CN115927288A

CN115927288A - Immobilized fat TLL enzyme and application of solid binding peptide in immobilization

Info

Publication number: CN115927288A
Application number: CN202210896280.3A
Authority: CN
Inventors: 李迅; 季骁彦; 许蕊; 王飞
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2023-04-07

Abstract

The invention discloses an immobilized fat TLL enzyme and application of a solid binding peptide in immobilization thereof. The preparation method has the advantages of mild process conditions, low energy consumption, simple and convenient operation and simple flow, and effectively overcomes the defects of weak surface adsorption of the enzyme and the solid carrier and easy enzyme loss of the traditional adsorption method. The immobilized enzyme prepared by the method has certain improvement in thermal stability and pH stability compared with free enzyme.

Description

Immobilized fat TLL enzyme and application of solid binding peptide in immobilization

Technical Field

The invention belongs to the technical field of enzyme immobilization, and particularly relates to an immobilized fat TLL enzyme and application of a solid binding peptide in immobilization.

Background

Thermomyces lanuginosus lipase (Thermomyces lanuginosus lipase) is a thermophilic lipase widely used in various industries. In recent years, heat-resistant lipase attracts more and more attention based on the characteristic that it has good heat resistance with mesophilic bacteria and thermophilic bacteria. TLL lipases are used in the form of soluble enzymes in industrial biocatalysts in a number of different fields: detergent, cosmetics, modification and reconstruction of grease, production and recovery of biodiesel, organic chemistry, environmental protection and the like. However, industrial application of free enzymes is often challenged by a technical hurdle to recovery and reuse of the enzyme due to poor long-term operational stability. Furthermore, the high cost of enzyme purification is another key factor affecting the economic sustainability of the process. Compared with free enzyme, the immobilized enzyme overcomes the defects of the free enzyme while keeping the characteristics of high efficiency, specificity and mild enzyme catalytic reaction, and has a series of advantages of high storage stability, easy separation and recovery, repeated use, continuous and controllable operation, simple and convenient process and the like. In order to achieve these goals, immobilization technology is one of the most innovative and studied production methods to achieve industrial scale with higher productivity.

Solid Binding Peptides (SBPs) are short amino acid sequences that selectively recognize and bind strongly to the surface of a variety of Solid materials, including metals, carbonaceous materials, silica-based materials, and minerals. These unique peptides can act as molecular linkers, directing the targeted immobilization of biomolecules (such as proteins and enzymes) onto solid matrices, and binding to the corresponding solids under mild conditions without the need for any chemical modification cations or physical treatments, which not only ensures enzyme integrity, but also enables environmentally friendly biocatalytic processes. .

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.

Therefore, the present invention aims to overcome the disadvantages of the prior art and provide an application of a solid binding peptide in the immobilization of lipase TLL.

In order to solve the technical problems, the invention provides the following technical scheme:

integrating the solid binding peptide with lipase TLL to obtain fusion enzyme;

mixing the obtained fusion enzyme with pretreated zeolite and incubating to obtain the immobilized fat TLL enzyme.

As a preferable aspect of the present invention, wherein: the solid binding peptide has a specific adsorption effect on zeolite, and the lipase TLL is derived from Thermomyces lanuginosus.

As a preferable aspect of the present invention, wherein: the solid binding peptide is an amino acid sequence shown in SEQ ID NO. 1.

As a preferable aspect of the present invention, wherein: the solid binding peptide is integrated with the lipase TLL to be connected to the C-terminal of the lipase TLL through a flexible connecting peptide. As a preferable aspect of the present invention, wherein: the flexible connecting peptide SEQ ID NO. 2.

As a preferable aspect of the present invention, wherein: the enzyme carrying amount of the zeolite is 3-4U/mg.

It is a further object of the present invention to overcome the deficiencies of the prior art and to provide an immobilized fat TLL enzyme.

As a preferable aspect of the present invention, wherein: the optimum storage pH of the immobilized fat TLL enzyme is 5-9.

As a preferable aspect of the present invention, wherein: the optimal storage temperature of the immobilized fat TLL enzyme is 45-65 ℃.

As a preferable aspect of the present invention, wherein: the optimal reaction temperature of the immobilized fat TLL enzyme during the enzymatic reaction is 45 ℃, and the optimal reaction pH is 9.0.

The invention has the beneficial effects that:

(1) The invention integrates the solid binding peptide and the TLL lipase by utilizing the genetic engineering technology to form the fusion protein, and endows the enzyme protein with the capability of directionally binding to a solid material on the premise of not modifying an immobilized carrier, thereby realizing the immobilization of the enzyme.

(2) The preparation method has the advantages of mild process conditions, low energy consumption, simple and convenient operation and simple flow, and effectively overcomes the defects of weak surface adsorption of the enzyme and the solid carrier and easy enzyme loss of the traditional adsorption method. The immobilized enzyme prepared by the method has certain improvement in thermal stability and pH stability compared with free enzyme.

(3) According to the invention, the specific type of solid binding peptide is combined with the carrier, impurity components with weak binding capacity with the carrier can be removed by repeated washing of buffer solution after immobilization, the amount of impurities adsorbed on the carrier is relatively small, the purification effect is achieved, and the removal effect of the impurities and the quality of the immobilized enzyme are effectively ensured. After each catalytic reaction of the immobilized enzyme, the immobilized enzyme is washed by a similar method, and then the immobilized enzyme can be used for the next catalytic reaction, so that the operation is simple, and the economy is high.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a schematic diagram of the construction of plasmid pPICZ α A-TLL-seg linker-SBP EctP1 in example 1 of the present invention.

FIG. 2 is a construction diagram of a DNA gel electrophoresis chart of the KM 71H-TLL-seguinker-SBP EctP1 plasmid in example 2 of the present invention.

FIG. 3 is a graph showing the quantitative analysis of the binding capacity of different kinds of zeolite and enzyme, i.e., the difference between them, in example 4 of the present invention.

FIG. 4 is a graph showing the optimal enzyme activity of free and immobilized TLL-EctP1 at different pH and temperature in examples 5 and 6 of the present invention.

FIG. 5 is a graph showing the pH and thermal stability of free and immobilized TLL-EctP1 of examples 7 and 8 of the present invention.

FIG. 6 is a graph showing the stability of the multiple-repeat enzyme activity assay of the immobilized TLL-EctP1 of example 9 of the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein, and it will be appreciated by those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the present invention and that the present invention is not limited by the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

SEQ ID NO.1：SSRSSSHRRHDHHDHRRGS

SEQ ID NO.2：GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS

The gene synthesis step in the invention is completed by Shanghai Czeri bioengineering GmbH.

The raw materials used in the invention are as follows:

the zeolite materials were all purchased from Tianjin south chemical catalyst, inc.; lauric acid 4-nitrophenol was purchased from Shanghai Allantin Biotechnology Ltd; escherichia coli top10 and Pichia pastoris KM71H are all commercial strains and are deposited in the laboratory.

The method for calculating the immobilization rate comprises the following steps:

and collecting the mixed liquid of the supernatant and the washing liquid after incubation, measuring the residual total enzyme activity in the mixed liquid, comparing with the initial total enzyme activity, and calculating the ratio of the immobilized total enzyme activity on the carrier to the initial total enzyme activity.

The method comprises the following steps of:

the reaction system is 200 μ l, a 1.5ml centrifuge tube containing 180 μ l PBS (pH7.5) buffer solution and 10 μ l 20mM lauric acid 4-nitrophenol solution is placed in a 40 ℃ water bath to be preheated for 5min, then 10 μ l immobilized enzyme suspension liquid is added to continue the reaction in the 40 ℃ water bath for 5min, 600 μ l Na is added immediately after the reaction is finished ₂ CO ₃ The solution stops the reaction. Centrifuge at 12000rpm for 3min. A sample treated under the same conditions without immobilized enzyme was replaced with 10. Mu.l of an unloaded zeolite suspension. And detecting the light absorption value of the sample at the wavelength of 410nm by using a microplate reader, and calculating the enzyme activity (U/ml) according to a standard curve by using the detected value.

Example 1

Construction of a fusion enzyme fusing lipase TLL with a solid binding peptide:

referring to FIG. 1, pPICZ alpha A-TLL is used as a template for gene synthesis, solid binding peptide is connected to the C-terminal of lipase TLL through a section of flexible connecting peptide to obtain recombinant plasmid pPICZ alpha A-TLL-seg linker-EctP1 dry powder, and the obtained dry powder is dissolved in TE buffer solution.

Screening of recombinant plasmid positive clones:

carrying out ice bath on the escherichia coli top10 competent cells introduced with the recombinant plasmids for 15min, then carrying out heat shock at 42 ℃ for 45-90 s, adding 850 mu l of SOC culture medium into a sterile operation platform, and carrying out shake culture at 37 ℃ and 200rpm for 45min;

coating a proper amount of the cultured bacterial liquid on an LB plate with bleomycin resistance, and putting the LB plate in an incubator at 37 ℃ for overnight culture;

randomly pick 2-3 single colonies from the overnight-cultured plate to 5ml shake tubes with bleomycin resistance, and shake-culture at 37 deg.C and 200rpm for 8-12h.

And extracting the plasmid in the bacterial liquid by using a plasmid extraction kit to finally obtain the plasmid pPICZ alpha A-TLL-seg linker-EctP1, and delivering the plasmid to a company for sequencing.

Example 2

Transformation of recombinant plasmids-selection of correctly sequenced recombinant plasmids transformed into pichia pastoris KM71H competence:

firstly, linearizing the recombinant plasmid by SacI, wherein the reaction system comprises the following steps: cut 1.5. Mu.l, 10xQ. Cut buffer 5. Mu.l, pPICZ alpha A-TLL-seglinker-EctP1 plasmid 43.5. Mu.l, reaction conditions: at 37 ℃ for 40min;

after linearization, taking part of the sample for agarose gel electrophoresis detection, and referring to fig. 2, the electrophoresis result is a single band, and the size of the band is correct, which indicates that the plasmid linearization is successful;

purifying the rest samples by using a PCR (polymerase chain reaction) cleaning kit, adding a proper amount of purified linearized plasmids into a pichia pastoris KM71H competence, uniformly mixing, transferring into a precooled electric rotating cup, standing for 2min in an ice manner, performing electric shock transformation, immediately adding precooled sorbitol and a YPD liquid culture medium after the electric shock transformation is finished, and incubating for 1H in an incubator at the temperature of 30 ℃ to obtain a YPDS plate; and finally, coating the bacterial liquid on a YPDS plate, and culturing for 3-5d until a single colony grows out.

Expression of the fusion enzyme:

picking several single colonies from YPDS plates to 2ml YPD shake tubes with bleomycin resistance, and culturing in a shaker at 30 ℃ and 250rpm for 16-20h;

inoculating 1ml of the culture solution in a shake tube, and shake culturing at 37 deg.C and 250rpm in 25ml of sterilized BMGY liquid medium to OD ₆₀₀ Is 2 to 6;

collecting bacterial liquid, centrifuging for 5min at 1500g, discarding supernatant, taking a proper amount of thallus precipitate, re-suspending, inoculating into 50ml of fresh sterilized BMMY liquid culture medium, carrying out shake culture at 30 ℃ and 250rpm, adding 0.5% methanol for induction every 24h, collecting bacteria after 96h, centrifuging, collecting supernatant enzyme liquid, and carrying out ultrafiltration concentration to obtain the fusion enzyme liquid.

Example 3

Pretreatment of the support material:

preparing an elution buffer solution: mixing 10mM Tris-HCl, 50mM NaCl, 1% Triton X-100, under the condition of pH 7.5;

1mg of zeolite is soaked in 1ml of elution buffer solution, washed in an ultrasonic washer for 10min, then washed with 50mM Tris-HCl (pH7.5) buffer solution for three times, and each time, the zeolite is shaken and washed in a vortex machine for 1min, and then centrifuged at 12000rpm for 1min to remove supernatant, so that the carrier is obtained.

Example 4

In the case of zeolites ZSM-5 and SAR-100, pretreatment was carried out according to the method of example 3, which compares the adsorption levels of TLL and TLL-EctP1 on different carriers and explores the maximum enzyme loading of the carriers.

Respectively taking enzyme liquid with the total enzyme activity of 1-10U and 1ml of Tris-HCl buffer solution, uniformly mixing, adding the mixed solution into a 2ml centrifuge tube filled with a cleaned carrier, placing the centrifuge tube on a rotary incubator for rotary incubation at room temperature, centrifuging the sample after rotary incubation at 12000rpm for 1min, washing the sample with Tris-HCl buffer solution for three times to obtain the immobilized fat TLL enzyme, and determining the immobilization rate of the immobilized fat TLL enzyme.

Referring to FIG. 3, showing the adsorption capacities of different carriers for immobilized enzymes, it can be seen from FIG. 3 that TLL and TLL-EctP1 have adsorption capacities for both zeolite carrier materials, wherein the carrier used in FIG. 3A is zeolite SAR-100 and the carrier used in FIG. 3B is zeolite ZSM-5.

Due to the nature of TLL, background adsorption exists on the carrier material, but the adsorption capacity is weak, and the immobilization rate is far lower than that of TLL-EctP1. As can be seen from FIG. 3, when the enzyme dosage is lower, the immobilization rate of TLL-EctP1 can reach more than 90% on both carriers, while the immobilization rate of TLL without immobilized binding peptide on zeolite SAR-100 is only about 40%, and the immobilization rate on zeolite SAR-100 is only about 20%. This indicates that the solid binding peptide plays a role in promoting the binding of the enzyme protein and the carrier, and the immobilization rate is improved. With the increase of the enzyme dosage, the immobilized enzyme dosage tends to be stable, and more enzyme protein can not be immobilized on the carrier, which shows that the carrying capacity of the carrier material is limited, while when the enzyme dosage is increased to 5-10U, the immobilization rate is gradually reduced, but the immobilized enzyme dosage is maintained at 3-4U, which shows that the maximum carrying capacity of the two zeolite materials is 3-4U/mg.

It can be seen that, under the same enzyme dosage, when zeolite ZSM-5 is used as a carrier, the immobilization rate of TLL-EctP1 is higher than that of zeolite SAR-100, which proves that the effect of using zeolite ZSM-5 immobilized enzyme is better than that of zeolite SAR-100, and zeolite ZSM-5 is used as an immobilized carrier in the subsequent examples.

Example 5

This example measures the effect of pH on the activity of TLL enzymes for free and immobilized fat.

Free and immobilized TLL-EctP1 was subjected to enzymatic reaction at 40 ℃ in buffers of different pH, and the results obtained by performing enzymatic reaction of free TLL under the same conditions were used as a control. The highest enzyme activity is taken as the relative enzyme activity of 100%.

Referring to FIG. 4A, the relative enzyme activity was highest for both the free and immobilized enzymes at pH9.0, indicating that the optimum pH of the TLL was not changed after immobilization.

Example 6

This example measures the effect of temperature on the activity of free and immobilized fat TLL enzymes.

The reaction system is subjected to enzymatic reaction at different temperatures (30-60 ℃), the highest enzyme activity obtained by determination is taken as the relative enzyme activity of 100%, and the result obtained by performing enzymatic reaction on free TLL under the same conditions is taken as a reference.

As shown in FIG. 4B, free TLL and TLL-EctP1 had the highest relative enzyme activity at 40 ℃ and immobilized TLL-EctP1 had the highest relative enzyme activity at 45 ℃, and the optimum temperature of immobilized TLL-EctP1 was increased by 5 ℃ as compared with TLL containing no linker peptide.

Example 7

This example explores the pH stability of the immobilized fat TLL enzyme.

Putting the immobilized enzyme into solutions with different pH values (pH value of 5.0-9.0), incubating for 1h at room temperature, determining the residual relative enzyme activity of the immobilized enzyme after incubation, taking the enzyme activity of the immobilized enzyme before incubation as the relative enzyme activity of 100%, and taking the result obtained after TLL which is not connected with the solid binding peptide is treated under the same condition as a control.

As can be seen from FIG. 5A, the solid binding peptide EctP1 had no effect on the pH stability of TLL, and the curve trend of free TLL was similar to that of TLL-EctP1, wherein free TLL and TLL-EctP1 were the most stable at pH7.0, and immobilized TLL-EctP1 was the most stable at pH 8.0. In addition, the trend of the curve of the immobilized TLL-EctP1 at pH5.0-9.0 is more gradual than that of the free enzyme, which indicates that the immobilized enzyme has better pH stability as a whole.

Example 8

This example explores the thermostability of the immobilized fat TLL enzyme.

And (2) placing the immobilized enzyme at different temperatures (45-65 ℃), incubating for 1h, determining the residual relative enzyme activity of the immobilized enzyme after incubation, taking the enzyme activity of the immobilized enzyme before incubation as the relative enzyme activity of 100%, and taking the result obtained after TLL (TLL) without connecting the solid binding peptide is treated under the same conditions as a control.

As can be seen from FIG. 5B, the relative enzyme activities of the immobilized TLL-EctP1 after incubation at 45-65 ℃ for 1 hour are all higher than those of the free enzyme, and the residual relative enzyme activities can still be maintained by more than 50% after incubation at 65 ℃. Compared with free enzyme, the thermal stability of TLL is further improved after immobilization.

Example 9

This example is a study of the reuse of immobilized TLL-EctP1

ZSM-5 was selected as an immobilization carrier, the immobilized enzyme TLL-EctP1 was prepared by the same immobilization method as in example 3, and the initial enzyme activity was determined as the relative enzyme activity of 100%. The immobilized enzyme was repeatedly used in the catalytic enzymatic reaction cycle, wherein after each enzymatic reaction was completed, the immobilized enzyme was washed with PBS buffer for the following cycle.

The reusability of the immobilized enzyme was evaluated in this way, and the results obtained after treatment of the TLL without solid binding peptide under the same conditions were used as a control.

As shown in FIG. 6, after 3 times of repeated use, the residual relative enzyme activity of the immobilized enzyme is gradually reduced, while the relative enzyme activity retained by the immobilized TLL-EctP1 can still reach more than 50%, and the enzyme activity of the immobilized TLL is basically completely lost under the same conditions. Therefore, the reusability of the immobilized enzyme TLL-EctP1 is far higher than that of the pure immobilized TLL.

Example 10

This example was conducted to investigate the elution stability of the immobilized enzyme TLL-EctP1.

The protein concentration of the supernatant before and after immobilization was measured, and the protein amount of the immobilized enzyme was calculated. The immobilized enzyme was then eluted with buffers of different conditions and the stability of the immobilized TLL-EctP1 was assessed by measuring the protein concentration in the eluate.

TABLE 1 stability of immobilized TLL-EctP1 under different elution conditions

As shown in Table 1, when the elution solution was 1 to 3M NaCl solution, only a very small amount of protein was eluted from the carrier, and when the concentration of NaCl solution was increased to 5M, only 14% of protein was eluted. Whereas when the eluent was a higher concentration of 3% Tween-20 solution, only 12% of the protein was eluted. When the eluate was a high-concentration 3-vol SDS solution, almost all of the proteins were eluted.

Therefore, the immobilized enzyme is basically kept stable when the NaCl solution and the Tween-20 solution with the hydrophobic damage function are treated, the SDS solution can block all non-covalent bond functions between the protein and the carrier, and in the case, the solid binding peptide does not play an adsorption function any more, the immobilized enzyme loses stability, and the enzyme protein is completely fallen off from the carrier.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. The application of the solid binding peptide in immobilization of lipase TLL is characterized in that: comprises the steps of (a) preparing a substrate,

integrating the solid binding peptide with lipase TLL to obtain fusion enzyme;

2. Use of a solid binding peptide according to claim 1 for TLL immobilization of lipases, characterized in that: the solid binding peptide has a specific adsorption effect on zeolite, and the lipase TLL is derived from Thermomyces lanuginosus.

3. Use of a solid binding peptide according to claim 1 or 2 for immobilization of a lipase TLL, wherein: the solid binding peptide is an amino acid sequence shown in SEQ ID NO. 1.

4. Use of a solid binding peptide according to claim 1 for TLL immobilization of lipases, characterized in that: the solid binding peptide is integrated with the lipase TLL to be connected to the C-terminal of the lipase TLL through a flexible connecting peptide.

5. Use of a solid binding peptide according to claim 4 for TLL immobilization of lipases, characterized in that: the flexible connecting peptide is an amino acid sequence shown in SEQ ID NO. 2.

6. Use of a solid binding peptide according to claim 1 for TLL immobilization of lipases, characterized in that: the maximum enzyme carrying amount of the zeolite is 3-4U/mg.

7. Use of a solid binding peptide according to claim 1 for TLL immobilization of lipases, characterized in that: the pretreatment comprises immersing 1mg of zeolite in 1ml of elution buffer, treating in an ultrasonic cleaning apparatus for 10min, then washing three times with 50mM Tris-HCl buffer, each time for 1min, and finally centrifuging at 12000rpm for 1min to discard the supernatant.

8. The immobilized fat TLL enzyme of claim 1, wherein: the optimum storage pH of the immobilized fat TLL enzyme is 5-9.

9. Use of a solid binding peptide according to claim 1 or 8 for immobilization of a lipase TLL, wherein: the optimal storage temperature of the immobilized fat TLL enzyme is 45-65 ℃.

10. The immobilized fat TLL enzyme of claim 8, wherein: the optimal reaction temperature of the immobilized fat TLL enzyme during the enzymatic reaction is 45 ℃, and the optimal reaction pH is 9.0.