CN112779186B - Intermediate-temperature bacterium for producing ester hydrolase simultaneously resisting high salinity, organic solvent and detergent and application thereof - Google Patents
Intermediate-temperature bacterium for producing ester hydrolase simultaneously resisting high salinity, organic solvent and detergent and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microorganisms, and discloses a mesophilic bacterium for producing ester hydrolase which can tolerate high salinity, organic solvent and detergent simultaneously and application thereof. The mesophilic bacteria SIOC 00125 have the function of secreting ester hydrolase, and the secreted ester hydrolase has high catalytic activity; has strong tolerance to high salinity. At the same time, the enzyme can tolerate Sr2+、Mg2+、Ba2+Plasma metal ions; EDTA has promoting effect on enzyme activity; at the same time, the enzyme can resist various organic solvents and detergents. The ester hydrolase has high catalytic activity on short-chain fatty acid, and the most suitable substrate is p-nitrophenol caproate. The thermal stability and the strong adaptability to high salinity of the ester hydrolase can be applied to industrial production under the salt-containing conditions of wastewater treatment, fine chemical engineering, pharmacy, environmental remediation and the like.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a mesophilic bacterium for producing ester hydrolase which can simultaneously tolerate high salinity, organic solvent and detergent and application thereof.
Background
Ester hydrolases are widely found in microorganisms, animals and plants and are a generic term for a class of hydrolases capable of catalyzing the hydrolysis or synthesis of fatty acid ester bonds. The ester hydrolase participates in a plurality of metabolic processes of organisms, plays an important role in ester transportation, cell structure construction and energy metabolism, and is one of enzymes necessary for maintaining the survival of the living body. The sixth family consists of phospholipases and carboxylesterases, with high homology (40%) to lysophospholipases from eukaryotes, with broad substrate specificity. The wide substrate spectrum and functional diversity make the family of hydrolytic enzymes have wide potential application values in the fields of food, medicine, textile, washing, sewage treatment, environmental remediation and the like.
However, in some application scenarios where the hydrolysis conditions are severe, such as high salinity, organic solvent-containing and detergent-removing agents, the enzyme activity of most ester hydrolases in the prior art is severely inhibited in these hydrolysis environments, thereby limiting the practical application thereof. Although some ester hydrolases can resist high salinity or organic solvents, the ester hydrolases with single characteristic cannot meet the practical application requirements for some application scenes with high salinity, organic solvents and detergents. Therefore, there is a need to develop more ester hydrolases with various characteristics to meet the requirements of different application scenarios.
Disclosure of Invention
The invention provides a mesophilic bacterium for producing ester hydrolase which can resist high salinity, organic solvent and detergent simultaneously and application thereof. The invention separates a strain of mesophilic bacteria SIOC 00125 from deep seawater of the western Pacific ocean, and the ester hydrolase produced by the strain has high catalytic activity and high tolerance to high salinity, organic solvents and detergents, and can be used for ester degradation under various severe conditions and biocatalysis and transformation of other ester compounds. Can be used in the industrial fields of fine chemistry industry, pharmacy, washing, wastewater treatment, environmental remediation and the like.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a mesophilic bacterium that produces an ester hydrolase that is tolerant to both high salinity, organic solvents and detergents, isolated from deep seawater in the western pacific ocean, designated SIOC 00125, which has been deposited at the guangdong provincial collection of microorganisms at 29.12.2020, with the deposition address of building 5, condemnation No. 59, courtyard 100, condemnation, guangzhou city, with the deposition number gdmccno: 61385, respectively; the microorganism is classified and named as mesophilic bacteriaMuricauda oceanensis。
The mesophilic bacterium SIOC 00125 has the function of secreting ester hydrolase, the secreted ester hydrolase has high catalytic activity, high activity (more than 50% of the maximum enzyme activity) is kept between the pH value =6.0-10.5, and the optimal pH value is 9.0; the temperature range is 15-70 ℃, and the optimal temperature is 55 ℃; incubating for 4 hours at 15-50 ℃, and still keeping more than 80% of activity; has stronger tolerance to high salinity, the enzyme activity reaches the highest under the NaCl concentration of 1.5M, about 2 times is improved, and when the NaCl concentration reaches 5M, about 100 percent of the enzyme activity can still be maintained. At the same time, the enzyme can tolerate Sr2+、Mg2+、Ba2+Divalent metal ions; EDTA has promoting effect on enzyme activity; meanwhile, the enzyme can also tolerate various organic solvents and detergents (DMSO, glycerol, methanol, Tween 20, Tween 80 and TritonX-100), and even the enzyme activity is improved to different degrees in the environments of DMSO, glycerol, methanol, Tween 20, Tween 80 and the like. The ester hydrolase has high catalytic activity on short-chain fatty acid, and the most suitable substrate is p-nitrophenol caproate. The thermal stability and the strong adaptability to high salinity of the ester hydrolase can be applied to industrial production under the salt-containing conditions of wastewater treatment, fine chemical engineering, pharmacy, environmental remediation and the like.
In a second aspect, the present invention provides a mutant of mesophilic bacteria producing an esterhydrolase that is tolerant to high salinity, organic solvents and detergents simultaneously, said mutant being obtained by subjecting said mesophilic bacteria to mutagenesis, acclimation, genetic recombination or natural mutation.
Preferably, the mutant has a nucleotide sequence at least 90% homologous to the mesophilic bacterium, and the mutant has at least 90% ester hydrolase activity with an ester hydrolase secreted by the mesophilic bacterium.
Further preferably, the mutant has a nucleotide sequence at least 95% homologous to the mesophilic bacterium and has at least 95% or more ester hydrolase activity with an ester hydrolase secreted by the mesophilic bacterium.
Most preferably, the mutant has a nucleotide sequence at least 99% homologous to the mesophilic bacterium and has at least 99% or more ester hydrolase activity with an ester hydrolase secreted by the mesophilic bacterium.
In a third aspect, the present invention provides a bacterial culture comprising said mesophilic bacterium or comprising said mutant.
Preferably, the bacterial culture is a bacterial solution or a bacterial agent.
In a fourth aspect, the present invention provides an ester hydrolase secreted by said mesophilic bacterium or said mutant.
In a fifth aspect, the present invention provides a method for preparing the ester hydrolase, comprising the steps of:
(1) culturing said mesophilic bacterium or said mutant under conditions conducive to the production of an ester hydrolase.
(2) Recovering, separating and purifying the ester hydrolase.
In the production method of the present invention, the strain is cultured in a nutrient medium suitable for producing the ester hydrolase using a method known in the art. For example, the strain may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium. The cultivation takes place using methods known in the art in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or may be prepared according to published compositions.
Preferably, in step (2), the resulting ester hydrolase can be recovered using methods known in the art. For example, recovery from the nutrient medium may be by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.
Preferably, in step (2), purification can be accomplished by a variety of methods known in the art including, but not limited to, chromatographic (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion) or differential solubility (e.g., ammonium sulfate precipitation) methods and the like.
In a sixth aspect, the invention provides the use of said mesophilic bacterium or said mutant or said cell culture or said ester hydrolase in catalyzing the hydrolysis of esters.
The invention also provides the industrial application of the substances, such as the substances used for catalyzing ester hydrolysis. Esterase activity assays indicate that the ester hydrolase has esterase activity and can be used for hydrolyzing C2-C10 fatty acid esters, such as p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8) and p-nitrophenol decanoate (C10).
The determination shows that the ester hydrolase has better catalytic activity on short acyl carbon chain esters and hydrolysis activity on short chain esters is better than that of long chain esters. Thus, it is preferred to use for the catalytic hydrolysis of C2-C8 short chain fatty acid esters, such as p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8), the most suitable short chain fatty acid ester substrate being a p-nitrophenol ester having a short carbon chain of C6, such as p-nitrophenol hexanoate.
Compared with the prior art, the invention has the beneficial effects that:
(1) the mesophilic bacterium SIOC 00125 has the function of secreting ester hydrolase, the secreted ester hydrolase has high catalytic activity, high activity (more than 50% of the maximum enzyme activity) is kept between the pH value =6.0-10.5, and the optimal pH value is 9.0; the temperature range is 15-70 ℃, and the optimal temperature is 55 ℃; incubating for 4 hours at 15-50 ℃, and still keeping more than 80% of activity; has stronger tolerance to high salinity, the enzyme activity reaches the highest under the NaCl concentration of 1.5M, about 2 times is improved, and when the NaCl concentration reaches 5M, about 100 percent of the enzyme activity can still be maintained. Can resist Sr2+、Mg2+、Ba2+Divalent metal ions; EDTA has promoting effect on enzyme activity; at the same time, the enzyme can resist various organic solvents and detergents. The enzyme activity is improved to different degrees in DMSO, glycerol, methanol, Tween 20, Tween 80 and other environments.
(2) The ester hydrolase produced by the mesophilic bacteria SIOC 00125 has high catalytic activity on short-chain fatty acid, and the most suitable substrate is p-nitrophenol caproate. The ester hydrolase has high thermal stability and strong adaptability to high salinity, can be applied to environments including acidic, neutral and alkaline hydrolysis environments, and provides the low-cost thermal stable hydrolase for subsequent industrial application. The production of the enzyme can show important economic and social values in production processes of detergents, wastewater treatment, fine chemical industry, pharmacy, environmental remediation and other environments with different pH values.
Drawings
FIG. 1 is a diagram showing the substrate specificity of ester hydrolase Muo 3. Wherein, C2: p-nitrophenol acetate; c4: p-nitrophenol butyrate, C6: p-nitrophenol hexanoate; c8: p-nitrophenol octanoate; c10: p-nitrophenol decanoate; c12, p-nitrophenol dodecanoate; c14 p-nitrophenol myristate; c16 p-nitrophenol palmitate. The measurement was 100% when the substrate was defined as C2.
FIG. 2 is a pH diagram of the optimum reaction of the ester hydrolase Muo 3.
FIG. 3 is a graph showing the optimum reaction temperature of the ester hydrolase Muo 3.
FIG. 4 is a graph showing the thermal stability of the ester hydrolase Muo3 at different temperatures.
FIG. 5 is a graph showing the effect of divalent cations on the activity of ester hydrolase Muo 3.
FIG. 6 is a graph showing the effect of organic solvents and detergents on the activity of ester hydrolase Muo 3.
FIG. 7 is a graph showing the effect of NaCl on the activity of ester hydrolase Muo 3.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
A mesophilic bacterium which produces an ester hydrolase that is tolerant to both high salinity, organic solvents and detergents, is isolated from deep seawater in the Western Pacific ocean, is designated SIOC 00125, and has been deposited at the Guangdong province collection of microorganisms at 29 months 12-2020, with the deposit number GDMCC NO: 61385, respectively; the microorganism is classified and named as mesophilic bacteriaMuricauda oceanensis。
A mutant of mesophilic bacteria producing an ester hydrolase which is tolerant to high salinity, organic solvents and detergents simultaneously, said mutant being obtained by mutagenesis, acclimation, genetic recombination or natural mutation of said mesophilic bacteria.
Preferably, the mutant has a nucleotide sequence at least 90% homologous to the mesophilic bacterium, and the mutant has at least 90% ester hydrolase activity with an ester hydrolase secreted by the mesophilic bacterium. Further preferably, the mutant has a nucleotide sequence at least 95% homologous to the mesophilic bacterium and has at least 95% or more ester hydrolase activity with an ester hydrolase secreted by the mesophilic bacterium. Most preferably, the mutant has a nucleotide sequence at least 99% homologous to the mesophilic bacterium and has at least 99% or more ester hydrolase activity with an ester hydrolase secreted by the mesophilic bacterium.
A bacterial cell culture containing said mesophilic bacterium or said mutant. Preferably, the bacterial cell culture is a bacterial solution or a bacterial agent.
An ester hydrolase secreted by said mesophilic bacterium or said mutant.
A method for preparing the ester hydrolase, comprising the steps of:
(1) culturing said mesophilic bacterium or said mutant under conditions conducive to the production of an ester hydrolase.
(2) Recovering, separating and purifying the ester hydrolase.
In the production method of the present invention, the strain is cultured in a nutrient medium suitable for producing the ester hydrolase using a method known in the art. For example, the strain may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium. The cultivation is carried out using methods known in the art in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or may be prepared according to published compositions.
Preferably, in step (2), the resulting ester hydrolase can be recovered using methods known in the art. For example, recovery from the nutrient medium may be by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In addition, purification can be achieved by a variety of methods known in the art, including but not limited to chromatographic (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion) or differential solubility (e.g., ammonium sulfate precipitation) methods and the like.
The invention provides the application of the mesophilic bacteria, the mutant, the thallus culture or the ester hydrolase in catalyzing ester hydrolysis. The invention also provides the industrial application of the substances, such as the substances used for catalyzing ester hydrolysis. Esterase activity assays indicate that the ester hydrolase has esterase activity and can be used for hydrolyzing C2-C10 fatty acid esters, such as p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8) and p-nitrophenol decanoate (C10). The determination shows that the ester hydrolase has better catalytic activity on short acyl carbon chain esters and hydrolysis activity on short chain esters is better than that of long chain esters. Thus, it is preferred to use for the catalytic hydrolysis of C2-C8 short chain fatty acid esters, such as p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8), the most suitable short chain fatty acid ester substrate being a p-nitrophenol ester having a short carbon chain of C6, such as p-nitrophenol hexanoate.
Example 1: activity detection of ester hydrolase Muo3 produced by mesophilic bacterium SIOC 00125
The activity of the purified ester hydrolase Muo3 was determined by the p-nitrophenol hexanoate method. The method comprises the following specific operations: a1 ml reaction system including 1 mM of p-nitrophenol caproate, 100 mM of CHES-NaOH buffer (pH 9.0) and 0.63. mu.g of pure enzyme protein was used to continuously measure the absorbance A at 55 ℃ using an ultraviolet-visible spectrophotometer (Beckman DU800, USA)405For 2 min, the inactivated enzyme solution was used as a control for zeroing. One unit of enzyme activity is defined as the amount of enzyme required to catalytically produce l μmol of p-nitrophenol from p-nitrophenol ester per minute. The esterase activity was found to be 1434662.29U/mg.
Example 2: substrate specificity assay for ester hydrolase Muo3
The substrate specificity of the ester hydrolase Muo3 was analyzed using the system (1 ml): 100 mM CHES-NaOH buffer (pH 9.0), 1 mM substrate, 0.63. mu.g of pure enzyme protein was added, and absorbance A was continuously measured at 55 ℃405And 2 min. The substrates used for the assay were: p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8), p-nitrophenol decanoate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol tetradecanoate (C14), p-nitrophenol hexadecanoate (C16). The measurement shows that Muo3 has higher catalytic activity to p-nitrophenol ester (C2, C4, C6 and C8) with shorter acyl carbon chains, wherein the catalytic activity is the highest when the substrate is p-nitrophenol acetate (C2) (figure 1). The result shows that the hydrolase Muo3 has better catalytic activity on esters with shorter acyl carbon chains, and the hydrolytic activity on short-chain esters is better than that on long-chain esters.
Example 3: analysis of optimum reaction conditions for ester hydrolase Muo3
The optimum reaction pH of the ester hydrolase Muo3 is determined in the range of 4.0 to 10.5. The specific operation is as follows: the absorbance A was continuously measured at 45 ℃ by adding 1 mM p-nitrophenol acetate and 0.63. mu.g pure enzyme protein to buffers of different pH348And 2 min. The buffers used for the assay were: 100 mM citric acid-sodium citrate buffer (pH 3.0-6.0), 100 mM potassium dihydrogen phosphate-sodium hydroxide buffer (pH 6.0-8.0), 100 mM Tris hydrochloric acid buffer (pH 7.5-9.0) and 50 mM 2-cyclohexylaminoethanesulfonic acid-sodium hydroxide buffer (pH 9.0-10.5). The measurement results showed that Muo3 had an optimum reaction pH of 9.0 and activity in the pH range of 5.0 to 10.5 (FIG. 2).
The optimum reaction temperature of the ester hydrolase Muo3 is measured within the range of 15-70 ℃. The specific operation is as follows: 1 ml of the reaction system was added with 1 mM of p-nitrophenol acetate, 100 mM of CHES-NaOH buffer (pH 9.0) and 0.63. mu.g of pure enzyme protein, and absorbance A was continuously measured at 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 ℃ respectively405And 2 min. The measurement results show that the reaction temperature range of Muo3 is 15-70 ℃, and the optimum reaction temperature is 55 ℃ (FIG. 3).
Example 4: enzymatic stability analysis of ester hydrolase Muo3
The thermal stability analysis of the ester hydrolase Muo3 was specifically performed by: a temperature gradient was established for every 10 ℃ in the temperature interval 20 to 60 ℃. Respectively incubating the enzyme solution for 1 h, 2 h and 4h under each temperature gradient condition, and determining the activity of the enzyme; the living body measuring system comprises the following steps: 1 ml of the reaction system was added with 1 mM of p-nitrophenol acetate, 100 mM of CHES-NaOH buffer (pH 9.0) and 0.63. mu.g of pure enzyme protein, and the absorbance A was continuously measured at 55 ℃405And 2 min. The result shows that Muo3 can still maintain more than 80% of activity under the condition of incubation for 4 hours at 20-50 ℃ (figure 4), which indicates that Muo3 has better thermal stability.
The determination of the effect of divalent cations on the activity of hydrolase Muo3 was performed by: 10 mM Ba was added to the reaction system2+、Ca2+、Cd2+、Co2+、Cu2+、Mg2+、Mn2+、Ni2+、Sr2+、Zn2+And ethylenediaminetetraacetic acid (EDTA), and measuring the enzyme activity. The enzyme activity measuring system comprises: 1 ml of the reaction system was charged with 1 mM of p-nitrophenol acetate, 100 mM of CHES-NaOH buffer (pH 9.0) and 0.63. mu.g of pure enzyme protein, and the absorbance A was continuously measured at 55 ℃405And 2 min. The determination result shows that Muo3 activity is changed by Zn2+、Ni2+、Mn2+、Cu2+、Co2+And Cd2+Significantly inhibit, Sr2+、Mg2+And Ba2+The enzyme activity of Muo3 was hardly affected, and EDTA promoted the enzyme activity (FIG. 5).
The determination of the effect of organic solvents and detergents on the activity of the hydrolase Muo3 was performed by: organic solvents are added into the reaction system respectively to determine the activity of the enzyme. The amount and kind of the added organic solvent are 15% (v/v): acetone (Acetone), Acetonitrile (Acetonitrile), Ethanol (Ethanol), Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), Glycerol (Glycerol), Isopropanol (Isopropanol) and Methanol (Methanol); the amount and type of detergent added was 1% (v/v): tween 20 (Tween 20), Tween 80 (Tween 80), TritonX-100 and SDS, and the enzyme activity measuring system comprises: 1 ml of the reaction system was charged with 1 mM of p-nitrophenol acetate, 100 mM of CHES-NaOH buffer (pH 9.0) and 0.63. mu.g of pure enzyme protein, and the absorbance A was continuously measured at 55 ℃405And 2 min. The test results show that acetone, acetonitrile, ethanol and SDS have obvious inhibition effect on Muo3 activity (figure 6).
The specific operation of the NaCl effect on the activity of the hydrolase Muo3 is as follows: NaCl was added to the reaction system at various concentrations to measure the enzyme activity. The NaCl concentration is 0-5M. The enzyme activity measuring system comprises: 1 ml of the reaction system was added with 1 mM of p-nitrophenol acetate, 100 mM of CHES-NaOH buffer (pH 9.0) and 0.63. mu.g of pure enzyme protein, and the absorbance A was continuously measured at 55 ℃405And 2 min. The experimental results show that the enzyme activity of the esterase Muo3 is improved to different degrees after NaCl is added (figure 7), and the enzyme activity reaches the highest at 1.5M and is improved by about 2 timesWhen the NaCl concentration reaches 5M, the enzyme activity can still be maintained at about 100%.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (5)
1. A mesophilic bacterium that produces an ester hydrolase that is tolerant to high salinity, organic solvents, and detergents at the same time, characterized in that: the mesophilic bacteria are named as SIOC 00125 and have been preserved in Guangdong provincial microorganism culture collection center at 29 months 12 in 2020, and the preservation numbers are GDMCC NO: 61385, respectively; the microorganism is classified and named as mesophilic bacteriaMuricauda oceanensis。
2. A bacterial cell culture comprising the mesophilic bacterium of claim 1.
3. The microbial culture according to claim 2, which is a microbial inoculum.
4. The bacterial culture according to claim 3, which is a bacterial solution.
5. Use of a mesophilic bacterium according to claim 1 or a bacterial culture according to one of claims 2 to 4 for catalysing the hydrolysis of short chain fatty acid esters of p-nitrophenol, C2-C10.
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| CN101603037A (en) * | 2009-07-23 | 2009-12-16 | 安徽师范大学 | A kind of thermostable carboxylesterase gene, proteins encoded and application thereof |
| CN101735967A (en) * | 2009-11-11 | 2010-06-16 | 南京工业大学 | Organic solvent resisting lipase, application thereof and bacteria for producing same |
| CN112094767A (en) * | 2020-06-09 | 2020-12-18 | 浙江万里学院 | Marine sediment-derived lignin degrading bacterium and application thereof in lignin degradation |
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| CN101603037A (en) * | 2009-07-23 | 2009-12-16 | 安徽师范大学 | A kind of thermostable carboxylesterase gene, proteins encoded and application thereof |
| CN101735967A (en) * | 2009-11-11 | 2010-06-16 | 南京工业大学 | Organic solvent resisting lipase, application thereof and bacteria for producing same |
| CN112094767A (en) * | 2020-06-09 | 2020-12-18 | 浙江万里学院 | Marine sediment-derived lignin degrading bacterium and application thereof in lignin degradation |
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| Muricauda maritima sp. nov., Muricauda aequoris sp. nov. and Muricauda oceanensis sp. nov., three marine bacteria isolated from seawater;Guo et al.;《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》;20201023;第70卷(第12期);6240–6250 * |
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