Rhodococcus qing Sheng XWSJ-14 and application thereof in cellulose degradation
Through the screening of Rheumatoideae XWSJ-14 from Baimafeng rot wood in Jinzhai County, Lu'an City, Anhui Province, the problem of low cellulase activity in the prior art was solved, and efficient straw cellulose degradation was achieved. Promote the resource utilization of crop straw.
Patent Information
- Application Number
- CN202411405962.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2024-12-27
AI Technical Summary
The cellulase activity obtained in the prior art is generally low, which cannot meet the needs of straw resource utilization, and limits the comprehensive utilization of straw.
Rheumatoideae-producing Rheumatoideae XWSJ-14 from Baimafeng rot wood in Jinzhai County, Lu'an City, Anhui Province was screened. Through screening and identification, a strain with high cellulose degradation efficiency was obtained to promote crop straws. Resource utilization.
Rheumatoideae XWSJ-14 has strong cellulase activity at 28°C, reaching 17.2 U/mg, which significantly improves the degradation efficiency of straw cellulose and has important application value.
Smart Images
Figure CN119193399A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to Rhodococcus qingshengii XWSJ-14 and application thereof in cellulose degradation, belonging to the field of biotechnology. Background Art
[0002] The straw produced in the agricultural production process is an important renewable resource. my country is the world's largest agricultural country with the largest straw production, but the current utilization efficiency of straw is low. Traditional straw burning not only pollutes air quality, but also causes waste of resources. Therefore, research on the resource utilization of straw is imminent. At present, the resource utilization of straw mainly includes feed, fertilizer, and energy. However, the straw contains a large amount of difficult-to-degrade lignin and cellulose, which has become a key problem in the resource utilization of straw and seriously limits the comprehensive utilization of straw. Therefore, improving the efficiency of straw cellulose decomposition has become the key to the resource utilization of straw.
[0003] In current research, cellulose-degrading functional bacteria with high cellulase production are often added to improve the process of straw into feed, fertilizer, and energy, which have the advantages of green, high efficiency, low cost, and less pollution, including bacteria, fungi, and actinomycetes. Among them, actinomycetes have a wide range of adaptability to temperature, but their growth and reproduction speed is slow; the cellulase system of fungi is a complete enzyme system, which is also the commonly used cellulose-degrading functional bacteria, such as Trichoderma reesei, Trichoderma viride, Penicillium, etc., but fungi grow slowly, have poor thermal stability and alkali resistance; bacteria grow fast, have strong adaptability, simple structure, and wide biological activity, and are the dominant bacterial groups for cellulose degradation. However, the cellulase activity of the strains currently obtained is generally low, which cannot meet the needs of straw resource utilization.
[0004] Therefore, screening and isolating functional bacteria with high cellulase production from different environments, especially screening and isolating cellulose-degrading bacteria from cellulose-rich straw and rotten wood, and determining their degradation ability and biological activity, and finally obtaining functional bacteria that efficiently and rapidly degrade straw cellulose, are of great significance for the resource utilization of straw. Summary of the invention
[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a Rhodococcus qingshengensis XWSJ-14 and its application in cellulose degradation. The Rhodococcus qingshengensis provided by the present invention is derived from the rotten wood of Baimafeng, Jinzhai County, Lu'an City, Anhui Province, from which strains capable of decomposing cellulose are screened, and the functional strain XWSJ-14 with high cellulose degradation efficiency is screened out through identification such as cellulase activity, and is used for cellulose degradation to promote the resource utilization of crop straw.
[0006] The present invention is a kind of Rhodococcus qingshengii XWSJ-14, which is classified and named as Rhodococcus qingshengii Rhodococcus qingshengii, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31245 and the deposit date July 9, 2024.
[0007] The screening method of the high-yield cellulase-producing Rhodococcus qingshengii XWSJ-14 of the present invention comprises the following steps: Step 1: Initial screening Weigh 4 g of rotten wood sample from Baimafeng, Jinzhai County, Lu'an City, Anhui Province, place it in a 50 mL centrifuge tube, add 36 mL of sterile water, and mix overnight. Use the gradient dilution method to dilute the sample solution to 10 -1 ~10 -7 , spread on the screening medium (CMC medium) plate, invert and culture at 20-30℃, and preliminarily identify the colonies according to their shape, color, wetness, convexity and concavity, transparency, number and size. Preliminarily screen the strains with strong cellulose degradation ability through colony size and hydrolysis circle, and streak the selected strains on the screening medium plate for one purification, repeat the streaking for secondary purification, then number them and store them in a refrigerator at 4℃.
[0008] Step 2: Rescreen The strain obtained in the initial screening was inoculated into 40 mL LB medium, and cultured at 20-30°C and 100-200 rpm for 2-3 days. The seed liquid of the expanded culture was inoculated into 100 mL of liquid fermentation enzyme production medium (CE medium) at an inoculum size of 1-5%, and cultured at 20-30°C and 100-200 rpm to determine the cellulase activity.
[0009] Step 3: Identification Morphological identification: Pick the monoclonal strain after rescreening and inoculate it into LB liquid culture medium. After fermentation for 1-7 days, take a small amount of bacteria on a sterile glass slide and dissolve it in 1 drop of sterile water. After drying, stain it with crystal violet, mordant it with Lugo's iodine solution, decolorize it with 95% ethanol, wash it with water, stain it with Tsar, wash it with water, and dry it. Then observe the morphology of the bacteria under a 40x microscope.
[0010] Molecular biological identification: The strain genome was extracted using the AxyPrep bacterial genome extraction kit, and the strain 16S gene was amplified using primers 27F and 1492R. The PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequence detection, and the measured sequence was compared by Blast on NCBI.
[0011] The culture medium used in the present invention is: Screening medium (CMC medium): weigh 2 g (NH4)2SO4, 0.5 g MgSO4, 1 g K2HPO4·3H2O, 0.5 g NaCl, and 5 g CMC into a beaker, add deionized water to make up to 1 L, adjust the pH to 7.0, and sterilize at 121°C for 20 min.
[0012] Expansion medium (LB): 5 g yeast extract, 10 g peptone, 10 g NaCl, add deionized water to 1 L, sterilize at 115°C for 30 min.
[0013] Fermentation enzyme production medium (CE medium): 3 g peptone; 2 g (NH4)2SO4; 0.3 g CaCl2·2H2O; 0.3 g MgSO4; 4 g KH2PO4; 1% Tween-80; 40 g microcrystalline cellulose; add deionized water to 1 L, adjust the pH to 5.5, and sterilize at 121°C for 20 min.
[0014] Filter paper strip disintegration medium: 1.0 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 1 g K2HPO4, 1 g yeast extract powder, add deionized water to 1 L, filter paper strip (1×6 cm 2 ) 3 strips / conical flask, sterilize at 121℃ for 20 min.
[0015] The invention discloses an application of the Rhodococcus qingshengii XWSJ-14 in cellulose degradation.
[0016] The inoculation amount of the Rhodococcus qingshengii XWSJ-14 is 0.5%-5% by volume, inoculated into the filter paper strip disintegration culture medium, and cultured at 20-30° C. and 100-200 rpm.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The cellulose degrading bacteria of the present invention are derived from the rotten wood environment of Baimafeng, Jinzhai County, Lu'an City, Anhui Province. A bacterial-derived cellulose degrading bacteria obtained by efficient screening is identified as Rhodococcus qingshengensis with cellulose degradation ability, and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.31245 and a deposit date of July 9, 2024. The Rhodococcus qingshengensis XWSJ-14 of the present invention has a strong cellulase activity at 28°C, reaching 17.2 U / mg, and has important application value for promoting the resource utilization of cellulose-rich crop straw. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1Growth morphology of strains in cellulose screening medium.
[0019] Figure 2 Glucose concentration standard curve.
[0020] Figure 3 Comparison of filter paper cellulose degradation abilities of screened strains. DETAILED DESCRIPTION
[0021] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. In the following examples, various processes and methods not described in detail are conventional methods known in the art. The specific embodiments of the present invention described below are to supplement the purpose, technical solutions and effects of the present invention, but they are not intended to limit the scope of protection of the present invention. Within the scope of the present invention, simple changes, modifications or replacements can be made, which are all considered to be within the scope of protection of the present invention.
[0022] The culture medium used in the present invention is: Screening medium (CMC medium): weigh 2 g (NH4)2SO4, 0.5 g MgSO4, 1 g K2HPO4·3H2O, 0.5 g NaCl, and 5 g CMC into a beaker, add deionized water to make up to 1 L, adjust the pH to 7.0, and sterilize at 121°C for 20 min.
[0023] Expansion medium (LB): 5 g Yeast extract, 10 g Peptone, 10 g NaCl, add deionized water to 1 L, sterilize at 115℃ for 30 min.
[0024] Fermentation enzyme production medium: 3 g Peptone; 2 g (NH4)2SO4; 0.3 g CaCl2.2H2O; 0.3 g MgSO4; 4 g KH2PO4; 1% Tween-80; 40 g microcrystalline cellulose; add deionized water to 1 L, adjust the pH to 5.5, and sterilize at 121℃ for 20 min.
[0025] Filter paper strip disintegration medium: 1.0 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 1 g K2HPO4, 1 g yeast extract powder, add deionized water to 1 L, filter paper strip (1×6 cm 2 ) 3 strips / conical flask, sterilize at 121℃ for 20 min.
[0026] The reagents used in the present invention are prepared as follows: DNS reagent: Accurately weigh 10.6 g of 3,5-dinitrosalicylic acid and 19.8 g of NaOH and place them in a beaker. Add 1416 mL of deionized water and heat to 45°C in a water bath. Stir with a glass rod to dissolve. After dissolution, add 306 g of potassium sodium tartrate, 7.6 ml of phenol, and 8.3 g of sodium sulfite. Continue to stir and dissolve at 45°C. After all the added substances are dissolved, place in a brown bottle away from light for one week before use. It can be stored at room temperature for 6 months.
[0027] Citric acid buffer (0.05 M, pH 4.8): Accurately weigh 210 g of citric acid monohydrate and place it in a beaker. Add 750 mL of deionized water and stir to dissolve. After all the solution is dissolved, add 60 g of NaOH until the pH value is about 4.3, and then dilute to 1 L with deionized water.
[0028] Whatman No. 1 filter paper strips: Cut Whatman No. 1 filter paper into 1×6 cm strips.
[0029] 1% glucose standard solution: Accurately weigh 0.1 g of glucose that has been dried to a constant weight, dilute to 10 mL with deionized water, and prepare before use.
[0030] Crystal violet solution: Solution A: 2g crystal violet, 20mL ethanol (95%); Solution B: 0.8g ammonium oxalate, 80mL distilled water. Mix solution A and solution B and set aside.
[0031] Lugo's iodine solution: 1g iodine tablet, 2g potassium iodide. First dissolve the potassium iodide in a small amount of distilled water, then add the iodine tablet. After dissolution, add water to 300mL.
[0032] 0.5% Shahuang solution: 20mL Shahuang alcohol solution (2.5%), 80mL distilled water, 2.5% Shahuang alcohol solution is stored in a brown bottle as the mother solution and diluted when used. Example 1: Sample processing
[0033] Weigh 4 g of the rotten wood sample from Baimafeng, Jinzhai County, Lu'an City, Anhui Province, dissolve it in sterile water, mix it well and place it at 28°C overnight. Take a sterile centrifuge tube and treat the sample with the gradient dilution method, diluting the sample concentration to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 For backup. Example 2: Preliminary Screening
[0034] Take 100 μL of each of the 7 dilution samples, apply them to the screening medium, and invert and culture at 28°C for 1 to 7 days. The 7 gradient-coated plates are used to preliminarily identify the colonies according to their shape, color, wetness, concavity, transparency, and number and size in the plate. The larger the colony morphology, the stronger the ability to decompose cellulose, which is a potential high-yield cellulase screening strain ( Figure 1 ). Use an inoculation loop to pick the strain to be selected, streak it on the screening medium plate for one purification, repeat the streak for a second purification, and then number it and store it in a 4℃ refrigerator. Example 3: Rescreening
[0035] The strains obtained in the initial screening were inoculated into 50 mL of LB medium and expanded at 28°C, 120 rpm for 3 days. The seed liquid of the expanded culture was inoculated into 100 mL of liquid enzyme production medium at a 5% inoculum and cultured at 28°C, 120 rpm. The cellulose filter paper enzyme activity was measured and the high cellulase-producing strains were selected based on the changes in enzyme activity.
[0036] Preparation of crude enzyme solution: inoculate the screened strain into the seed culture medium, culture at 28°C, 120rpm shaking, take 2 mL of fermentation liquid every day, centrifuge at 12000rpm for one minute, and take the supernatant as the crude enzyme solution.
[0037] Cellulose filter paper enzyme activity test: Prepare the standard solution of glucose according to the dilution ratio in Table 1 below.
[0038]
[0039] Take 1.00 mL of glucose solution in Table 1 and add it to the graduated test tubes, add 2.00 mL of deionized water and 2.00 mL of DNS, vortex for 5 seconds, boil in a water bath for 5 minutes, cool to room temperature with running water, make up to 25 mL, adjust the blank sample to zero, measure the absorbance of the solution at 540 nm, and draw a glucose standard curve ( Figure 2 ).
[0040] Take 0.5 mL of enzyme solution and add it to a 5 mL EP tube containing a filter paper strip (60 mm × 10 mm), add 2.0 mL of citric acid buffer, react at 50°C for 30 min, add 2.5 mL of DNS reagent, vortex and oscillate for 5 seconds, and place in a water bath at 100°C for 5 min.
[0041] OD 540 Determine its absorbance and calculate the enzyme activity in U / g. After 9 days of fermentation, the enzyme activity of strain XWSJ-14 reached 17.2U / g. The enzyme activity calculation formula is:
[0042] Where: X—cellulose filter paper enzyme activity (U / g); m—mass of glucose (mg); M—glucose molar mass; t—reaction time (min); 1000—conversion factor; n—sample dilution multiple. Example 4: Identification
[0043] 1. Morphological identification of strains Bacterial cells are extremely small and transparent. When observed under an optical microscope using a water-immersion slide, only their general shape and movement can be seen. If you want to observe their detailed shape and main structure under an optical microscope, they must be stained.
[0044] (1) The target strain XWSJ-14 was inoculated into LB liquid culture medium and cultured at 28°C and 200 rpm for 20 h.
[0045] (2) Place a drop of sterile water in the center of a clean glass slide. Use a sterile inoculation loop to take a small amount of the bacteria to be observed, mix it thoroughly with the water drop, and apply it to form a very thin bacterial film.
[0046] (3) After the smear has dried in the air, hold the slide at one end with the biofilm facing up and pass it over low heat three times to cool.
[0047] (4) Staining: Primary staining: add crystal violet solution and stain for 1 to 1.5 minutes; Mordanting: add Lugo's iodine solution and stain for 1 to 2 minutes; wash with water; Decolorization: add 95% ethanol, shake, pour off the ethanol, repeat 2 to 3 times; wash with water; Restaining: add safranin and stain for 1 to several minutes; wash with water; dry.
[0048] (5) Microscopic examination.
[0049] 2. Molecular biological identification of strains The fermentation broth of XWSJ-14 strain cultured overnight at 28°C and 200 rpm was centrifuged to obtain the bacteria, which were extracted using the AxyPrep bacterial genome extraction kit, and the 16S gene of the strain was amplified using primers 27F (5′~3′ sequence: AGAGTTTGATCTGGCTCAG) and 1492R (5′~3′ sequence: GGCTACCTTGTTACGACTT). The PCR amplification program was: 94°C pre-denaturation reaction for 5 minutes, 55°C annealing for 2 minutes, 72°C extension for 2 minutes, 94°C denaturation for 1 minute, 55°C annealing for 50 seconds, 72°C extension for 55 seconds, the number of cycles was set to 30 times, 72°C reaction for 5 minutes, and stored at 4°C. The PCR product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequence detection, and the measured sequence was compared by Blast on NCBI. The results showed that XWSJ-14 was Rhodococcus qingshengii . It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31245 and the deposit date July 9, 2024. Example 5: Degradation ability of XWSJ-14
[0050] XWSJ-14 was inoculated into LB liquid culture medium, and after culturing at 28°C and 200 rpm for 20 hours, it was transferred to 100 mL of filter paper strip disintegration culture medium at a 1% inoculation amount. The filter paper was weighed before preparing the filter paper strip disintegration culture medium. After the cultivation was completed, the filter paper in the fermentation liquid was taken out and dried and weighed. The filter paper degradation rate was calculated based on the weight loss of the filter paper. After inoculation of XWSJ-14, the fermentation was terminated by culturing at 28°C and 120 rpm for 10 days. The filter paper degradation rate of XWSJ-14 was calculated to be 78.5%. At the same time, compared with other strains screened from the same batch of samples, the cellulose degradation ability of XWSJ-14 was significantly better than that of other strains ( Figure 3 ).
Claims
1. A Rhodococcus qingshengii XWSJ-14, characterized in that: The Qingshengshi Rhodococcus XWSJ-14 is classified and named Qingshengshi Rhodococcus Rhodococcus qingshengii , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31245 and the deposit date July 9, 2024.
2. Use of the Rhodococcus qingshengii XWSJ-14 described in claim 1 in cellulose degradation.
3. The use according to claim 2, characterized in that: The temperature of the degradation system is controlled at 20-30°C.