the other components were glucose 10g/L. Dipotassium hydrogen phosphate 1g/L, magnesium sulfate 0.5g/L, and the initial pH of the medium was 6.0. The spore suspension of the strain was inoculated into the fermentation medium at an inoculation ratio of 6%, cultured at a constant temperature of 30° C. and 160 rpm for 96 hours, and the mycelium was separated by filtration, and the supernatant was the extracellular enzyme. The mycelium was crushed with a mortar, dissolved in 50mM PBS buffer, centrifuged at 12000rpm at 4°C, the supernatant was taken as intracellular enzymes, and the enzyme activities of intracellular enzymes and extracellular enzymes were measured respectively. The wet cells were dried at a constant temperature of 105°C to constant weight, and the biomass was measured. The results are shown in Table 2 below.

Table 2

Among them, when the nitrogen source is yeast extract, the extracellular enzyme activity is the highest, which is 1.31U/mL, the intracellular enzyme activity is 0.09U/mL, and the biomass can reach 8.10g/L. It can be seen that the influence of nitrogen source on enzyme production is greater than that of carbon source.

Example 4

The screening experiment of inorganic salt in Aspergillus versicolor (Aspergillus versicolor) X production medium of chitin deacetylase. Magnesium sulfate, manganese sulfate, calcium chloride, ferrous chloride, zinc sulfate and copper sulfate were respectively selected as medium inorganic salts, the concentration was 0.4g/L, other components were glucose 8g/L, yeast extract 2g/L , dipotassium hydrogen phosphate 1g/L, and the initial pH of the medium was 6.5. The spore suspension of the strain was inoculated into the fermentation medium at an inoculation ratio of 6%, cultured at a constant temperature of 30° C. and 160 rpm for 96 hours, and the mycelium was separated by filtration, and the supernatant was the extracellular enzyme. The mycelium was crushed with a mortar, dissolved in 50mM PBS buffer, centrifuged at 12000rpm at 4°C, the supernatant was taken as intracellular enzymes, and the enzyme activities of intracellular enzymes and extracellular enzymes were measured respectively. The wet cells were dried at a constant temperature of 105°C until constant weight, and the biomass was measured. Among them, when manganese sulfate is used as inorganic salt, the extracellular enzyme activity is the highest, which is 0.44U/mL, the intracellular enzyme activity is 1.21U/mL, and the biomass can reach 8.8g/L.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. A strain producing chitin deacetylase bacterial strain, is characterized in that, this bacterial strain is Aspergillus versicolor X, and described bacterial strain has been preserved in China Center for Type Culture Collection, preservation number is CCTCC NO:M2014459, preservation date is 2014 October 8. 2. the application of bacterial strain described in claim 1 in fermenting and producing chitin deacetylase. 3. application according to claim 1, is characterized in that, after Aspergillus versicolor (Aspergillus versicolor) X is activated, its spore is made into spore suspension and inoculated in fermentation medium, initial pH is 5.0-9.0, The temperature is 25-35 DEG C and the rotating speed is 120-200rpm on a shaker to ferment for 72-100h, and the chitin deacetylase crude enzyme liquid is obtained through separation. 4. The application according to claim 3, characterized in that the initial pH value of the fermentation medium is 6.0-7.0, the culture temperature is 28-30° C., and the rotation speed is 160-200 rpm. 5. The application according to claim 3, characterized in that the incubation time is 90-96h. 6 . The application according to claim 3 , characterized in that, the spore suspension is 10 ⁶ spores/mL, and is connected to the fermentation medium with an inoculum size of 2-6%. 7. according to the application described in claim 3 or 4 or 5 or 6, it is characterized in that, described fermentation medium comprises following components: The carbon source is one or more of glucose, sucrose, xylose, lactose, maltose and soluble starch; The nitrogen source is one or more of yeast extract, tryptone, soybean peptone, bactopeptone, urea, ammonium nitrate, ammonium chloride and ammonium sulfate; The inorganic salt is one or more of magnesium sulfate, manganese sulfate, calcium chloride, ferrous chloride, zinc sulfate and copper sulfate. 8. application according to claim 7, is characterized in that, the composition of described fermentation medium comprises: sucrose 6-12g/L, manganese sulfate 0.3-0.7g/L, yeast extract 2.0-12.5g/L and Urea 1-3g/L. 9. application according to claim 8, is characterized in that, also comprises the dipotassium hydrogen phosphate of 0.5-3g/L in the described fermentation medium. 10. The application according to claim 9, wherein the composition of the fermentation medium is 10g/L of sucrose, 10g/L of yeast extract, 2g/L of urea, 0.5g/L of manganese sulfate, dihydrogen phosphate Potassium 1g/L.