CN108004186A - A kind of bacterial strain and application produced hydrolyzable and prepare high polymerization degree XOS zytases - Google Patents

Abstract

In the present invention, a strain producing xylo-oligosaccharide xylanase that can be hydrolyzed to prepare a high degree of polymerization is screened from the collected soil by using the transparent circle method, named Paenibacillus sp. B1709, and was released in November 2017 No. 8 was deposited in the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microbial Cultures, the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, and the preservation numbers are CGMCC NO.14870. Its discovery enriches the microbial resources of xylanase production, and through the investigation of the physical and chemical properties of the xylanase, it is proved that its enzymatic characteristics and hydrolysis properties have certain application potential. The invention relates to a bacterial strain capable of hydrolyzing and preparing xylooligosaccharides with a high degree of polymerization, and the products are mainly xylotriose and xylotetraose (no xylose and xylobiose are produced). The invention opens up a new way to produce xylo-oligosaccharides with a high degree of polymerization (DP value 3‑4), can effectively promote economic and social benefits, and has good industrialized production prospects and practical value.

Description

A strain producing XOS xylanase that can be hydrolyzed to prepare a high degree of polymerization and its application

technical field

The invention relates to the fields of microbial fermentation technology, bioengineering technology and biological enzymes, in particular how to obtain and prepare a hydrolysis catalytic enzyme capable of producing xylooligosaccharides with a polymerization degree of 3-4 and its application.

Background technique

Xylanase (Xylanase, EC.3.2.1.8) exists widely in organisms in nature, and bacteria, fungi, molds, etc. can all produce xylanase. In actual industrial production, xylanase is a widely used enzyme preparation, which has shown great practical commercial value in many industries such as food, paper making, and aquaculture. In the food industry, xylanase is used as a clarifying agent in the beverage industry, in the production of fruit juice and liquor. In health food, xylanase can produce functional product xylooligosaccharide by enzymatic method, and xylooligosaccharide can promote the growth and reproduction of beneficial bacteria such as bifidobacteria in the human body, and has a significant effect on improving human immunity, lowering blood fat and blood pressure. effect. In the paper industry, xylanase can improve the bleaching effect of pulp, deink recycled waste paper, and change the fiber properties of pulp. In the aquaculture industry, xylanase can be used as a feed additive and added to animal feed. It can degrade anti-nutritional factors such as non-starch polysaccharides in the animal intestines, reduce the viscosity of chyme in the small intestine of animals, and promote the absorption of animal body in the feed. Nutrients are digested and absorbed, enhancing immunity.

At present, fungi and molds produce more xylanase, while bacteria produce less xylanase, because the xylanase produced by bacteria has good thermal stability, high enzymatic hydrolysis efficiency, and high content of endoxylanase , Efficient degradation of insoluble xylan and other characteristics. Therefore, more and more people pay attention to the xylanase industry produced by bacteria. In addition, the research on bacterial xylanase production not only enriches the resources of xylanase-producing strains, but also provides a more important theory for further understanding the physical and chemical characteristics of bacterial xylanase to meet the different needs of actual production and application. in accordance with.

Most studies have shown that when xylooligosaccharides are prepared by enzymatic hydrolysis, various components such as xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, etc. are often produced. Separate and purify one by one with activated carbon and other media, the steps are cumbersome, and it is not conducive to industrial production and application, which increases the cost of the enterprise and reduces the social benefits. However, when the enzyme involved in the present invention uses xylan as a substrate for enzymolysis, the products are only xylotriose and a small amount of xylotetraose, and no xylose and xylobiose are produced. The production and preparation of sugar greatly improves the extraction efficiency of the target product during the preparation process and reduces the cost of separation and purification, bringing new hope for the preparation and industrial application of high-polymerization xylo-oligosaccharides.

Contents of the invention

Aiming at the problems involved in the background technology, the present invention screens a strain of Paenibacillus sp. B1709 from the soil through the flat transparent circle method. ; Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC NO.14870. This strain can be effectively applied to the research of bacterial xylanase production, which not only enriches the strain resources of xylanase-producing bacteria, but also provides a basis for further understanding the physical and chemical characteristics of bacterial xylanase and meeting the different needs of actual production and application. research basis. The bacterial strain involved in the present invention can produce special xylanase, and the enzyme catalyzes the substrate to produce xylooligosaccharides with a degree of polymerization of 3-4, which brings huge application value for the industrial production of xylotriose and xylotetraose, and reduces its The preparation cost is reduced, and economic and social benefits are increased. The scheme adopted in the present invention has the following steps:

Preparation of fermentation broth: Inoculate xylanase-producing Paenibacillus sp. B1709 into the fermentation medium at an inoculum size of 2%, shake the flask at 30°C and 160 r/min for 72 hours to obtain xylanase fermentation broth; The obtained enzyme fermentation broth was centrifuged at 10,000 r/min at 4°C for 5 min, and the supernatant was taken.

Purification of special xylanase XynA produced by strain B1709: Paenibacillus sp. B1709 was cultured in shake flasks at appropriate temperature, pH value, rotation speed, and liquid volume, using corncobs and beef extract as carbon and nitrogen sources to produce enzymes, and extracted The enzyme solution is roughly purified by two-phase extraction of polymer PEG4000, 18% ammonium sulfate, etc., first Sephacryl S-200 gel filtration chromatography with acetic acid buffer solution as the balance liquid, and then ion exchange with citric acid buffer solution as the balance liquid Further purification by chromatography.

Study on enzymatic and hydrolytic properties of special xylanase: the optimum pH value and temperature of special xylanase produced by strain Paenibacillus sp. Enzyme stability is good. By Line Weaver-Burk double reciprocal plotting method (beech xylan as substrate), the enzymatic kinetic parameters Km of this xylanase was calculated to be 20.41 (mg/mL), and Vmax was 80 (units/min). Add acetic acid-sodium acetate buffer to the purified enzyme solution, use beech wood xylan as substrate, react in a water bath shaker at 55°C, add anion and cation resin after centrifugation, and use thin layer chromatography after overnight treatment ( TLC) for qualitative analysis.

Description of drawings

Figure 1 Purification diagram of xylanase XynA produced by Paenibacillus sp. B1709.

Fig. 2 Analysis results of enzymatic hydrolyzate by thin-layer chromatography (TLC).

Fig. 3 Analysis diagram of enzyme-catalyzed hydrolysis process of beech xylan.

Detailed ways

The specific examples of the present invention will be further described in detail below in conjunction with examples. The following examples are illustrative, not restrictive, and the protection scope of the present invention cannot be limited by the following examples.

Experiment 1 Preparation of Paenibacillus sp. B1709 fermentation broth

Preparation of fermentation broth: Inoculate xylan-producing Paenibacillus sp. B1709 into the fermentation medium at an inoculum size of 2% by mass, shake the flask at 30°C and 160 r/min for 72 hours to obtain xylan fermentation broth;

The fermentation medium is prepared by mixing by weight: 80 mesh corncob powder 8 g, beef extract 10 g, K2HPO4 2.5 g, MgSO4 0.5 g, NaCl 5 g, pH 7.2 and 1000 mL of water.

The prepared enzyme fermentation broth was centrifuged at 10000 r/min at 4°C for 5 min, and the supernatant was taken.

Purification of special xylanase XynA produced by strain Paenibacillus sp. B1709 in experiment 2

The supernatant obtained in Experiment 1 was subjected to aqueous two-phase extraction at a pH of 6.0, 2% sodium chloride, 22% polymer PEG4000, and 18% ammonium sulfate to obtain the active ingredient collected for the first time.

Sephacryl S-200 Gel Filtration Chromatography: Pack the first active ingredient into a column with 1 ml of column type, use 0.02 mol/L acetic acid buffer solution with pH 5.5 as the balance solution, and perform filtration chromatography at a flow rate of 0.4 mL/min. A second collection of active ingredients was obtained.

SP-SepharoseTM Fast Flow Ion Exchange Chromatography: Pack the second active ingredient into the column, column type 1ml, use 0.05 mol/L citric acid buffer solution with pH 3.8 as the balance solution, flow rate: 0.8mL/min, use 1 mol /L of the equilibrium buffer of sodium chloride solution for linear gradient elution to obtain the third collection of active ingredients.

Purification results are shown in Figure 1, 1: standard protein Maker; 2, 5: fermented crude enzyme liquid; 3, 6: preliminary purification concentrated crude enzyme liquid; 4, 7: SP purified enzyme liquid.

Experiment 3 Study on enzymatic properties of xylanase XynA

The optimal pH value and temperature of special xylanase produced by strain Paenibacillus sp. B1709 were 5.5 and 60°C, respectively, and the stability of enzyme activity was better in the range of pH 5.0-6.0 and 40°C-50°C. By Line Weaver-Burk double reciprocal plotting method (beech xylan as substrate), the enzymatic kinetic parameters Km of this xylanase was calculated to be 20.41 (mg/mL), and Vmax was 80 (units/min).

Experiment 4 Study on the Hydrolysis Properties of Xylanase XynA

(1) Xylan enzymatic hydrolysis test

Add 100 µL of purified xylanase solution to 1.0 mL (pH 5.5, 50 mM acetic acid-sodium acetate buffer), use 2% beech wood xylan as a substrate, and react in a water bath shaker at 55 °C, press Time (0.5h, 1h, 1.5h, 2h, 2.5h, 3, 3.5h, 4h) Take out the sample for inactivation, centrifuge, add anion and cation resin, treat overnight, and use thin layer chromatography (TLC) for qualitative analyze.

The results are shown in Figure 2. M is the standard sample of xylose-xylpentaose, and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 respectively represent the hydrolyzed solution obtained after hydrolysis for 0.5 4 h. The results show that, When Paenibacillus sp. B1709 uses beech wood xylan as substrate for enzymatic hydrolysis, the product is mainly xylotriose, and a small amount of xylotetraose is produced at the same time, and xylose and xylobiose are not produced, which is different from the reported xylan There are significant differences in enzymatic hydrolysis properties.

(2) Determination of non-xylan substrate enzyme activity

With the concentration of 2% (α-glucan, β-galactose, soluble starch, chitosan, β-glucan, carboxymethylcellulose (CMC) as the substrate, in 1.0 mL (pH5.5 , 50 mM acetic acid-sodium acetate buffer) to the reaction system, add 4.41U, XynA pure enzyme, and react for 1h. Check whether reducing sugar is produced.

Table 1 is the result of non-xylan substrate enzyme activity assay

Note: "+" means enzyme activity; "-" means no enzyme activity.

Experiments show that Paenibacillus sp. B1709 has no activity on these non-xylan substrates, indicating that its catalytic domain is relatively single.

The invention provides a new bacterial strain producing special xylanase, and at the same time greatly enriches the strain resource of producing xylanase. The invention relates to the xylanase XynA produced by the bacterial strain, which has the characteristics of single catalytic domain and no activity to common non-xylan substrates. The strain can efficiently produce special xylanase by using the waste corn cob as a fermentation carbon source. When xylan is hydrolyzed, it can specifically produce xylotriose and xylotetraose, but not xylose and xylobiose. This catalytic property brings hope for the separation and purification of xylo-oligosaccharides with a low degree of polymerization of 3-4 in industrial production, and brings innovation to the production of xylanase. At the same time, it also reduces the production cost of enterprises and has huge economic and social benefits.

Claims (5)

1. A bacterial strain producing high-molecular-weight xylanase, which was screened from soil by the transparent circle method, was named Paenibacillus sp. B1709, and was preserved in the General Microorganisms of China Microbiological Culture Collection Committee Center (CGMCC), deposit number is CGMCC NO.14870, deposit date: November 7, 2017, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Yard 1, Beichen West Road, Chaoyang District, Beijing. 2. A strain capable of hydrolyzing and preparing high-polymerization-degree xylo-oligo-xylanase as claimed in claim 1 and its application. 3. The bacterial strain described in claim 1 can be effectively applied to the research of bacterial xylanase production, which can not only enrich the microbial resources of xylanase production, but also provide potential follow-up applications by further understanding the physical and chemical characteristics of bacterial xylanase The research basis is provided, and the experiment confirms that the xylanase produced by the strain can hydrolyze and prepare high-polymerization xylooligosaccharides (xylotriose and xylotetraose). The present invention brings huge benefits for the production of xylotriose and xylotetraose. Practical application value (improving the purity of the target product and reducing the cost of separation and purification). 4. the scheme described in claim 3 has the following steps: (1) Preparation of fermentation broth: Inoculate xylanase-producing Paenibacillus sp. B1709 into the fermentation medium at an inoculum size of 2% by mass, shake the flask at 30°C and 160 r/min for 72 hours to obtain xylan Enzyme fermentation broth; centrifuge the prepared enzyme fermentation broth at 10000r/min and 4°C for 5min, take the supernatant, (2) Purification of the special xylanase XynA produced by the strain B1709: the crude enzyme liquid (ie, the fermentation pretreatment liquid) was subjected to two-phase extraction of polymer PEG4000, 18% ammonium sulfate and other aqueous phases for rough purification. Sephacryl S-200 gel filtration chromatography as the equilibrium liquid, and further purification by ion-exchange chromatography using citric acid buffer solution as the equilibrium liquid, (3) Study on the enzymatic and hydrolytic properties of special xylanase: the optimal pH value and temperature of special xylanase produced by strain Paenibacillus sp. B1709 are 5.5 and 60°C, respectively. The stability of the enzyme activity is good in the range of 50°C. The enzymatic kinetic parameters Km of xylanase measured with beech wood xylan as the substrate is 20.41 (mg/mL), Vmax is 80 (units/min), after purification Add acetic acid-sodium acetate buffer to dissolve the enzyme solution, use beech wood xylan as the substrate, react in a water bath shaker at 55°C, add anion and cation resins to the hydrolyzate after centrifugation, and use thin layer chromatography (TLC) after overnight treatment ) for qualitative analysis. 5. when beech wood xylan as described in claim 4 is substrate, institute's enzymatic hydrolysis product only has xylotriose and xylotetraose, does not produce xylose and xylobiose, can be used for high polymerization degree xylooligosaccharide (DP value 3-4) targeted and efficient preparation.