Preparation method of trehalose hydrolase

Abstract

The invention belongs to the technical field of preparation of trehalose hydrolase, and particularly relates to a preparation method of trehalose hydrolase, which comprises the following steps: S1, screening strains; s2, cloning a strain gene; s3, enzyme extraction and purification; s4, enzyme property research: enzyme property research and optimization: the trehalose hydrolase obtained through purification is subjected to property research including the optimum temperature, the optimum pH value, the enzyme stability and the like, and the preparation conditions of the enzyme can be optimized so as to improve the yield and the activity of the enzyme. According to the method disclosed by the invention, the most suitable temperature and the most suitable pH value are obtained and researched by controlling variables, and then are applied to yield control to control the cost of the preparation process of the trehalose hydrolase, so that the maximization of the final yield of trehalose is kept.

Description

Technical Field

[0001] The invention belongs to the technical field of trehalose hydrolase preparation, and specifically relates to a method for preparing trehalose hydrolase. Background Art

[0002] Trehalose hydrolase is an enzyme substance that can break down trehalose into glucose. Trehalose is a disaccharide found in seaweed, which is composed of two glucose molecules connected by α-1,3-glycosidic bonds. Trehalose hydrolase can break down this complex trehalose into two glucose molecules, thereby providing energy and carbon source for cells to use.

[0003] Trehalose hydrolase exists in many organisms, especially those that feed mainly on seaweed. For example, some microorganisms and seaweed in the ocean, as well as some marine animals, contain trehalose hydrolase. These organisms synthesize and secrete trehalose hydrolase to utilize trehalose as an energy and carbon source, enabling them to adapt to survival in the marine environment.

[0004] Trehalose hydrolase is an enzyme that can decompose trehalose into glucose. It has highly specific catalytic activity and can help organisms use trehalose as energy and carbon source.

[0005] However, the current extraction process of trehalose hydrolase is relatively complicated. Since trehalose hydrolase needs to be mass-produced to meet the needs of existing industrial use, the production process of trehalose hydrolase needs to control its preparation cost. Since there are many reaction steps in the preparation process of trehalose hydrolase, and the temperature and pH value have a great influence on each other, it is difficult to ensure the balance of trehalose hydrolase production.

[0006] Therefore, a method for preparing trehalose hydrolase is needed to solve the above-mentioned problems. Summary of the invention

[0007] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a method for preparing trehalose hydrolase, which effectively solves the problems raised in the above background technology.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for preparing trehalose hydrolase, the preparation method comprising the following steps:

[0009] S1. Screening strains: First, strains capable of producing trehalose hydrolase need to be screened out from various microbial resources. During the strain screening process, attention needs to be paid to the integrity of the strains to ensure that the strains are viable strains.

[0010] S2. Cloning strain genes: After the above steps, the enzyme-producing strain is determined, and the gene encoding trehalose hydrolase needs to be cloned. Molecular biological techniques such as PCR are used to amplify the gene fragment and insert it into a suitable expression vector. During the operation, the integrity of the original strain gene needs to be guaranteed.

[0011] S3. Enzyme extraction and purification: After the above steps, after successful expression in the expression host, the enzyme needs to be extracted and purified, which usually includes cell disruption, centrifugation, filtration and other steps to remove cell debris and impurities. Cell disruption, centrifugation, filtration and other steps need to ensure that the cell extract in the test tube does not leak. Then, a series of chromatographic techniques, such as ion exchange chromatography, gel filtration chromatography, etc., are used to purify the enzyme protein to obtain high-purity trehalose hydrolase.

[0012] S4. Study on the properties of enzymes: Study on the properties and optimization of enzymes. The properties of the purified trehalose hydrolase were studied, including the optimum temperature, optimum pH value, enzyme stability, etc. The preparation conditions of the enzyme can be optimized to increase the yield and activity of the enzyme.

[0013] In the context of increasing the yield of trehalose hydrolase, the relationship between temperature, pH value and enzyme can be approximately described by some mathematical models, and its mathematical relationship can be expressed in the form of a multivariate quadratic function:

[0014] Y=aT 2 +bT+cpH 2 +dpH+eTpH+f,

[0015] Where: Y represents the yield of trehalose hydrolase;

[0016] T represents temperature;

[0017] pH stands for acidity or alkalinity;

[0018] a, b, c, d, e, and f are constants, and this formula represents the combined effect of temperature and pH on enzyme production, where the quadratic term takes into account their nonlinear effects and the cross term eTpH takes into account the interaction between temperature and pH.

[0019] Furthermore, in S4, the method for increasing the yield of trehalose hydrolase is as follows:

[0020] a. Optimize gene expression: Select strong promoters and efficient terminators to enhance gene transcription efficiency. Optimize codons to make them more suitable for the codon preference of the expression host and improve translation efficiency.

[0021] b. Optimize host strains: Screen or modify host strains with high growth rates and high metabolic capacity to support more enzyme synthesis.

[0022] c. Optimization of fermentation conditions: Adjust the composition of the culture medium to provide sufficient nutrients, such as carbon source, nitrogen source, inorganic salts and trace elements, etc. Control the culture conditions such as temperature, pH value, dissolved oxygen, etc. to keep them in the optimal range.

[0023] Furthermore, in S4, the relationship between the enzymatic reaction rate and the temperature and pH value cannot usually be accurately described by a simple mathematical formula because their relationship is relatively complex and nonlinear.

[0024] Furthermore, in S4, under a stable environment, the relationship between temperature and enzymatic reaction rate is often described using a variant of the Arrhenius equation: Where v is the reaction rate, A is the pre-exponential factor, Ea is the activation energy of the reaction, R is the gas constant, and T is the absolute temperature;

[0025] The relationship between pH and the rate of an enzymatic reaction may be approximated by a function similar to a bell curve, for example: V max is the maximum reaction rate, pH opt is the optimum pH value, K m is a constant.

[0026] It should be noted that these formulas are only a simplification and approximation of the actual situation. The actual enzymatic reaction is affected by a variety of factors, and these formulas cannot fully and accurately reflect all situations.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention obtains the most suitable temperature and the most suitable pH value by controlling variables, and then applies them to yield control to control the cost of the production process of trehalose hydrolase, thereby maximizing the final yield of trehalose. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0030] Figure 1 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] Embodiment 1, by Figure 1 The present invention discloses a method for preparing trehalose hydrolase, which comprises the following steps:

[0033] S1. Screening strains: First, strains capable of producing trehalose hydrolase need to be screened out from various microbial resources. During the strain screening process, attention needs to be paid to the integrity of the strains to ensure that the strains are viable strains.

[0034] S2. Cloning strain genes: After the above steps, the enzyme-producing strain is determined, and the gene encoding trehalose hydrolase needs to be cloned. Molecular biological techniques such as PCR are used to amplify the gene fragment and insert it into a suitable expression vector. During the operation, the integrity of the original strain gene needs to be guaranteed.

[0035] S3. Enzyme extraction and purification: After the above steps, after successful expression in the expression host, the enzyme needs to be extracted and purified, which usually includes cell disruption, centrifugation, filtration and other steps to remove cell debris and impurities. Cell disruption, centrifugation, filtration and other steps need to ensure that the cell extract in the test tube does not leak. Then, a series of chromatographic techniques, such as ion exchange chromatography, gel filtration chromatography, etc., are used to purify the enzyme protein to obtain high-purity trehalose hydrolase.

[0036] S4. Study on the properties of enzymes: Study on the properties and optimization of enzymes. The properties of the purified trehalose hydrolase were studied, including the optimum temperature, optimum pH value, enzyme stability, etc. The preparation conditions of the enzyme can be optimized to increase the yield and activity of the enzyme.

[0037] In the context of increasing the yield of trehalose hydrolase, the relationship between temperature, pH value and enzyme can be approximately described by some mathematical models, and its mathematical relationship can be expressed in the form of a multivariate quadratic function:

[0038] Y=aT 2 +bT+cpH 2 +dpH+eTpH+f,

[0039] Where: Y represents the yield of trehalose hydrolase;

[0040] T represents temperature;

[0041] pH stands for acidity or alkalinity;

[0042] a, b, c, d, e, and f are constants. This formula represents the combined effect of temperature and pH on enzyme production, where the quadratic term takes into account their nonlinear effects and the cross term eTpH takes into account the interaction between temperature and pH;

[0043] Specifically, through the above steps, trehalose hydrolase can be improved, and the preparation conditions of the enzyme can be optimized by controlling the temperature, pH value, and enzyme stability to increase the yield and activity of the enzyme;

[0044] Specifically, in S4, the relationship between the rate of enzymatic reaction and temperature and pH value cannot usually be accurately described by simple mathematical formulas because their relationship is relatively complex and nonlinear;

[0045] Embodiment 2, by Figure 1 Given, in S4, under stable conditions, the relationship between temperature and enzymatic reaction rate is often described using a variant of the Arrhenius equation: Where v is the reaction rate, A is the pre-exponential factor, Ea is the activation energy of the reaction, R is the gas constant, and T is the absolute temperature;

[0046] The relationship between pH and the rate of an enzymatic reaction may be approximated by a function similar to a bell curve, for example: V max is the maximum reaction rate, pH opt is the optimum pH value, K m is a constant.

[0047] Through the above steps, the most suitable temperature and the most suitable pH value are obtained to maintain the maximum rate of the enzymatic reaction;

[0048] Then, the most suitable temperature and the most suitable pH value are brought into Y=aT in Example 1. 2 +bT+cpH 2 +dpH+eTpH+f, and finally the final yield of trehalose is obtained, thereby maximizing the final yield of trehalose;

[0049] However, it should be noted that the real biological reaction system is far more complex than this simple mathematical model. Different enzymes may have different characteristics. The actual effect of temperature and pH on yield may present a more complex curve, and there may even be multiple peaks or irregular changes.

[0050] For example, in some cases, small changes in temperature and pH may have a significant effect on yield, while in other cases there may be a wide temperature or pH range with relatively little effect on yield. This requires a lot of experimental data and more precise mathematical analysis to determine the specific relationship.

[0051] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0052] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing trehalose hydrolase, characterized in that: The preparation method comprises the following steps: S1. Screening strains: First, strains capable of producing trehalose hydrolase need to be screened out from various microbial resources. During the strain screening process, attention needs to be paid to the integrity of the strains to ensure that the strains are viable strains. S2. Cloning strain genes: After the above steps, the enzyme-producing strain is determined, and the gene encoding trehalose hydrolase needs to be cloned. Molecular biological techniques such as PCR are used to amplify the gene fragment and insert it into a suitable expression vector. During the operation, the integrity of the original strain gene needs to be guaranteed. S3. Enzyme extraction and purification: After the above steps, after successful expression in the expression host, the enzyme needs to be extracted and purified, which usually includes cell disruption, centrifugation, filtration and other steps to remove cell debris and impurities. Cell disruption, centrifugation, filtration and other steps need to ensure that the cell extract in the test tube does not leak. Then, a series of chromatographic techniques, such as ion exchange chromatography, gel filtration chromatography, etc., are used to purify the enzyme protein to obtain high-purity trehalose hydrolase. S4. Study on the properties of enzymes: Study on the properties and optimization of enzymes. The properties of the purified trehalose hydrolase were studied, including the optimum temperature, optimum pH value, enzyme stability, etc. The preparation conditions of the enzyme can be optimized to increase the yield and activity of the enzyme. In the context of increasing the yield of trehalose hydrolase, the relationship between temperature, pH value and enzyme can be approximately described by some mathematical models, and its mathematical relationship can be expressed in the form of a multivariate quadratic function: Y=aT 2 +bT+cpH 2 +dpH+eTpH+f, Where: Y represents the yield of trehalose hydrolase; T represents temperature; pH stands for acidity or alkalinity; a, b, c, d, e, and f are constants, and this formula represents the combined effect of temperature and pH on enzyme production, where the quadratic term takes into account their nonlinear effects and the cross term eTpH takes into account the interaction between temperature and pH.

2. The method for preparing trehalose hydrolase according to claim 1, characterized in that: In S4, the method for increasing the yield of trehalose hydrolase is as follows: a. Optimize gene expression: Select strong promoters and efficient terminators to enhance gene transcription efficiency. Optimize codons to make them more suitable for the codon preference of the expression host and improve translation efficiency. b. Optimize host strains: Screen or modify host strains with high growth rates and high metabolic capacity to support more enzyme synthesis. c. Optimization of fermentation conditions: Adjust the composition of the culture medium to provide sufficient nutrients, such as carbon source, nitrogen source, inorganic salts and trace elements, etc. Control the culture conditions such as temperature, pH value, dissolved oxygen, etc. to keep them in the optimal range.

3. The method for preparing trehalose hydrolase according to claim 1, characterized in that: In S4, the relationship between the enzymatic reaction rate and the temperature and pH value cannot usually be accurately described by a simple mathematical formula because their relationship is relatively complex and nonlinear.

4. The method for preparing trehalose hydrolase according to claim 3, characterized in that: In S4, under a stable environment, the relationship between temperature and enzymatic reaction rate is often described using a variant of the Arrhenius equation: Where v is the reaction rate, A is the pre-exponential factor, Ea is the activation energy of the reaction, R is the gas constant, and T is the absolute temperature; The relationship between pH and the rate of an enzymatic reaction may be approximated by a function similar to a bell curve, for example: V max is the maximum reaction rate, pH opt is the optimum pH value, K m is a constant. It should be noted that these formulas are only a simplification and approximation of the actual situation. The actual enzymatic reaction is affected by a variety of factors, and these formulas cannot fully and accurately reflect all situations.

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