Strain capable of efficiently expressing xylanase and application of strain

Abstract

The invention discloses a strain capable of efficiently expressing xylanase, the strain is preserved in Guangdong Microbial Culture Collection Center on October 23, 2024, the preservation date is Pichia pastoris, and the preservation number is GDMCC NO65325. The invention further discloses a preparation method of the strain capable of efficiently expressing xylanase, and the strain is preserved in Guangdong Microbial Culture Collection Center on October 23, 2024. According to the invention, a strain with excellent genetic stability for producing xylanase is screened by means of ultraviolet light induction of the strain and component optimization and synergistic induction of a primary screening culture medium, the strain can efficiently express xylanase, and in addition, histidine and nicotinic acid are added into an enzyme production culture medium, so that the expression of xylanase is further promoted, and the yield of xylanase is improved. And the xylanase has more stable enzyme activity, so that the xylanase has better utilization value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain capable of efficiently expressing xylanase and an application thereof. Background Art

[0002] Xylanase belongs to the class of hydrolases, which can randomly cut the main chain of polyxylan in plant cell walls. It is a complex enzyme system that can degrade xylan into xylose or oligoxylose. Xylanase is widely distributed in nature. It exists in marine algae microorganisms and terrestrial bacteria, fungi, actinomycetes, yeast, snails, crustaceans, plant tissues and some invertebrates. Currently, the xylanases secreted by microorganisms are more studied. Since xylanases in nature exist in different forms, they often have different characteristics, such as mechanism of action, folding mode, enzyme substrate specificity, hydrolysis activity and physicochemical properties.

[0003] In the prior art, there are dozens of genera and more than one hundred species of microorganisms that have the ability to produce xylanases, many of which can release and produce extracellular xylanases, including Aspergilli, Trichodermi, Penicillium, Fusarium, Streptomycetes, Bacilli, etc. The properties of xylanases produced by different microorganisms are also different. Bacteria can generally produce high molecular weight acid xylanases and low molecular weight alkaline xylanases, while most fungi produce low molecular weight alkaline xylanases. However, on the whole, the activity of xylanases produced by fungi is higher than that of bacteria, but there is a disadvantage of poor thermal stability. Therefore, in order to make xylanases have a wider application, it is of great significance to provide a method that can efficiently express xylanases and has stable activity. Summary of the invention

[0004] Based on this, in order to solve the technical problems that the strains in the prior art have poor expression effects of xylanase and poor stability of xylanase activity, the present invention provides a strain that efficiently expresses xylanase and its application, and the specific technical scheme is as follows:

[0005] A strain that efficiently expresses xylanase, the strain is deposited in Guangdong Microbial Culture Collection Center, the preservation date is October 23, 2024, the taxonomic name is Pichia pastoris, and the preservation number is GDMCC NO65325.

[0006] Further, the strain was identified and named as Pichia pastoris HNC-06, and the Pichia pastoris HNC-06 was obtained by the following method:

[0007] The preserved Pichia pastoris was inoculated into LB liquid culture medium, cultured at 150 r / min and 30°C for 24 h, then centrifuged, the supernatant was discarded, sterile saline was added, shaken, centrifuged again, sterile saline was added again, shaken to obtain a bacterial suspension;

[0008] The bacterial suspension was placed in a culture dish and treated under ultraviolet light for 30s to 120s, then spread on the primary screening culture medium, cultured in a dark environment at 30°C for 24h to 48h, and then Congo red was added for staining to screen out the strain with the largest hydrolysis circle. This method was repeated for 3 rounds of mutagenesis, and then rescreened to obtain the strain;

[0009] The screened strain was inoculated into a seed culture medium, cultured at 30°C for 24 to 48 hours, and stably cultured for 10 generations to obtain Pichia pastoris HNC-06.

[0010] Furthermore, the concentration of the bacterial suspension is 1×10 6 ~10 8 .

[0011] Furthermore, the ultraviolet light condition is a 15W to 20W ultraviolet light at a distance of 30cm to 40cm.

[0012] Furthermore, the strain inoculation amount in the seed culture medium is 3% to 5%.

[0013] Furthermore, the primary screening culture medium includes the following components: corn cob xylan (8-12) g / L, magnesium sulfate (0.1-0.5) g / L, manganese sulfate (0.5-0.8) g / L, calcium sulfate (1-5) g / L, sodium chloride (6-8) g / L, potassium hydrogen phosphate (1-3) g / L, yeast extract (3-7) g / L, agar (12-15) g / L, diethyl sulfate (0.01-0.05) mL / L, sodium nitrite (3-5) g / L and diammonium citrate (1-2) g / L.

[0014] In addition, the present invention also provides an application of a strain that efficiently expresses xylanase, wherein the application is to use the strain as a production strain of a xylanase product.

[0015] Furthermore, the xylanase product is as follows: Pichia pastoris HNC-06 strain is inoculated into an enzyme production medium, cultured at 30°C for 48h to 72h, fermented liquid is centrifuged at 3000rpm for 10min to 15min at 4°C, supernatant is taken, ammonium sulfate is added to make the saturation of the solution 50%, after standing for 12h to 20h, centrifuged at 10000r / min for 10min to 15min, supernatant is taken, ammonium sulfate is continuously added to make the saturation of the solution 80%, after standing for 12h to 20h, centrifuged at 10000r / min for 10min to 15min, salting out and chromatography treatment are performed, and xylanase dry powder is obtained after freeze-drying.

[0016] Furthermore, the inoculation amount of the strain in the enzyme production medium is 8% to 10%.

[0017] Furthermore, the enzyme production culture medium comprises the following components: bran (55-60) g / L, histidine (1-3) g / L, nicotinic acid (1-2) g / L, potassium hydrogen phosphate (5-7) g / L, magnesium sulfate (0.1-0.5) g / L, manganese sulfate (0.1-0.5) g / L, calcium chloride (0.1-0.3) g / L, yeast extract powder (0.5-0.8) g / L and Tween-80 (3-5) mL / L.

[0018] Compared with the prior art, this application has the following beneficial effects:

[0019] The present invention optimizes the composition of the strain and the primary screening culture medium through ultraviolet light induction, and the method of synergistic induction is used to screen the strains with good genetic stability of xylanase production, and the strains can efficiently express xylanase, which not only enriches the strain resource library of xylanase production, but also can be used as the applied strains of xylanase production to obtain xylanase with more stable activity. Specifically, diethyl sulfate and sodium nitrite are added to the primary screening culture medium. Under the premise of ultraviolet light induction, diethyl sulfate and sodium nitrite can synergistically act on DNA molecules that are being replicated or at the end of replication, and the amino groups in the bases are removed from the DNA molecules to become ketone groups, changing the potential of the hydrogen bonds in the bases, causing base conversion after adenine and cytosine deamination, and hindering the cross-linking between the two single strands of DNA, thereby affecting the replication of DNA molecules. In addition, the present invention adds histidine and nicotinic acid to the enzyme production culture medium to further promote the expression of xylanase, and make the xylanase have a more stable enzyme activity, so that it has a better utilization value. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0021] A strain of bacteria that efficiently expresses xylanase in one embodiment of the present invention is deposited in Guangdong Microbiological Culture Collection Center on October 23, 2024, with a taxonomic name of Pichia pastoris and a collection number of GDMCC NO65325.

[0022] In one embodiment, the strain was identified and named as Pichia pastoris HNC-06, and the Pichia pastoris HNC-06 was obtained by the following method:

[0023] The preserved Pichia pastoris was inoculated into LB liquid culture medium, cultured at 150 r / min and 30°C for 24 h, then centrifuged, the supernatant was discarded, sterile saline was added, shaken, centrifuged again, sterile saline was added again, shaken to obtain a bacterial suspension;

[0024] The bacterial suspension was placed in a culture dish and treated under ultraviolet light for 30s to 120s, then spread on the primary screening culture medium, cultured in a dark environment at 30°C for 24h to 48h, and then Congo red was added for staining to screen out the strain with the largest hydrolysis circle. This method was repeated for 3 rounds of mutagenesis, and then rescreened to obtain the strain;

[0025] The screened strain was inoculated into a seed culture medium, cultured at 30°C for 24 to 48 hours, and stably cultured for 10 generations to obtain Pichia pastoris HNC-06.

[0026] In one embodiment, the concentration of the bacterial suspension is 1×10 6 ~10 8 .

[0027] In one embodiment, the ultraviolet light condition is a 15W to 20W ultraviolet light at a distance of 30cm to 40cm.

[0028] In one embodiment, the strain inoculation amount in the seed culture medium is 3% to 5%.

[0029] In one embodiment, the primary screening culture medium comprises the following components: corn cob xylan (8-12) g / L, magnesium sulfate (0.1-0.5) g / L, manganese sulfate (0.5-0.8) g / L, calcium sulfate (1-5) g / L, sodium chloride (6-8) g / L, potassium hydrogen phosphate (1-3) g / L, yeast extract (3-7) g / L, agar (12-15) g / L, diethyl sulfate (0.01-0.05) mL / L, sodium nitrite (3-5) g / L and diammonium citrate (1-2) g / L.

[0030] In addition, the present invention also provides an application of a strain that efficiently expresses xylanase, wherein the application is to use the strain as a production strain of a xylanase product.

[0031] In one embodiment, the xylanase product is: inoculating Pichia pastoris HNC-06 strain into an enzyme-producing medium, culturing at 30°C for 48h to 72h, taking the fermentation broth and centrifuging it at 4°C and 3000rpm for 10min to 15min, taking the supernatant, adding ammonium sulfate to make the solution saturation 50%, standing for 12h to 20h, centrifuging at 10000r / min for 10min to 15min, taking the supernatant, continuing to add ammonium sulfate to make the solution saturation 80%, standing for 12h to 20h, centrifuging at 10000r / min for 10min to 15min, and obtaining xylanase dry powder after salting out and chromatography treatment and freeze drying.

[0032] In one embodiment, the inoculation amount of the strain in the enzyme production medium is 8% to 10%.

[0033] In one embodiment, the enzyme production culture medium comprises the following components: bran (55-60) g / L, histidine (1-3) g / L, niacin (1-2) g / L, potassium hydrogen phosphate (5-7) g / L, magnesium sulfate (0.1-0.5) g / L, manganese sulfate (0.1-0.5) g / L, calcium chloride (0.1-0.3) g / L, yeast extract powder (0.5-0.8) g / L and Tween-80 (3-5) mL / L.

[0034] The strain Pichia pastoris HNC-06 screened by the above scheme can efficiently express xylanase, and the activity of the xylanase is better and more stable.

[0035] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0036] Embodiment 1:

[0037] The screening culture medium included the following ingredients: 10 g / L corncob xylan, 0.3 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 2 g / L calcium sulfate, 6 g / L sodium chloride, 1 g / L potassium hydrogen phosphate, 5 g / L yeast extract, 13 g / L agar, 0.02 mL / L diethyl sulfate, 4 g / L sodium nitrite, and 1 g / L diammonium citrate;

[0038] The enzyme production medium includes the following ingredients: bran 60 g / L, histidine 3 g / L, nicotinic acid 1 g / L, potassium hydrogen phosphate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.1 g / L, calcium chloride 0.3 g / L, yeast extract powder 0.8 g / L and Tween-80 3 mL / L;

[0039] The preserved Pichia pastoris was inoculated into LB liquid culture medium, cultured at 150 r / min and 30°C for 24 h, then centrifuged, the supernatant was discarded, sterile saline was added, shaken, centrifuged again, sterile saline was added again, shaken to obtain a bacterial suspension;

[0040] The bacterial suspension was placed in a culture dish and treated at a distance of 30 cm under a 15W ultraviolet lamp for 60 seconds, then spread on the primary screening culture medium, cultured in a dark environment at 30°C for 24 hours, and then Congo red was added for staining to screen out the strain with the largest hydrolysis circle. This method was repeated for 3 rounds of mutagenesis, and then rescreened to obtain the strain;

[0041] The screened strain was inoculated into a seed culture medium at an inoculum size of 5%, cultured at 30°C for 48 hours, and stably cultured for 10 generations to obtain Pichia pastoris HNC-06;

[0042] Inoculate Pichia pastoris HNC-06 strain into enzyme production medium at 10% inoculation amount, culture at 30°C for 72h, centrifuge the fermentation broth at 4°C and 3000rpm for 10min-15min, and take the supernatant;

[0043] Ammonium sulfate was added to make the saturation of the solution 50%. After standing for 12 hours, the solution was centrifuged at 10,000 r / min for 10 minutes. The supernatant was taken and ammonium sulfate was continued to be added to make the saturation of the solution 80%. After standing for 12 hours, the solution was centrifuged at 10,000 r / min for 10 minutes. After salting out and chromatography treatment and freeze-drying, xylanase dry powder was obtained.

[0044] Embodiment 2:

[0045] The screening medium included the following ingredients: 12 g / L corncob xylan, 0.4 g / L magnesium sulfate, 0.8 g / L manganese sulfate, 1 g / L calcium sulfate, 6 g / L sodium chloride, 1 g / L potassium hydrogen phosphate, 6 g / L yeast extract, 14 g / L agar, 0.03 mL / L diethyl sulfate, 5 g / L sodium nitrite, and 2 g / L diammonium citrate;

[0046] The enzyme production medium includes the following ingredients: bran 60 g / L, histidine 2 g / L, nicotinic acid 2 g / L, potassium hydrogen phosphate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.1 g / L, calcium chloride 0.3 g / L, yeast extract powder 0.8 g / L and Tween-80 3 mL / L;

[0047] The preserved Pichia pastoris was inoculated into LB liquid culture medium, cultured at 150 r / min and 30°C for 24 h, then centrifuged, the supernatant was discarded, sterile saline was added, shaken, centrifuged again, sterile saline was added again, shaken to obtain a bacterial suspension;

[0048] The bacterial suspension was placed in a culture dish and treated at a distance of 30 cm under a 15W ultraviolet lamp for 60 seconds, then spread on the primary screening culture medium, cultured in a dark environment at 30°C for 24 hours, and then Congo red was added for staining to screen out the strain with the largest hydrolysis circle. This method was repeated for 3 rounds of mutagenesis, and then rescreened to obtain the strain;

[0049] The screened strain was inoculated into a seed culture medium at an inoculum size of 5%, cultured at 30°C for 48 hours, and cultured stably for 10 generations to obtain Pichia pastoris HNC-06;

[0050] The Pichia pastoris HNC-06 strain was inoculated into the enzyme production medium at an inoculum of 10%, and cultured at 30°C for 72 hours. The fermentation broth was centrifuged at 4°C and 3000 rpm for 15 minutes, and the supernatant was collected.

[0051] Ammonium sulfate was added to make the saturation of the solution 50%. After standing for 12 hours, the solution was centrifuged at 10,000 r / min for 10 minutes. The supernatant was taken and ammonium sulfate was continued to be added to make the saturation of the solution 80%. After standing for 12 hours, the solution was centrifuged at 10,000 r / min for 10 minutes. After salting out and chromatography treatment and freeze-drying, xylanase dry powder was obtained.

[0052] Embodiment 3:

[0053] The screening medium included the following ingredients: 12 g / L corncob xylan, 0.3 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 2 g / L calcium sulfate, 6 g / L sodium chloride, 1 g / L potassium hydrogen phosphate, 5 g / L yeast extract, 13 g / L agar, 0.02 mL / L diethyl sulfate, 4 g / L sodium nitrite, and 1 g / L diammonium citrate;

[0054] The enzyme production medium includes the following ingredients: bran 60 g / L, histidine 3 g / L, nicotinic acid 1 g / L, potassium hydrogen phosphate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.1 g / L, calcium chloride 0.3 g / L, yeast extract powder 0.8 g / L and Tween-80 3 mL / L;

[0055] The preserved Pichia pastoris was inoculated into LB liquid culture medium, cultured at 150 r / min and 30°C for 24 h, then centrifuged, the supernatant was discarded, sterile saline was added, shaken, centrifuged again, sterile saline was added again, shaken to obtain a bacterial suspension;

[0056] The bacterial suspension was placed in a culture dish and treated at a distance of 30 cm under a 15W ultraviolet lamp for 60 seconds, then spread on the primary screening culture medium, cultured in a dark environment at 30°C for 24 hours, and then Congo red was added for staining to screen out the strain with the largest hydrolysis circle. This method was repeated for 3 rounds of mutagenesis, and then rescreened to obtain the strain;

[0057] The screened strain was inoculated into a seed culture medium at an inoculum size of 5%, cultured at 30°C for 48 hours, and stably cultured for 10 generations to obtain Pichia pastoris HNC-06;

[0058] The Pichia pastoris HNC-06 strain was inoculated into the enzyme production medium at an inoculum of 10%, and cultured at 30°C for 72 hours. The fermentation broth was centrifuged at 4°C and 3000 rpm for 15 minutes, and the supernatant was collected.

[0059] Ammonium sulfate was added to make the saturation of the solution 50%. After standing for 12 hours, the solution was centrifuged at 10,000 r / min for 10 minutes. The supernatant was taken and ammonium sulfate was continued to be added to make the saturation of the solution 80%. After standing for 12 hours, the solution was centrifuged at 10,000 r / min for 10 minutes. After salting out and chromatography treatment and freeze-drying, xylanase dry powder was obtained.

[0060] Comparative Example 1:

[0061] The difference between Comparative Example 1 and Example 3 is that Comparative Example 1 is not treated with ultraviolet light conditions, and the rest is the same as Example 3.

[0062] Comparative Example 2:

[0063] The difference between Comparative Example 2 and Example 3 is that diethyl sulfate is not added to the primary screening culture medium in Comparative Example 2, and the rest is the same as Example 3.

[0064] Comparative Example 3:

[0065] The difference between Comparative Example 3 and Example 3 is that sodium nitrite is not added to the primary screening culture medium in Comparative Example 3, and the rest is the same as Example 3.

[0066] Comparative Example 4:

[0067] The difference between Comparative Example 4 and Example 3 is that histidine is not added to the enzyme production medium in Comparative Example 4, and the rest is the same as Example 3.

[0068] Comparative Example 5:

[0069] The difference between Comparative Example 5 and Example 3 is that nicotinic acid is not added to the enzyme production medium in Comparative Example 5, and the rest is the same as Example 3.

[0070] The supernatants obtained after culturing for 72 hours in Examples 1 to 3 and the supernatants obtained after culturing for 72 hours in Comparative Examples 1 to 5 were tested, and the protein content of the supernatants was determined by the Bradford method. The results are shown in Table 1 below.

[0071] Table 1: Test results

[0072] Group Xylanase activity (U / mL) Example 1 456.8 Example 2 462.1 Example 3 459.3 Comparative Example 1 386.9 Comparative Example 2 375.7 Comparative Example 3 408.3 Comparative Example 4 411.7 Comparative Example 5 417.5

[0073] From the data analysis of Table 1, it can be seen that the present application promotes the expression of xylanase by synergistic induction through ultraviolet light combined with diethyl sulfate and sodium nitrite, and the addition of histidine and nicotinic acid in the culture medium can also synergistically promote the expression of xylanase, thereby achieving the purpose of efficiently expressing xylanase.

[0074] In addition, in order to test the stability of xylanase, the following test was performed. The conditions and results are shown in Table 2 below.

[0075] Table 2: Test results

[0076]

[0077]

[0078] From the data analysis of Table 2, it can be seen that the xylanase of the present application has better pH stability, thermal stability and ability to resist protease degradation, and can be used in feed additives to resist degradation by proteases in the animal stomach and intestines, while effectively reducing the viscosity of gastrointestinal chyme and promoting feed utilization.

[0079] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain that efficiently expresses xylanase, characterized in that: The strain is deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, with the taxonomic name Pichia pastoris and the collection number GDMCCNO 65325.

2. The strain for efficiently expressing xylanase according to claim 1, characterized in that: The strain was identified and named as Pichia pastoris HNC-06, and the Pichia pastoris HNC-06 was obtained by the following method: The preserved Pichia pastoris was inoculated into LB liquid culture medium, cultured at 150 r / min and 30°C for 24 h, then centrifuged, the supernatant was discarded, sterile saline was added, shaken, centrifuged again, sterile saline was added again, shaken to obtain a bacterial suspension; The bacterial suspension was placed in a culture dish and treated under ultraviolet light for 30s to 120s, then spread on the primary screening culture medium, cultured in a dark environment at 30°C for 24h to 48h, and then Congo red was added for staining to screen out the strain with the largest hydrolysis circle. This method was repeated for 3 rounds of mutagenesis, and then rescreened to obtain the strain; The screened strain was inoculated into a seed culture medium, cultured at 30°C for 24 to 48 hours, and stably cultured for 10 generations to obtain Pichia pastoris HNC-06.

3. The strain for efficiently expressing xylanase according to claim 2, characterized in that: The concentration of the bacterial suspension was 1×10 6 ~10 8 .

4. The strain for efficiently expressing xylanase according to claim 2, characterized in that: The ultraviolet light condition is 15W-20W ultraviolet light at a distance of 30cm-40cm.

5. The strain for efficiently expressing xylanase according to claim 2, characterized in that: The inoculation amount of the strain in the seed culture medium is 3% to 5%.

6. The strain for efficiently expressing xylanase according to claim 2, characterized in that: The primary screening culture medium comprises the following components: corn cob xylan (8-12) g / L, magnesium sulfate (0.1-0.5) g / L, manganese sulfate (0.5-0.8) g / L, calcium sulfate (1-5) g / L, sodium chloride (6-8) g / L, potassium hydrogen phosphate (1-3) g / L, yeast extract (3-7) g / L, agar (12-15) g / L, diethyl sulfate (0.01-0.05) mL, sodium nitrite (3-5) g / L and diammonium citrate (1-2) g / L.

7. An application of a strain for efficiently expressing xylanase, characterized in that: The application is that the strain described in any one of claims 1 to 6 is used as a strain for producing xylanase products.

8. The use according to claim 7, characterized in that: The xylanase product comprises the following steps: inoculating Pichia pastoris HNC-06 strain into an enzyme production medium, culturing for 48 to 72 hours at 30°C, centrifuging the fermented liquid at 4°C and 3000 rpm for 10 to 15 minutes, taking the supernatant, adding ammonium sulfate to make the saturation of the solution 50%, standing for 12 to 20 hours, centrifuging at 10000 r / min for 10 to 15 minutes, taking the supernatant, continuing to add ammonium sulfate to make the saturation of the solution 80%, standing for 12 to 20 hours, centrifuging at 10000 r / min for 10 to 15 minutes, and obtaining xylanase dry powder after salting out and chromatography treatment and freeze drying.

9. The use according to claim 7, characterized in that: The inoculation amount of the strain in the enzyme production medium is 8% to 10%.

10. The use according to claim 7, characterized in that: The enzyme production culture medium comprises the following components: bran (55-60) g / L, histidine (1-3) g / L, nicotinic acid (1-2) g / L, potassium hydrogen phosphate (5-7) g / L, magnesium sulfate (0.1-0.5) g / L, manganese sulfate (0.1-0.5) g / L, calcium chloride (0.1-0.3) g / L, yeast extract powder (0.5-0.8) g / L and Tween-80 (3-5) mL / L.

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