Intermediate-temperature bacterium for producing alkali-resistant, metal ion-resistant and organic solvent ester hydrolase and application thereof
Technical Field
The invention relates to the field of microorganisms, in particular to a medium-temperature bacterium for producing alkali-resistant, metal ion-resistant and organic solvent ester hydrolase and application thereof.
Background
Ester hydrolases are widely found in microorganisms, animals and plants, and are a generic term for a class of hydrolases that can catalyze hydrolysis or synthesis of fatty acid ester bonds. The ester hydrolase is involved 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 living bodies.
The ester hydrolase has the characteristics of broad substrate spectrum, enantioselectivity, less reaction by-products, no need of coenzyme or cofactor participation in the substrate hydrolysis process and the like when participating in the catalytic reaction, so that the ester hydrolase is widely applied to the industrial production fields of food, medicine, chemical industry, energy, sewage treatment and the like and plays an important role in industrial production.
However, in some application scenarios with harsh hydrolysis conditions, such as high alkaline environment, metal ion-containing environment, organic solvent environment, etc., most of the ester hydrolases in the prior art have severely inhibited enzyme activity in these hydrolysis environments, thereby limiting their practical applications and being difficult to meet the practical application requirements. Therefore, there is a need to develop more ester hydrolases that can simultaneously resist high alkalinity, metal ions and organic solvents to meet the requirements of different application scenarios.
Disclosure of Invention
The invention provides a medium-temperature bacterium for producing alkali-resistant, metal ion-resistant and organic solvent ester hydrolase and application thereof. The invention separates a strain of mesophilic bacteria SIOC 00170 from seawater, and the ester hydrolase produced by the strain has high catalytic activity and high tolerance to strong basicity, metal ions and organic solvents, and can be used for ester degradation and biocatalysis and conversion of other ester compounds under various severe conditions. Can be used in the industrial fields of fine chemical 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 medium-temperature bacterium for producing alkali-resistant, metal ion-resistant and organic solvent ester hydrolase, which is separated from seawater, is named as SIOC 00170, is preserved in Guangdong province microorganism strain preservation center in 12-22 months 2020, and has the preservation address of No. 59 building 5 of Michelia Tokyo No. 100, guangzhou city, and the preservation number is GDMCC 61380; the microorganism is classified and named as mesophilic bacteriaRhodophyticola sp.。
The mesophilic bacteria SIOC 00170 have 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 and 11, the optimum pH value is 9.5, and the mesophilic bacteria SIOC 00170 have strong tolerance to alkalinity; the temperature range is 15 to 60 ℃, and the optimal temperature is 50 ℃; the culture medium can be incubated for 4 hours at the temperature of 15 to 50 ℃ and still can keep more than 80 percent of activity; at the same time, the enzyme can tolerate Ca 2+ 、Mn 2+ 、Sr 2+ 、Mg 2+ 、Ba 2+ Plasma metal ions; EDTA has promoting effect on enzyme activity; meanwhile, the enzyme can also tolerate various organic solvents (DMSO, glycerol, methanol and the like), and the enzyme activity is improved even in a glycerol environment. 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 strong alkali and metal ion-containing environments of the ester hydrolase enable the ester hydrolase to be applied to industrial production of detergents, wastewater treatment, fine chemical engineering, pharmacy, environmental remediation and the like.
In a second aspect, the invention provides a mutant of an alkali-resistant, metal ion-resistant and organic solvent ester hydrolase-resistant mesophilic bacterium, which is obtained by mutagenesis, domestication, gene recombination or natural mutation of the mesophilic bacterium.
Preferably, the mutant has a nucleotide sequence at least 90% homologous to the mesophilic bacterium, and the mutant has at least 90% or more 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, it may be recovered from the nutrient medium 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 present 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 to hydrolyze C2-C10-chain 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 esters with shorter acyl carbon chains and better hydrolysis activity on short-chain esters than 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 is a p-nitrophenol ester having a C6 short carbon chain, such as p-nitrophenol hexanoate.
Compared with the prior art, the invention has the beneficial effects that:
(1) The mesophilic bacterium SIOC 00170 has the function of secreting ester hydrolase, the secreted ester hydrolase has high catalytic activity, and the high activity (50% of the maximum enzyme activity) is kept between the pH value =6.0-11Above), the optimum pH is 9.5, and the alkali resistance is strong; the temperature range is 15 to 60 ℃, and the optimal temperature is 50 ℃; the culture medium can be incubated for 4 hours at the temperature of 15 to 50 ℃ and still can keep more than 80 percent of activity; at the same time, the enzyme can tolerate Ca 2+ 、Mn 2+ 、Sr 2+ 、Mg 2+ 、Ba 2+ Plasma metal ions; EDTA has promoting effect on enzyme activity; meanwhile, the enzyme can also tolerate various organic solvents (DMSO, glycerol, methanol and the like), and the enzyme activity is improved even in a glycerol environment.
(2) The ester hydrolase produced by the mesophilic bacteria SIOC 00170 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 strong alkali and metal ion-containing environments of the ester hydrolase enable the ester hydrolase to be applied to industrial production of detergents, wastewater treatment, fine chemical engineering, pharmacy, environmental remediation and the like.
Drawings
FIG. 1 is a substrate specificity chart of an ester hydrolase Aln 10. Wherein, C2: p-nitrophenol acetate; c4: p-nitrophenol butyrate, C6: p-nitrophenol caproate; c8: p-nitrophenol octanoate; c10: p-nitrophenol decanoate; c12, p-nitrophenol dodecanoate; c14 p-nitrophenol myristate; c16, p-nitrophenol hexadecanoate. The measurement was 100% when the substrate was defined as C6.
FIG. 2 is a pH diagram of the optimum reaction of the ester hydrolase Aln 10.
FIG. 3 is a diagram showing the optimum reaction temperature of the ester hydrolase Aln 10.
FIG. 4 is a graph showing the thermal stability of the ester hydrolase Aln10 at different temperatures.
FIG. 5 is a graph showing the effect of divalent cations on the activity of Aln10, an ester hydrolase.
FIG. 6 is a graph showing the effect of organic solvents and detergents on the activity of Aln 10.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
An intermediate temperature bacterium for producing alkali-resistant, metal ion-resistant and organic solvent ester hydrolase,isolated from seawater, named SIOC 00170, and deposited in Guangdong province microorganism culture collection center at 22/12/2020 with the collection number GDMCC 61380; the microorganism is classified and named as mesophilic bacteriaRhodophyticola sp.。
A mutant of an alkali-resistant, metal ion-resistant and organic solvent ester hydrolase-resistant intermediate-temperature bacterium is obtained by carrying out mutagenesis, domestication, gene recombination or natural mutation on the intermediate-temperature bacterium.
Preferably, the mutant has a nucleotide sequence at least 90% homologous to the mesophilic bacterium, and the mutant has at least 90% or more 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 the mesophilic bacterium or the 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 by a variety of methods known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion) or differential solubility (e.g., ammonium sulfate precipitation), among others.
The invention provides 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 to hydrolyze C2-C10-chain 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 esters with shorter acyl carbon chains and better hydrolysis activity on short-chain esters than 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 is a p-nitrophenol ester having a C6 short carbon chain, such as p-nitrophenol hexanoate.
Example 1: activity detection of ester hydrolase Aln10 produced by mesophilic bacteria SIOC 00170
The activity of the purified ester hydrolase Aln10 was determined by the p-nitrophenol hexanoate method. The method comprises the following specific operations: 1 ml of the reaction system contained 1 mM p-nitrophenol hexanoate, 100 mM CHES-NaOH buffer (pH 9.5) and 0.36. Mu.g of pure enzyme protein, and the absorbance A was continuously measured at 50 ℃ for 405 min with an ultraviolet-visible spectrophotometer (Beckman DU800 model, USA) using the inactivated enzyme solution 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 13553.39U/mg.
Example 2: analysis of substrate specificity of Aln10 for ester hydrolase
Substrate specificity analysis of the ester hydrolase Aln10 using the system (1 ml): 100 mM CHES-NaOH buffer (pH 9.5), 1 mM substrate, 0.36. Mu.g of pure enzyme protein was added, and absorbance A was continuously measured at 50 ℃ for 405 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 determination shows that Aln10 has higher catalytic activity to p-nitrophenol esters (C2, C4, C6 and C8) with shorter acyl carbon chains, wherein the catalytic activity is the highest when the substrate is p-nitrophenol caproate (C6) (figure 1). The results show that the hydrolase Aln10 has better catalytic activity on acyl carbon chain shorter lipid substances and hydrolysis activity on short-chain lipids is better than that on long-chain lipids.
Example 3: analysis of optimum reaction conditions for Aln10 ester hydrolase
The optimum reaction pH of the ester hydrolase Aln10 is determined in the range of 4.0 to 11.0. The specific operation is as follows: 1 mM p-nitrophenol caproate and 0.36. Mu.g pure enzyme protein were added to buffers of different pH and the absorbance A348 was measured continuously at 50 ℃ for 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 9.0-11.0). The measurement results showed that Aln10 had an optimum reaction pH of 9.5 and an activity in the pH range of 6.0 to 11.0 (FIG. 2).
The optimal reaction temperature of the ester hydrolase Aln10 is measured within the range of 15-70 ℃. The specific operation is as follows: to 1 ml of the reaction system, 1 mM of p-nitrophenol hexanoate, 100 mM of CHES-NaOH buffer (pH 9.5) and 0.36. Mu.g of pure enzyme protein were added, and absorbance A was continuously measured at 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 ℃ for 405 min, respectively. The measurement results showed that the reaction temperature range of Aln10 was 15 to 60 ℃ and the optimum reaction temperature was 50 ℃ (FIG. 3).
Example 4: enzymatic stability analysis of ester hydrolase Aln10
The thermal stability analysis of the ester hydrolase Aln10 is 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: to 1 ml of the reaction system, 1 mM of p-nitrophenol hexanoate, 100 mM CHES-NaOH buffer (pH 9.5) and 0.36. Mu.g of pure enzyme protein were added, and the absorbance A was continuously measured at 50 ℃ for 405 min. The result shows that the Aln10 can still keep more than 80% of activity under the condition of incubation for 4 hours at 20 to 50 ℃ (figure 4), which indicates that the Aln10 has better thermal stability.
The specific operation of the determination of the influence of the divalent cation on the activity of the ester hydrolase Aln10 is as follows: 10 mM Ba was added to the reaction system 2+ 、Ca 2+ 、Cd 2+ 、Co 2+ 、Cu 2+ 、Mg 2+ 、Mn 2+ 、Ni 2+ 、Sr 2+ 、Zn 2+ And ethylenediaminetetraacetic acid (EDTA), to determine enzyme activity. The enzyme activity measuring system comprises: 1 ml of the reaction system was added with 1 mM of p-nitrophenol caproate, 100 mM of Tris-HCl buffer (pH 7.5) and 0.36. Mu.g of pure enzyme protein, and the absorbance A was continuously measured at 50 ℃ for 405 min. The results of the measurement showed that the activity of Aln10 was hardly affected by Ba 2+ 、Ca 2+ 、Mg2+、Mn 2+ And Sr 2+ The enzyme activity was promoted by EDTA (FIG. 5).
The determination of the effect of organic solvents and detergents on the activity of the hydrolase Aln10 was carried out in particular by: adding organic solvent into the reaction system respectively, and measuring 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 is as follows: to 1 ml of the reaction system, 1 mM of p-nitrophenol hexanoate, 100 mM CHES-NaOH buffer (pH 9.5) and 0.36. Mu.g of pure enzyme protein were added, and the absorbance A was continuously measured at 50 ℃ for 405 min. The measurement result shows that Aln10 can resist organic solvents such as DMSO, glycerol and methanol (figure 6), and the activity of Aln10 in 5% glycerol is improved compared with that of a blank control.
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 any simple modifications, alterations and equivalent changes made to the above embodiment according to the technical spirit of the present invention still belong to the protection scope of the technical solution of the present invention.