Esterase-producing strain and application thereof
By providing the esterase-producing strain Acinetobacter soli WCO-12, the problem of insufficient substrate specificity and environmental adaptability of esterases is solved, and efficient catalysis of short-chain ester and application in environmental protection is achieved.
Patent Information
- Application Number
- CN202510514626.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-23
- Publication Date
- 2025-07-25
AI Technical Summary
The existing esterases have limited substrate specificity in catalytic ester bond hydrolysis and synthesis reactions, especially the catalytic efficiency of short-chain esters is not high, and their application potential in the field of environmental protection has not been fully realized.
An esterase-producing strain Acinetobacter soli WCO-12 is provided, which has extensive substrate specificity, is able to catalyze the hydrolysis of p-nitrophenyl ester of C2-C18 chain length, and remains active under high temperature and alkaline conditions. The specific method includes reacting in Tris-HCl buffer and termination with ethanol, and determining the esterase activity by the p-nitrophenol method.
It has achieved efficient hydrolysis of C2-C18 chain length p-nitrophenyl ester, especially at 90°C and pH 9, and is suitable for waste oil treatment and environmental protection.
Smart Images
Figure CN120366129A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microbial technology. More specifically, the present invention relates to an esterase-producing strain and its application. Background Art
[0002] Esterase is a class of enzymes that can catalyze the hydrolysis and synthesis of ester bonds and widely exist in nature, including microorganisms (bacteria, fungi, and actinomycetes), plants, and animal tissues. According to substrate specificity, esterases can be divided into lipases (EC 3.1.1.3) and narrow-sense esterases (EC 3.1.1.1). Lipases usually act on long-chain carboxyl esters (≥C10), while narrow-sense esterases mainly hydrolyze short-chain carboxyl esters (≤C10).
[0003] Esterase (EC 3.1.1.1) is a class of hydrolases widely existing in nature, which can catalyze the hydrolysis and synthesis reactions of ester bonds. Due to its advantages such as wide substrate specificity, mild reaction conditions, and environmental friendliness, esterase shows great application potential in the fields of food, medicine, chemical industry, environmental protection, etc. Summary of the Invention
[0004] One object of the present invention is to solve at least the above problems and / or defects and provide at least the advantages described hereinafter.
[0005] To achieve these objects and other advantages of the present invention, an esterase-producing strain is provided. The esterase-producing strain is WCO-12, and its taxonomic name is Acinetobacter soli. It was registered and preserved in the Guangdong Provincial Microbial Culture Collection Center on March 12, 2025, and the preservation number is GDMCC No: 65974.
[0006] Preferably, the 16S rDNA sequence of the esterase-producing strain is as shown in SEQ ID NO.1.
[0007] An application of the above-mentioned esterase-producing strain in the production of esterase.
[0008] An application of the above-mentioned esterase-producing strain in the preparation of a product for producing esterase.
[0009] Preferably, the esterase-producing strain is used to catalyze the hydrolysis of p-nitrophenyl esters with a C2-C 18 chain length.
[0010] Preferably, Tris-HCl buffer solution and reaction substrate solution are mixed evenly, then the crude enzyme solution obtained by culturing the esterase-producing strain is added, reacted in a 40°C water bath for 5-15 min, 95 wt% ethanol is added to terminate the reaction, and the light absorption value is measured at 410 nm. The esterase activity is measured by the p-nitrophenol method.
[0011] Preferably, the concentration of the Tris-HCl buffer solution is 40 to 60 mmol / L, and the pH is 7.0 to 11.0; the volume ratio of the Tris-HCl buffer solution, the reaction substrate solution and the crude enzyme solution is 4 to 5:1:0.3 to 0.7; the volume ratio of the reaction substrate solution and 95 wt% ethanol is 1:8 to 10.
[0012] Preferably, the preparation method of the reaction substrate solution is: adding p-nitrophenyl ester into isopropanol and mixing evenly to obtain the reaction substrate solution; wherein, the mass-volume ratio of the p-nitrophenyl ester and isopropanol is 1 to 5 mg:1 mL.
[0013] Preferably, the p-nitrophenyl ester is any one of 4-Nitrophenyl acetate (ρ-NPC2), 4-Nitrophenyl butyrate (ρ-NPC4), 4-Nitrophenyl hexanoate (ρ-NPC6), 4-Nitrophenyl octanoate (ρ-NPC8), 4-Nitrophenyl decanoate (ρ-NPC 10 ), 4-Nitrophenyl laurate (ρ-NPC 12 ), 4-Nitrophenyl myristate (ρ-NPC 14 ), 4-Nitrophenyl palmitate (ρ-NPC 16 ), 4-Nitrophenyl stearate (ρ-NPC 18 ).
[0014] Preferably, the preparation method of the crude enzyme solution is: streaking and activating the esterase-producing strain, picking a single colony into 10 to 30 mL of LB liquid medium, culturing in a shaker flask at 30 °C and 150 to 300 r / min for 12 to 24 h to prepare a seed solution, inoculating the seed solution into a fermentation flask containing 30 to 80 mL of LB medium at an inoculation amount of 0.5 to 2%, culturing with shaking for 36 to 72 h to obtain a fermentation broth; after the fermentation broth is shaken well, centrifuging at 6000 to 10000 r / min for 3 to 8 min, and taking the supernatant as the crude enzyme solution.
[0015] Preferably, the optimal reaction substrate of the esterase produced by the esterase-producing strain is 4-Nitrophenyl acetate.
[0016] The present invention has at least the following beneficial effects:
[0017] (1) The present invention provides an esterase-producing strain, Acinetobacter soli WCO-12. Its physical and chemical properties are as follows: The colony is white, flat and round, with an irregular and scattered edge. The colony is viscous and its surface is moist and opaque. Microscopic observation shows that it is spherical or short rod-shaped, without spores, non-motile, with diploids, and it is a Gram-negative bacterium. It grows well at 30 °C in LB medium under aerobic conditions and can grow to the logarithmic phase in 12 - 16 h.
[0018] (2) The esterase-producing strain Acinetobacter soli WCO-12 isolated in the present invention has a special isolation environment. This strain WCO-12 is an alkaline esterase-producing strain. The optimal reaction pH of the esterase it produces is 9, and it has hydrolytic activity against p-nitrophenyl esters with C2 - C 18 chain lengths, and the optimal reaction substrate is 4-Nitrophenylacetate (ρ-NPC2). The optimal enzyme reaction temperature is 90 °C, and it has good research and application value in aspects such as waste oil treatment and environmental protection.
[0019] Other advantages, objectives and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 shows the morphological characteristics of the strain WCO-12 of the present invention, where: a is the colony morphology of the strain WCO-12; b is the microscopic morphology diagram of the Gram staining of the strain WCO-12.
[0021] Figure 2 shows the phylogenetic analysis of the strain WCO-12 of the present invention based on the 16S rDNA sequence;
[0022] Figure 3 shows the standard curve drawn in Example 2 of the present invention;
[0023] Figure 4 shows the hydrolytic activity of the strain WCO-12 against p-nitrophenyl ester substrates with different chain lengths measured by the p-nitrophenol method in Example 2 of the present invention;
[0024] Figure 5 shows the relative enzyme activity of the esterase secreted by the strain WCO-12 at different temperatures in Example 3 of the present invention;
[0025] Figure 6 shows the relative enzyme activity of the esterase secreted by the strain WCO-12 at different pH values in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it according to the text of the specification.
[0027] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.
[0028] Example 1
[0029] An esterase-producing strain, the esterase-producing strain is WCO-12, and its taxonomic name is Acinetobacter soli. It was deposited in the Guangdong Microbial Culture Collection Center (Address: 5th Floor, Experimental Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province) on March 12, 2025, and the deposit number is GDMCC No: 65974.
[0030] The screening, purification and identification process of strain WCO-12:
[0031] The composition ratio of the enrichment medium is: yeast extract 0.2 g / L, NaCl 0.5 g / L, Na2HPO4 3.5 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.5 g / L;
[0032] The composition ratio of the rhodamine grease medium is: 175 mL of LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) + 25 mL of emulsion. Add 15 g / L of agar powder to the solid medium; Prepare a 10 wt% rhodamine B solution and filter it with four layers of gauze and a bacterial microporous filter respectively, and set aside; Add the rhodamine B solution according to the ratio of medium: 10 wt% rhodamine B = 1000:1 and shake well for staining;
[0033] Preparation of the emulsion: Prepare a 4 wt% polyvinyl alcohol (PVA) solution and filter it with four layers of gauze and a bacterial microporous filter respectively, and set aside; Mix 4 wt% PVA and olive oil at a volume ratio of 3:1, and treat it with an ultrasonic crusher on ice for 20 min. Take it out and stir well every 3-5 minutes until the solution becomes milky white and there are no yellow oil beads on the surface, which is a qualified emulsion (the emulsion is prepared and used immediately, and the validity period at 4°C is 1 week).
[0034] Collect soil samples that have been contaminated by oil for a long time within the scope of Southwest University of Science and Technology, and collect the soil 1-2 cm below the soil surface. Take 5 g of soil sample in 45 mL of physiological saline, shake well at 30°C and 200 r / min for 1 h. After standing, take 1 mL of the supernatant and put it into 20 mL of enrichment medium, shake and culture at 30°C and 200 r / min for 24 h. The bacterial liquid after overnight culture for 24 h is diluted by 10 -1 -10-8 Gradient dilution, select 10 -6 -10 -8 For each of the 3 gradients, take 100 μL and evenly coat it on the rhodamine lipid screening plate. Place the coated plate in a 30°C constant temperature incubator and incubate it in the dark for 2 days. Mark the colonies that produce clear zones. Pick all the colonies from each plate and streak-purify them on an LB plate 2 - 3 times. Pick a single colony and culture it in 20 mL of LB liquid medium until the OD 600 reaches 0.6 - 0.8, then preserve the strain with glycerol.
[0035] Pick a single colony of the purified strain and perform Gram staining to observe the colony morphology. As Figure 1 shown, its colonies are white, flat and round, with irregular edges and a scattered shape. The colonies are viscous and the surface is moist and opaque. Microscopic observation shows that they are spherical or short rod-shaped, without spores, non-motile, with the presence of diploids, and are Gram-negative bacteria.
[0036] Amplify and sequence the 16S rDNA sequence of strain WCO-12. Its 16S rDNA sequence is shown in SEQ ID NO.1. After BLAST alignment and N-J method clustering analysis ( Figure 2 ), it is determined that this strain is a bacterium of the genus Acinetobacter, and its taxonomic status is Acinetobacter soli. The Acinetobacter genus strain WCO-12 has a high genetic relationship with Acinetobacter baumannii in the same genus, and they are in two different evolutionary branches. It has a genetic relationship with Pseudomonas, and Pseudomonas is an important source of bacterial lipase.
[0037] Example 2
[0038] Determination of the esterase activity secreted by strain WCO-12 under different substrates:
[0039] Drawing of the standard curve: Weigh 0.08346 g of p-nitrophenol, first dissolve it with a small amount of 95% ethanol, and then make the volume up to 100 mL with water, with a concentration of 6 mmol / L. Add different amounts of p-nitrophenol solution and 50 mmol / L Tris-HCl (pH 8.0) buffer according to Table 1. Then add 0.9 mL of 95wt% ethanol to each tube, with a total volume of 1.5 mL. Measure the light absorption value at 410 nm and draw the standard curve, with 3 replicates for each group; The standard curve is as Figure 3 shown;
[0040] Table 1
[0041]
[0042] The test strain (strain WCO-12) was streaked and activated, and a single colony was picked and cultured in 20 mL of LB liquid medium at 30 °C with shaking at 200 r / min for 16 h to prepare a seed solution. The seed solution was inoculated into a fermentation flask containing 50 mL of LB medium at an inoculation amount of 1% (v / v) and cultured with shaking for 48 h to obtain a fermentation broth; after the fermentation broth was fully shaken, it was centrifuged at 8000 r / min for 5 min, and the supernatant was taken as the crude enzyme solution; using isopropanol as the solvent, 45 mg of p-nitrophenyl ester substrate was weighed into 15 mL of isopropanol and mixed evenly to prepare a reaction substrate solution (the substrate was prepared and used immediately); reaction system: first, 450 μL of 50 mmol / L Tris-HCl (pH 8.0) buffer solution and 100 μL of the reaction substrate solution were added and mixed evenly, then 50 μL of the crude enzyme solution was added, and the reaction was carried out in a 40 °C water bath for 5 min. Then, 900 μL of 95 wt% ethanol was quickly added to terminate the reaction, and the light absorption value was measured at 410 nm; each group was repeated 3 times, and the blank control was the inactivated enzyme solution treated with boiling water for 20 min. One enzyme activity unit (U) was defined as the amount of enzyme required to release 1 μmoL of p-nitrophenol per minute under the experimental conditions:
[0043] Enzyme activity calculation formula: A = ([A1 - A0] × K + C0) × V1 × n / (V2 × t)
[0044] In the formula: A--sample enzyme activity (U / mL); A1--absorbance OD value of the sample enzyme solution; A0--blank absorbance OD value of the corresponding enzyme solution; K--slope of the p-nitrophenol standard curve; C0--intercept of the p-nitrophenol standard curve; n--dilution factor; V1--volume of the reaction solution / mL; V2--volume of the enzyme solution / mL; t--reaction time / min;
[0045] Among them, the p-nitrophenyl ester substrates used were p-nitrophenyl acetate (4-Nitrophenyl acetate, ρ-NPC2), p-nitrophenyl butyrate (4-Nitrophenyl butyrate, ρ-NPC4), p-nitrophenyl caproate (4-Nitrophenyl butyrate, ρ-NPC6), p-nitrophenyl octanoate (4-Nitrophenyl octanoate, ρ-NPC8), p-nitrophenyl decanoate (4-Nitrophenyl decanoate, ρ-NPC 10 ), p-nitrophenyl laurate (4-Nitrophenyllaurate, ρ-NPC 12 ), p-nitrophenyl myristate (4-Nitrophenyl myristate, ρ-NPC 14 ), p-nitrophenyl palmitate (4-Nitrophenyl palmitate, ρ-NPC16 ) 4-Nitrophenyl stearate (ρ-NPC) 18 )
[0046] The results are as Figure 4 shown. The hydrolysis activity of the esterase of strain WCO-12 towards the C2 substrate (4-nitrophenyl acetate) reached 573.77 U / L, and the hydrolysis activities towards C4, C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 chain length substrates were 442.23 U / L, 383.77 U / L, 358.38 U / L, 325.31 U / L, 368.77 U / L, 235.31 U / L, 200.69 U / L and 163 U / L respectively. The results indicate that strain WCO-12 or the esterase it produces can be used to catalyze the hydrolysis of esters. Its esterase has hydrolysis activity towards p-nitrophenyl esters with C2-C 18 chain lengths and can be used to catalyze the hydrolysis of p-nitrophenyl esters with C2-C 18 chain lengths. The optimal reaction substrate is 4-nitrophenyl acetate.
[0047] Example 3
[0048] Determination of the activity of the esterase secreted by strain WCO-12 at different temperatures and pH values:
[0049] A single-factor experiment was used to analyze and determine the activity of the esterase produced by strain WCO-12 fermentation at different temperatures and pH values
[0050] Specific experimental method: The strain WCO-12 stored at -80 °C was taken out and streaked on an LB plate for activation. A single colony was picked and inoculated into an LB liquid medium and continuously cultured at 30 °C and 200 r / min for 12 h to prepare a seed solution. According to the initial OD 600 = 0.1 inoculation amount, the seed solution was inoculated into the LB medium. After culturing at 30 °C and 200 r / min for 36 h, the supernatant was taken by centrifugation as the crude enzyme solution. The activity of the crude enzyme solution was detected by the p-nitrophenol method (the substrate is 4-nitrophenyl acetate). Among them, the crude enzyme solution was reacted in a water bath at 30 - 100 °C for 5 min, and the activity of the esterase at each temperature was calculated, as Figure 5 shown; The crude enzyme solution was prepared in the same way as above. The activity of the crude enzyme solution was detected by the p-nitrophenol method (the substrate is 4-nitrophenyl acetate) at 40 °C. Among them, 50 mmol / L Tris-HCl buffer solutions with pH values of 7, 8, 9, 10, and 11 were added to the enzyme activity determination system to set a pH gradient, and the activity of the esterase under each pH condition was calculated, as Figure 6 shown.
[0051] FromFigure 5 It can be seen that the esterase produced by WCO-12 is active at 30-100 °C, and the optimal enzyme reaction temperature is 90 °C; from Figure 6 It can be seen that the esterase produced by WCO-12 is active at pH = 7-11, and the optimal reaction pH is 9. WCO-12 is a strain producing alkaline esterase. In summary, the esterase secreted by WCO-12 of the present invention has excellent high temperature and alkali resistance characteristics and can be applied to high temperature and alkaline environments.
[0052] The strain WCO-12 of the present invention can be used as an ideal source for developing novel esterases with excellent enzymatic properties, can be applied to daily production, especially for the production of esterases, and can be used to prepare products for producing esterases.
[0053] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.
Claims
1. An esterase-producing strain, characterized in that, The esterase-producing strain is WCO-12, which is taxonomically named Acinetobacter soli. It was registered and preserved in the Guangdong Provincial Culture Collection Center of Microorganisms on March 12, 2025, and the preservation number is GDMCC No: 65974.
2. The esterase-producing strain according to claim 1, characterized in that The 16S rDNA sequence of the esterase-producing strain is as shown in SEQ ID NO.
1.
3. Use of an esterase-producing strain as described in claim 1 in the production of esterase.
4. Use of an esterase-producing strain as described in claim 1 in the preparation of a product for producing esterase.
5. Use of the esterase-producing strain according to claim 3 in the production of esterase, characterized in that, The esterase-producing strain is used to catalyze the hydrolysis of p-nitrophenyl esters with a C2-C 18 chain length.
6. Use of the esterase-producing strain according to claim 5 in the production of esterase, characterized in that, Mix the Tris-HCl buffer solution and the reaction substrate solution, then add the crude enzyme solution obtained by culturing the esterase-producing strain, react in a 40°C water bath for 5 - 15 min, add 95 wt% ethanol to terminate the reaction, measure the light absorption value at 410 nm, and determine the esterase activity using the p-nitrophenol method.
7. Use of the esterase-producing strain according to claim 6 in the production of esterase, characterized in that, The concentration of the Tris-HCl buffer solution is 40 - 60 mmol / L, and the pH is 7.0 - 11.0; the volume ratio of the Tris-HCl buffer solution, the reaction substrate solution, and the crude enzyme solution is 4 - 5:1:0.3 - 0.7; the volume ratio of the reaction substrate solution and 95 wt% ethanol is 1:8 - 10.
8. Use of the esterase-producing strain according to claim 6 in the production of esterase, characterized in that, The preparation method of the reaction substrate solution is: add p-nitrophenyl ester to isopropanol and mix well to obtain the reaction substrate solution; wherein, the mass-volume ratio of the p-nitrophenyl ester and isopropanol is 1 - 5 mg:1 mL.
9. Use of the esterase-producing strain according to claim 8 in the production of esterase, characterized in that, The p-nitrophenyl ester is any one of p-nitrophenyl acetate, p-nitrophenyl butyrate, p-nitrophenyl caproate, p-nitrophenyl octanoate, p-nitrophenyl decanoate, p-nitrophenyl laurate, p-nitrophenyl myristate, p-nitrophenyl palmitate, p-nitrophenyl stearate.
10. Use of the esterase-producing strain according to claim 6 in the production of esterase, characterized in that, The preparation method of the crude enzyme solution is: streak and activate the esterase-producing strain, pick a single colony and culture it in 10 - 30 mL of LB liquid medium at 30°C with a shaking speed of 150 - 300 r / min for 12 - 24 h to prepare a seed solution. The seed solution is inoculated into a fermentation flask containing 30 - 80 mL of LB medium at an inoculation amount of 0.5 - 2%, and cultured with shaking for 36 - 72 h to obtain a fermentation broth; after the fermentation broth is fully shaken, centrifuge it at 6000 - 10000 r / min for 3 - 8 min, and take the supernatant as the crude enzyme solution.