Method for improving trehalose conversion rate by optimizing trehalase catalytic system

Abstract

The invention discloses a method for improving trehalose conversion rate by optimizing a trehalase catalytic system, and belongs to the technical field of genetic engineering. According to the method for improving the trehalose conversion rate by optimizing the trehalase catalytic system, disclosed by the invention, gene sequences of maltooligosyl trehalose hydrolase, maltooligosyl trehalose synthase and 4-alpha-glucosyltransferase are optimized, so that the expression level of enzymes and the content of trehalose in a reaction system are improved; the conversion rate reaches 83.3%, and a foundation is laid for further large-scale industrial production of the trehalose.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and more particularly to a method for optimizing a trehalase catalytic system to improve trehalose conversion rate. Background Art

[0002] Trehalose is a non-reducing disaccharide formed by two glucose molecules linked together by α, α-1, 1 glycosidic bonds. It is widely distributed in plants, algae, fungi, bacteria, insects and other invertebrates. Trehalose is one of the main osmotic protectants found in nature and plays an important role in the adaptive response of organisms to osmotic stress and extreme temperatures. The sweetness of trehalose is only 45% of that of sucrose. Due to its mild taste and refreshing taste, it is suitable for use in food seasonings; in the pharmaceutical industry, it can be used as a stabilizer for reagent drugs and diagnostic drugs; because trehalose has extremely strong moisturizing effects and multiple physiological effects such as sun protection and UV protection, it can be added to cosmetics as a moisturizer and protective agent.

[0003] Nowadays, the production methods of trehalose are divided into biological extraction, microbial fermentation and enzyme conversion. The biological extraction method uses yeast and mold as the extraction source, accumulates trehalose through drying and other treatments, and then uses organic solvents to extract it. The microbial fermentation method is to select high-yield strains of trehalose through mutagenesis or genetic modification, and then cultivate, ferment and extract trehalose. The enzyme conversion method uses glucose, starch or maltodextrin as the substrate, and the in vitro enzyme catalyzes the substrate into trehalose.

[0004] The biological extraction method uses the stress resistance of microorganisms to produce trehalose. It requires more abundant nutrients in adversity, which increases the cost. In addition, the trehalose yield of this method is low, the time consumption is long, and it is accompanied by great instability, which is not suitable for large-scale industrial production. The production process of the microbial fermentation method is complicated. Many by-products will be produced during the growth of microorganisms, resulting in many impurities and high costs in the product purification and refining stage. The time consumption is reduced compared to the biological extraction method, but it is still not conducive to large-scale industrial production. The reaction of the single enzyme method in the enzyme conversion method to produce trehalose is reversible, which limits the conversion rate of trehalose to a low level, and the substrate maltose used in the single enzyme method is relatively expensive.

[0005] Therefore, providing a method for optimizing the trehalase catalytic system to improve the trehalose conversion rate is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a method for optimizing the trehalase catalytic system to improve the trehalose conversion rate.

[0007] The present invention produces trehalose by a double enzyme method in an enzyme conversion method, has a simple process flow, uses a substrate maltodextrin with low cost, has strong specificity of enzyme catalysis, reduces the types of by-products, and is beneficial to the subsequent purification and refining of the product trehalose; the enzyme catalysis has high efficiency, greatly shortens the production time; and the enzyme reaction has strong stability, the output and conversion rate of trehalose are basically maintained at a high level, is suitable for industrial large-scale production, and has important economic value and social significance.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for optimizing the trehalase catalytic system to improve trehalose conversion rate, the specific steps are as follows:

[0010] (1) Optimizing the gene sequences of maltooligosyltrehalose hydrolase MTSase, maltooligosyltrehalose synthase MTHase ​​and 4-α-glucosyltransferase 4αGT; after optimization, the sequences MTSase-op, MTHase-op and 4αGT-op shown in SEQ ID NO.4-6 were obtained;

[0011] (2) constructing pBAD-MTSase-op, pBAD-MTHase-op, and pBAD-4αGT-op strains, and preparing crude enzyme solutions of MTSase-op, MTHase-op, and 4αGT-op, respectively;

[0012] (3) The crude enzyme solutions of MTSase-op, MTHase-op and 4αGT-op were reacted with maltodextrin in a ratio of 1:1:1.

[0013] Furthermore, the method is used in the production of trehalose.

[0014] Furthermore, the method is used to improve the conversion rate of trehalose.

[0015] Furthermore, the sequences MTSase-op, MTHase-op and 4αGT-op shown in SEQ ID NO. 4-6 are used to improve the conversion rate of trehalose.

[0016] Furthermore, the pBAD-MTSase-op, pBAD-MTHase-op and pBAD-4αGT-op strains were used to improve the trehalose conversion rate.

[0017] Furthermore, crude enzyme solutions of MTSase-op, MTHase-op and 4αGT-op were used to improve the conversion rate of trehalose.

[0018] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a method for optimizing the trehalase catalytic system to improve the trehalose conversion rate, which can improve the conversion rate of trehalose products produced by the Escherichia coli double enzyme method; through appropriate sequence optimization, the expression level of the enzyme and the content of trehalose in the reaction system are improved, and the conversion rate reaches 83.3%, which lays a foundation for the further large-scale industrial production of trehalose. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The attached drawing shows the plasmid vectors of maltooligosyltrehalose hydrolase MTSase-op, maltooligosyltrehalose synthase MTHase-op and 4-α-glucosyltransferase 4αGT-op after sequence optimization constructed by the present invention;

[0021] Figure 2 The accompanying drawings are SDS-PAGE of the maltooligosyltrehalose hydrolase, maltooligosyltrehalose synthase and 4-α-glucosyltransferase of the present invention before and after sequence optimization;

[0022] Where: op: enzyme after sequence optimization; S: supernatant; P: precipitate;

[0023] Figure 3 The accompanying drawings show the comparison of the enzyme activities of the maltooligosyltrehalose hydrolase MTSase, the maltooligosyltrehalose synthase MTHase ​​and the 4-α-glucosyltransferase 4αGT of the present invention and their sequence-optimized MTSase-op, MTHase-op and 4αGT-op. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The culture medium and buffer used in the present invention are:

[0026] LB liquid medium (g / L): yeast powder 5, peptone 10, sodium chloride 10, liquid volume 50mL per 250ml Erlenmeyer flask.

[0027] LB solid medium (g / L): yeast powder 5, peptone 10, sodium chloride 10, agarose 15.

[0028] Kanamycin LB liquid medium (g / L): yeast powder 5, peptone 10, sodium chloride 10, kanamycin 0.05, liquid volume 50mL per 250ml Erlenmeyer flask.

[0029] Kanamycin LB solid medium (g / L): yeast powder 5, peptone 10, sodium chloride 10, agarose 15, kanamycin 0.05.

[0030] Sterilization conditions: 115°C, 20 minutes. After sterilization, the culture medium containing kanamycin is cooled to 60°C before adding kanamycin.

[0031] PBS buffer: 0.02 mol / L disodium hydrogen phosphate and sodium dihydrogen phosphate, 0.5 mol / L sodium chloride, pH adjusted to 6.0 using phosphoric acid and sodium hydroxide.

[0032] The wild-type maltooligosyltrehalose hydrolase MTSase gene sequence is shown in SEQ ID NO.1.

[0033]

[0034] The wild-type maltooligosyltrehalose synthase MTHase ​​gene sequence is shown in SEQ ID NO.2.

[0035]

[0036] The wild-type 4-α-glucosyltransferase 4αGT gene sequence is shown in SEQ ID NO.3.

[0037]

[0038] The optimized maltooligosaccharide-trehalose hydrolase MTSase gene sequence is shown in SEQ ID NO.4.

[0039]

[0040] The optimized maltooligosyltrehalose synthase MTHase ​​gene sequence is shown in SEQ ID NO.5.

[0041]

[0042] The optimized 4-α-glucosyltransferase 4αGT gene sequence is shown in SEQ ID NO.6.

[0043]

[0044] Example 1

[0045] According to the sequences of genes such as MTSase and the vector pBAD, the primers in Table 1 were designed using the software SnapGene 6.0.2 according to the 15-25 bp homology arm and the basic principles of primer design.

[0046] Table 1

[0047]

[0048] In order to further improve the conversion rate of trehalose production by the dual enzyme method, sequence optimization of maltooligosyltrehalose hydrolase, maltooligosyltrehalose synthase and 4-α-glucosyltransferase was carried out, that is, the wild-type maltooligosyltrehalose hydrolase MTSase, maltooligosyltrehalose synthase MTHase ​​and 4-α-glucosyltransferase 4αGT sequences shown in SEQ ID NO.1-3 were optimized to sequences MTSase-op, MTHase-op and 4αGT-op as shown in SEQ ID NO.4-6.

[0049] The method for sequence optimization is as follows: based on the codon preference of Escherichia coli combined with the optimization of GC content and the removal of problematic codons, the sequences of maltooligosyltrehalosehydrolase, maltooligosyltrehalose synthase and 4-α-glucosyltransferase were optimized through the online website of Genscript, and then the genes maltooligosyltrehalosehydrolase MTSase, maltooligosyltrehalose synthase MTHase ​​and 4-α-glucosyltransferase 4αGT and the sequence-optimized genes maltooligosyltrehalosehydrolase MTSase-op, maltooligosyltrehalose synthase MTHase-op and 4-α-glucosyltransferase 4αGT-op were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0050] The purchased plasmid pBAD was used as a template and MTSase mb-F and MTSase mb-R were used as primers for PCR amplification to obtain the linear plasmid pBAD.

[0051] The target gene fragment MTSase was obtained by PCR amplification using the synthesized maltooligosaccharide-trehalose hydrolase MTSase gene as a template and MTSase-F and MTSase-R as primers.

[0052] PCR amplification reaction system: 1 μL each of upstream and downstream primers, 1 μL of template sequence, 7 μL of ddH2O, 10 μL of prime starmax, a total of 20 μL.

[0053] PCR amplification reaction conditions: pre-denaturation at 98°C for 5 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 90 seconds, 32 cycles; incubation at 12°C for 30 minutes.

[0054] After PCR is completed, agarose gel electrophoresis is performed to verify the band size. 5μL loading buffer is mixed with 5μL PCR reaction solution. 5μL of the mixture is taken with a pipette and added to the agarose gel well. After the sample is added, 4μL nucleic acid marker is added to another gel well. After running at 100V for 15 minutes, the nucleic acid gel is placed in a gel imaging instrument to observe whether the sample bands are consistent with the theoretical values.

[0055] The gene fragment pBAD and MTSase verified by nucleic acid electrophoresis were connected using a seamless cloning kit purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The reaction solution system was: 1μL MTSase fragment, 1μL linear plasmid vector pBAD, 10μL seamless cloning mix, 8μL ddH2O. After the reaction solution was placed in a 50℃ water bath for 15 minutes, 5μL of the reaction solution was gently mixed with the Escherichia coliBL21 competent cells that had been melted on ice in advance, and then placed in an ice bath for 45 minutes. The centrifuge tube containing the competent cells was placed in a 42℃ water bath for heat shock for 90 seconds, and then the centrifuge tube was transferred to an ice bath to cool the cells for 2 minutes, and then 800μL LB liquid culture medium was added and cultured in a shaking incubator at 37℃ for 1 hour. After the culture is completed, place the centrifuge tube in a centrifuge at 4000rpm for 2 minutes, carefully remove 600μL of supernatant, and then use a pipette to blow and thoroughly suspend the bacteria. Spread the bacterial solution on a kanamycin LB solid culture medium plate, invert and culture at 37℃ for 10-12 hours, observe the growth of the colony, pick the positive single colony in kanamycin LB liquid culture medium, culture overnight, extract the plasmid and perform sequencing verification, and name the single colony strain with correct sequencing as pBAD-MTSase.

[0056] The same method was used to obtain pBAD-MTHase, pBAD-4αGT, pBAD-MTSase-op, pBAD-MTHase-op and pBAD-4αGT-op strains (the primers used for linearization of plasmid pBAD were the same before and after gene optimization). The bacterial culture solution of the strain verified to be correct by sequencing was mixed with 50% glycerol in a 1:1 ratio and then transferred to a -80°C ultra-low temperature refrigerator for storage.

[0057] The plasmid vectors pBAD-MTSase-op, pBAD-MTHase-op and pBAD-4αGT-op of sequence-optimized maltooligosyltrehalose hydrolase MTSase-op, maltooligosyltrehalose synthase MTHase-op and 4-α-glucosyltransferase 4αGT-op are shown in Figure 1 .

[0058] The strains pBAD-MTSase, pBAD-MTHase, pBAD-4αGT, pBAD-MTSase-op, pBAD-MTHase-op and pBAD-4αGT-op were streaked onto kanamycin LB solid culture medium and cultured in a 30°C constant temperature incubator overnight. After a single colony grew, it was inoculated into kanamycin LB liquid culture medium at 30°C and shaken at 220 rpm until the bacterial solution OD 600 After reaching 2.0, 10% of the inoculum was inoculated into 50 mL of kanamycin LB liquid medium. After inoculation, the culture was continued at 30°C and 220 rpm for 24 hours. At this time, the OD of the bacterial solution of pBAD-MTSase, pBAD-MTHase, pBAD-4αGT, pBAD-MTSase-op, pBAD-MTHase-op and pBAD-4αGT-op was 600 The values ​​were 14.568, 14.214, 14.574, 14.214, 13.878 and 13.428 respectively. After the culture, the cells were collected by centrifugation at 6000 rpm for 2 minutes and the supernatant was discarded.

[0059] The cells collected by centrifugation were washed once with 50 mL of sterile water, centrifuged at 6000 rpm for 2 minutes, the supernatant was discarded, and the bacterial cells were collected; then washed once with 50 mL of PBS buffer, centrifuged again at 6000 rpm for 2 minutes, the supernatant was discarded, and the bacterial cells were collected; finally, the bacterial cells were thoroughly suspended with 50 mL of PBS buffer. After filtering the bacterial solution with a filter, the cells were broken with a high-pressure homogenizer, the pressure was set to 600 bar and run for 3 minutes, the broken bacterial solution was collected, and it was centrifuged at 4°C and 10000 rpm in a high-speed centrifuge for 30 minutes, the supernatant was collected as a crude enzyme solution, and the precipitate was suspended with an equal volume of PBS buffer before centrifugation for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0060] 30 μL crude enzyme solution and precipitate solution were mixed with 10 μL LDS sample buffer and heat denatured at 99°C for 10 minutes as sample solution. A prefabricated protein gel with a concentration of 12% was used in conjunction with a Bio-Rad electrophoresis tank. Each sample solution was added to the gel well at a volume of 20 μL. Finally, 4 μL of protein marker was added to another gel well. The electrophoresis operation conditions were: Tris as electrophoresis buffer, voltage 150V, and duration 30-40 minutes. After the electrophoresis was completed, the protein gel was placed on a decolorization shaker for Coomassie brilliant blue staining for 30 minutes, and then decolorized with a decolorizing solution until the protein gel background was clear. Finally, a protein gel imager was used to take pictures and record them to observe whether the protein electrophoresis bands were consistent with the theoretical values. See the results. Figure 2 .

[0061] Take 100 μL of crude enzyme solution of MTSase and MTSase-op respectively and mix them evenly with 900 μL of 1 mg / mL maltohexaose prepared in advance with PBS, react in a 40°C water bath for 30 minutes, and add an equal volume of 1 mol / L NaOH solution to terminate the reaction. Then add 1 mL of DNS solution to the reaction system, react in a boiling water bath for 7 minutes, and use a spectrophotometer to measure the absorbance of the sample at 540 nm. Definition of enzyme activity unit: The amount of enzyme required to consume 1 mmol of maltohexaose per minute at 52°C with other conditions constant.

[0062] Take 100 μL of crude enzyme solution of MTHase ​​and MTHase-op respectively and mix them evenly with 900 μL of 1 mg / mL maltotetraosyl trehalose prepared in advance with PBS, react in a 40°C water bath for 30 minutes, and add an equal volume of 1 mol / L NaOH solution to terminate the reaction. Then add 1 mL of DNS solution to the reaction system, react in a boiling water bath for 7 minutes, and use a spectrophotometer to measure the absorbance of the sample at 540 nm. Definition of enzyme activity unit: The amount of enzyme required to consume 1 mmol of maltotetraosyl trehalose per minute at 52°C with other conditions constant.

[0063] Take 100 μL of 4αGT and 4αGT-op crude enzyme solution and mix them with 900 μL of 1 mg / mL maltotriose prepared in advance with PBS, and react in a 40°C water bath for 10 minutes. After the reaction is finished, treat it in a boiling water bath for 10 minutes to terminate the reaction. Then use a 1mL syringe and a 0.22μm filter to filter the reaction solution and add it to a liquid phase bottle to detect the amount of glucose produced. The glucose high-performance liquid chromatography (HPLC) detection method: Agilent1260, RID detector, sugar-paktm1 column (Waters), mobile phase: ultrapure water, flow rate 0.5mL / min, column temperature 70°C, injection volume 10μL, each sample treatment for 20 minutes. Enzyme activity unit definition: the amount of enzyme required to produce 1mmol of glucose per minute under 52°C conditions, other conditions are constant.

[0064] The enzyme activities of maltooligosyltrehalose hydrolase MTSase, maltooligosyltrehalose synthase MTHase ​​and 4-α-glucosyltransferase 4αGT and their sequence-optimized MTSase-op, MTHase-op and 4αGT-op are shown in Figure 3 .

[0065] Add 1 mL each of the crude enzyme solutions of MTSase, MTHase ​​and 4αGT into a 250 mL shake flask containing 12 g of maltodextrin, and then add PBS buffer to make the reaction volume up to 50 mL. Incubate the mixture in a shaker at 40°C and 220 rpm for 24 hours, then transfer the shake flask to a boiling water bath for 10 minutes to completely inactivate the enzyme.

[0066] Add 1 mL each of the crude enzyme solutions of MTSase-op, MTHase-op and 4αGT-op into a 250 mL shake flask containing 12 g of maltodextrin, and then add PBS buffer to make the reaction volume up to 50 mL. Incubate the mixture in a shaker at 40°C and 220 rpm for 24 hours, and then transfer the shake flask to a boiling water bath for 10 minutes to completely inactivate the enzyme.

[0067] Use a 1 mL syringe and a 0.22 μm filter membrane to filter the reaction solution to obtain the filtrate and add it to a liquid phase bottle, then transfer the liquid phase bottle to a high performance liquid chromatograph, trehalose high performance liquid chromatography (HPLC) detection method: Agilent 1260, RID detector, sugar-pak TM1 column (Waters), mobile phase: ultrapure water, flow rate 0.5 mL / min, column temperature 70°C, injection volume 10 μL, each sample was processed for 20 minutes. According to the peak area of ​​the prepared trehalose standard, the trehalose content in the reaction solution with the addition of MTSase, MTHase ​​and 4αGT was calculated to be 9.7368g; and the trehalose content in the reaction solution with the addition of MTSase-op, MTHase-op and 4αGT-op was 9.996g. The conversion rate of trehalose = trehalose content in the reaction solution / initial maltodextrin content. Finally, the trehalose conversion rates of MTSase, MTSase and 4αGT were calculated to be 81.14%, and the trehalose conversion rates of MTSase-op, MTSase-op and 4αGT-op were 83.3%.

[0068] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for optimizing the trehalase catalytic system to improve the trehalose conversion rate, characterized in that: The specific steps are as follows: (1) Optimizing the gene sequences of maltooligosyltrehalose hydrolase MTSase, maltooligosyltrehalose synthase MTHase ​​and 4-α-glucosyltransferase 4αGT; after optimization, the sequences MTSase-op, MTHase-op and 4αGT-op shown in SEQ ID NO.4-6 were obtained; (2) constructing pBAD-MTSase-op, pBAD-MTHase-op, and pBAD-4αGT-op strains, and preparing crude enzyme solutions of MTSase-op, MTHase-op, and 4αGT-op, respectively; (3) The crude enzyme solutions of MTSase-op, MTHase-op and 4αGT-op were reacted with maltodextrin in a ratio of 1:1:

1.

2. Use of the method according to claim 1 in producing trehalose.

3. Use of the method according to claim 1 in improving trehalose conversion rate.

4. Application of the sequences MTSase-op, MTHase-op and 4αGT-op shown in SEQ ID NO. 4-6 in improving the conversion rate of trehalose.

5. Application of pBAD-MTSase-op, pBAD-MTHase-op and pBAD-4αGT-op strains in improving trehalose conversion rate.

6. Application of crude enzyme solutions of MTSase-op, MTHase-op and 4αGT-op in improving the conversion rate of trehalose.

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