Reagent and method for high-throughput plate screening of 3-sialyltransferase mutant

Abstract

The invention relates to the technical field of biology, in particular to a reagent and a method for screening a 3-sialyltransferase mutant through a high-throughput flat plate. According to the invention, by adopting a solid flat plate chromogenic screening technology and combining with a pH indicator, efficient screening of 3SL high-yield mutants in a large-scale mutation library is realized. According to the method, the target mutant is quickly identified by directly observing the color change on the flat plate, so that the tedious steps of strain construction, sequencing, transferring, shake-flask culture and the like in the traditional screening process are omitted, and the screening speed and efficiency are remarkably improved. Compared with the prior art, the method has the advantages that the experiment time is greatly shortened, the operation process is simplified, the requirements on human resources and equipment are reduced, and great convenience is provided for screening of the enzyme mutation library. The method has the technical advantages of rapidness, high efficiency and low cost, and is expected to be widely applied to the fields of biomedicine and food science.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a reagent and method for high-throughput plate screening of 3-sialyltransferase mutants. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk after lactose and fat. They are a complex mixture of many oligosaccharides with multiple structures and functions, and therefore have important biological functions. In addition, the importance of HMOs in improving the health and nutritional needs of infants and young children was emphasized, and it was pointed out that the safety and functionality of HMOs have been recognized worldwide. At the same time, the application of HMOs in food was also pointed out, especially the necessity of adding HMOs to infant formula to make up for the lack of HMOs in cow's milk.

[0003] Sialyl lactose (SL) is an acidic human milk oligosaccharide, which can be divided into 3-SL and 6-SL according to the position where sialic acid is combined with lactose. As one of the main components of human milk oligosaccharides, 3-SL has been proven to have several beneficial effects on infants: it can reach the distal small intestine and colon intact, stimulate the growth of beneficial bacteria in the intestine, and maintain the balance of intestinal flora. It can inhibit the growth of harmful bacteria, bind pathogens, and reduce the incidence of infection. It supports intestinal barrier function, promotes immune development and potential neurodevelopment.

[0004] The currently commonly used enzymatic method for producing 3-SL is to use sialic acid (NeuAc) as the original substrate, use CMP-NeuAc synthetase (CSS) to convert SA and CTP into CMP-NeuAc, and then use sialyltransferase (ST) to catalyze the transfer reaction between CMP-NeuAc and lactose to produce 3-SL.

[0005] Glycosyltransferases are a class of enzymes that catalyze glycosylation reactions in organisms. They transfer sugar groups from an activated glycosyl donor to an acceptor molecule to form a glycosidic bond. Although glycosyltransferases play an important role in biosynthesis and metabolic regulation, there are still some shortcomings in their research and application. For example, glycosyltransferases from natural sources may have low expression levels, limiting their potential for industrial-scale application, the catalytic efficiency of some glycosyltransferases may not be sufficient to meet the needs of large-scale production, some glycosyltransferases may have solubility problems in the expression system, and the stability and activity of the enzymes may be affected by environmental conditions (such as temperature and pH). In order to address these shortcomings, directed evolution, rational or semi-rational design are needed to improve their catalytic efficiency. Therefore, the establishment of a novel, sensitive and rapid 3-SL detection method has high market application value. Although some high-throughput screening methods have been developed for enzyme screening and optimization, these methods may need to be further improved to improve screening efficiency and accuracy.

[0006] The existing high-throughput screening methods include: screening by using the flow cytometer screening system, using the difference in permeability of galactose permease on the cell membrane surface to substrates and glycosylation products. And using CRISPR / Cas9 technology to insert the luciferase coding sequence into the 3' end of the target protein gene of the neuroblastoma cell line, and judging the effect of small molecules on the expression level of the target protein by detecting the fluorescence value. In addition, fluorescence-activated cell sorting (FACS) and droplet microfluidics sorting (DMFS) technologies are specifically mentioned. They can be combined with mutagenesis breeding to identify and separate living cells with high 3-SL production, further improving the screening effect. Although these methods have been widely used in the fields of biomedicine and food, they all have high requirements for detection instruments, cumbersome operations, and greatly increased costs.

[0007] Directed evolution includes in vivo chemical gene mutagenesis, error-prone polymerase chain reaction, and in vitro saturated / unsaturated site-directed mutagenesis. Through these methods, a library containing a large number of mutants can be easily established. Therefore, whether beneficial mutations can be efficiently and quickly screened out from a huge number of protein mutant sub-libraries is the key to determining the success rate of enzyme directed evolution. However, some traditional screening methods require a lot of manpower and time, and it is difficult to cope with the challenges faced by the field of enzyme engineering, but high-throughput screening methods are developing rapidly as an important technology in enzyme directed evolution.

[0008] For application as an efficient biocatalyst, many challenges must be overcome, such as limited substrate range, thermal stability, solubility, and low affinity for substrates. In addition to rational protein engineering methods, directed evolution remains the key to designing proteins to adapt to the manufacturing process, which inevitably leads to huge libraries containing mainly inactive or less active variants. Therefore, in order to speed up the directed evolution process, it is crucial to provide sensitive, efficient and low-cost detection methods. This avoids the time-consuming and technically demanding analysis of each variant by HPLC or GC, while saving the expensive instruments required for FACS and DMFS technologies. Summary of the invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a reagent and method for high-throughput plate screening of 3-sialyltransferase mutants.

[0010] The invention provides a reagent for screening 3-sialyltransferase mutants, which comprises a buffer, cytidine monophosphate-sialic acid, lactose and a pH indicator.

[0011] In the process of producing 3-SL by cell catalytic reaction, due to the production of cytidine monophosphate and the formation of glycosidic bonds, the pH value in the reaction system gradually decreases with the increase of 3-SL production. The 3-sialyltransferase mutant has strong activity, and can more efficiently catalyze the transfer reaction between CMP-NeuAc and lactose to generate more 3-SL, thereby reducing the pH value. The present invention utilizes this characteristic to make a preliminary judgment on the activity of the 3-sialyltransferase mutant by observing the change of pH value.

[0012] The reagent provided by the present invention can judge the colonies, omitting the need to construct each mutant strain in the traditional enzyme mutant screening process, or construct a mutant library to select a large number of bacteria for cultivation, and then screen after the enzyme is produced. Not only that, it also avoids the possibility of contamination caused by transfer and cumbersome steps such as cell breakage, greatly simplifying the screening steps.

[0013] In the present invention, the most important thing in the screening reagent is the selection of pH indicator. For this reason, the present invention screens feasible pH indicators in the prior art. In the present invention, the pH indicator needs to change color within the range of 6.0 to 7.5. For example, the color change range of the pH indicator is 6 to 8, or 6.2 to 8.2, or 6.5 to 8.5, or 6 to 7.6, or 6.8 to 8.8, or 6.0 to 7.0 or 7.0 to 9.0. In some embodiments, the pH indicator is at least one of bromothymol blue, neutral red or catechol purple. Preferably, the color change range of the pH indicator is 6 to 7.6. More preferably, the pH indicator is bromothymol blue.

[0014] In some embodiments, the concentration of bromothymol blue in the screening reagent is 0.1-1 mM, for example, 0.1-0.5 mM; or 0.3-0.5 mM; or 0.4-0.5 mM. Preferably, the concentration of bromothymol blue is 0.4 mM.

[0015] In the present invention, the buffer needs to be matched with a pH indicator, does not interfere with the occurrence of the reaction in the bacteria, and cannot affect the color change. In the reagent of the present invention, the buffer is preferably a buffer with a pH value of 7.5 to 8.0. For example, the buffer is Tris-HCl, phosphate buffer or TM buffer. It was found in the experiment that the best buffer is Tris-HCl buffer, wherein the concentration of Tris-HCl is 30 to 50mM, preferably 45 to 55mM, and in the more preferred screening reagent, the concentration of Tris-HCl is 50mM.

[0016] The present invention also optimizes and screens the ratio and concentration of lactose and cytidine monophosphate-sialic acid, and the lactose concentration that can make the reaction more fully and quickly occur is 20-30mM. For example, the concentration of lactose in the screening reagent is 20-25mM, or 22-27mM, or 25-30mM. Preferably, the concentration of lactose in the screening reagent is 23-25mM. More preferably, the lactose concentration is 24mM. The cytidine monophosphate-sialic acid concentration that can make the reaction more fully and quickly occur is 15-25mM. For example, the concentration of lactose in the screening reagent is 17-23mM, or 18-24mM, or 16-24mM. Preferably, the concentration of lactose in the screening reagent is 19-21mM. More preferably, the lactose concentration is 20mM.

[0017] In the present invention, the screening reagents include: Tris-HCl buffer, cytidine monophosphate-sialic acid, lactose and bromothymol blue.

[0018] In some embodiments, 30-50 mM Tris-HCl buffer, 15-25 mM cytidine monophosphate-sialic acid, 20-30 mM lactose and 0.1-1 mM bromothymol blue.

[0019] The screening reagent of the present invention is a solid reagent, which is not limited in the present invention. If it is a solid reagent, the reagent also includes agarose. The mass fraction of the agarose is 0.5% to 1.0%. In the embodiment, the mass fraction of agarose is 0.8%, which is more conducive to the penetration of the screening reagent.

[0020] In a specific embodiment, the screening reagent includes: 50 mM Tris-HCl pH=8.0 buffer, 24 mM lactose, 20 mM cytidine monophosphate-sialic acid, 0.4 mM bromothymol blue and 0.8% agarose.

[0021] The preparation of the screening reagent of the present invention comprises boiling Tris-HCl buffer, cytidine monophosphate-sialic acid, lactose, bromothymol blue and agarose to 100° C., cooling to about 50° C. and pouring on a plate to obtain a solid screening reagent.

[0022] In the present invention, the screening reagent also includes a nitrocellulose membrane.

[0023] The present invention does not limit the pore size of the nitrocellulose membrane, which can penetrate the culture medium or the screening reagent, so that the bacterial colony is in contact with the reagent or the culture medium.

[0024] The reagent provided by the present invention can carry out cultivation and screening simultaneously without affecting the growth of the colonies, and the colonies after screening can be directly picked, expanded and tested. Therefore, the present invention provides the use of the above-mentioned reagent in screening 3-sialyltransferase mutants.

[0025] The present invention also provides a method for screening 3-sialyltransferase mutants, which comprises contacting the colony with the reagent and judging the activity of the 3-sialyltransferase according to the color change.

[0026] The method described in the present invention specifically includes:

[0027] Step 1), applying the bacterial solution on a solid culture medium for cultivation;

[0028] Step 2), printing the bacterial colony on the nitrocellulose membrane, and then applying the membrane with the bacterial colony printed on it upwards on a solid culture medium for cultivation;

[0029] Step 3), stick the nitrocellulose membrane containing the colonies after the culture in step 2) on the reagent as described above, and observe the color change after the reaction.

[0030] In the present invention, the colonies are not in direct contact with the screening reagent, but are in contact with the permeated reagent through the nitrocellulose membrane, thereby causing color change.

[0031] In the present invention, the bacterial solution is Escherichia coli transformed with a mutant fragment, which is constructed by genetic engineering or obtained by mutagenesis, and the present invention does not limit this.

[0032] In the embodiment, it is constructed by genetic engineering means, and the main steps include transferring the obtained nucleic acid encoding the 3-sialyltransferase mutant into competent Escherichia coli via a plasmid, and inducing the expression of the 3-sialyltransferase mutant with IPTG after cultivation.

[0033] In the present invention, the solid culture medium for culture in step 1) is LB solid culture medium containing 50 μg / ml kanamycin.

[0034] In the present invention, the solid culture medium for the culture in step 2) is an LB solid culture medium containing 50 μg / ml kanamycin and 0.5 mM IPTG, and the culture conditions include culturing at 30° C. for 8 h;

[0035] In the present invention, the reaction conditions of step 3) include standing the reaction at 28° C. for 2 hours.

[0036] In the present invention, judging the activity of 3-sialyltransferase according to the color change includes: if the colonies are blue or green, the activity of 3-sialyltransferase is weak, and if the colonies turn yellow, the activity of 3-sialyltransferase is strong.

[0037] The present invention realizes efficient screening of 3SL high-yield mutants in large-scale mutation libraries by adopting solid plate colorimetric screening technology in combination with pH indicators. This method can quickly identify target mutants by directly observing the color changes on the plate, thereby omitting the cumbersome steps of strain construction, sequencing, transfer, and shake flask culture in the traditional screening process, and significantly improving the screening speed and efficiency. Compared with the prior art, the present invention not only greatly shortens the experimental time, simplifies the operation process, but also reduces the requirements for human resources and equipment, providing great convenience for the screening of enzyme mutation libraries. The technical advantages of the present invention are that it is fast, efficient, and low-cost, and is expected to be widely used in the fields of biomedicine and food science. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shows screening on a color plate containing 10 mM Tris-HCl;

[0039] Figure 2 Shows screening on a color plate containing 30 mM Tris-HCl;

[0040] Figure 3 Shows screening on a color plate containing 50 mM Tris-HCl;

[0041] Figure 4 The color development result of Example 1 is shown;

[0042] Figure 5 The color development results of Example 2 are shown;

[0043] Figure 6 The color development results of Example 3 are shown;

[0044] Figure 7 The liquid phase spectrum of the F314 mutant in the control group is shown;

[0045] Figure 8 Shown is the liquid phase spectrum of Y314 mutant. DETAILED DESCRIPTION

[0046] The present invention provides reagents and methods for high-throughput plate screening of 3-sialyltransferase mutants, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0047] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0048] In the present invention, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone.

[0049] In the present invention, "at least one" means one or more, and "more" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0050] In the present invention, "include", "comprising" and "having" are used interchangeably, and are intended to indicate the inclusiveness of the scheme, meaning that the scheme may have other elements besides the listed elements. At the same time, it should be understood that the descriptions of "include", "comprising" and "having" used in this article also provide "consisting of..." or "as shown in..." schemes.

[0051] The numerical ranges and parameters involved in the present invention have been presented in the specific embodiments as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have certain reasonable deviations within a certain range, such as: ±10%, ±5%, ±1% or ±0.5%.

[0052] In a specific embodiment of the present invention, the pH range of bromothymol blue is 6-7.6. When the initial pH value of the screening plate is about 7.5, it is found in preliminary experiments that if there is a better mutant, the pH value in the bacteria will gradually drop to below 7, and the bacteria will change from blue to green to yellow, showing obvious changes visible to the naked eye. This method omits the need to construct each mutant strain in the traditional enzyme mutant screening process, or to construct a mutant library and select a large number of bacteria for cultivation, and then screen after the enzyme is produced. This method requires several cultures and transfers, and also requires steps such as cell crushing. The present invention omits the method of selecting bacteria in large quantities and culturing on 96-well plates. The configured color development plate has good biocompatibility, combines induced expression, growth, catalytic reaction and enzyme activity color development detection, and performs them simultaneously on the plate. Rapid and efficient screening of effective mutant strains is achieved.

[0053] The amino acid sequence of the wild-type ASL involved in the present invention is:

[0054] MGLKKACLTVLCLIVFCFGIFYTFDRVNQGEPVNLIFCYTILQMKVAERIMAQHPGERFYVVLMSENRNEKYDYYFNQIKDKAERAYFFHLPYGLNKSFNFIPTMAELKVKAMLLPKVKRIYLASLEKVSIAAFLSTYPDAEIKIFDDGTGNLIQSSSYLGDEFSVNGTIKRNFARM MIGDWSIAKTRNASDEHYTIFKGLKNIMDDGRRKMTYLPLFDASELKAGDETGGTVRILLGSPDKEMKEISEKAAKNFNIQYVAPHPRQTYGLSGVTTLNSPYVIEDYILREIKKNPHTRYEIYTFFSGAALTMKDFPNVHVYALKPASLPKDYWLKPVYALFTQSGIPILTFDDKD

[0055] The nucleic acid sequence of the wild-type ASL involved in the present invention is:

[0056]

[0057] The test materials used in the present invention are all common commercial products and can be purchased on the market. It should be understood that in various embodiments of the present application, the size of the sequence number of each process does not mean the order of execution, some or all of the steps can be executed in parallel or in sequence, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application. The present invention is further described below in conjunction with the embodiments:

[0058] Example 1 Screening of color plate formula:

[0059] 1. Solution configuration:

[0060] 1) 500 mM tris(hydroxymethyl)aminomethane (Tris), adjusted to pH 8 with hydrochloric acid (HCl) to obtain 500 mM Tris-HCl buffer with pH=8.

[0061] 2) LB liquid culture medium: 1% peptone, 1% sodium chloride, 0.5% yeast powder.

[0062] 3) LB solid medium: 1% peptone, 1% sodium chloride, 0.5% yeast powder, 1.5% agar powder.

[0063] Second, three strains with different sialyltransferase activities were cultured separately in LB liquid medium containing 50 μg / ml kanamycin resistance. After the bacteria grew, they were counted according to OD 600 The measured absorbance values ​​were mixed in a 1:1:1 ratio and diluted to a suitable concentration, then spread on LB solid medium containing 50 μg / ml kanamycin resistance and cultured overnight at 37°C to obtain single colonies of suitable density.

[0064] 3. Divide an 80cm nitrocellulose membrane (0.45μm) into three parts and cover them on the above-mentioned overnight culture plate respectively (do not cover the same area repeatedly), then stick the side printed with colonies upwards to the induction plate containing 50μg / ml kanamycin and 0.5mM IPTG, and induce expression at 30℃ for 8h.

[0065] 4. Add 20mM cytidine monophosphate-sialic acid (CMP-SA), 24mM lactose, 0.4mM bromothymol blue solution (BTB) to 10mM, 30mM, 50mM Tris-HCl system at pH=8, and finally add 0.8% agarose powder. Mix the color-developing solution evenly and boil it to dissolve the agarose. When it cools to about 50°C, quickly pour it into a plate. After the plate is completely solidified, stick the nitrocellulose membrane with the induced bacteria on the color-developing plate (the side containing the colonies faces up). Observe the color development results after reacting at 28°C for 2h.

[0066] The results are as follows Figures 1 to 3 When the concentration of Tris-HCl was 10mM and 30mM, the sensitivity of color development was low, and the single colonies of the mutants that reacted all turned yellow, making it difficult to screen out mutants with higher enzyme activity. It is worth noting that when the concentration of Tris-HCl was 50mM, single colonies could show different colors, and the sensitivity was greatly improved.

[0067] Example 2 Mutant Construction and Screening

[0068] 1. Construction of ASL target gene. The target gene ASL was synthesized by gene synthesis. The amino acid and gene sequences are shown in Appendix 1. The synthesized gene was connected and inserted into the pET28a expression vector, using the two sites of NdeI and XhoI restriction endonucleases to form the pET28a-ASL original plasmid.

[0069] Solution configuration:

[0070] 1) 500 mM tris(hydroxymethyl)aminomethane (Tris), adjusted to pH 8 with hydrochloric acid (HCl) to obtain 500 mM Tris-HCl buffer with pH=8.

[0071] 2) LB liquid culture medium: 1% peptone, 1% sodium chloride, 0.5% yeast powder.

[0072] 3) LB solid medium: 1% peptone, 1% sodium chloride, 0.5% yeast powder, 1.5% agar powder.

[0073] Second, using Schrödinger to scan residue mutations, mutations at the L123 site are beneficial to enhancing the affinity of the ASL enzyme to lactose. This site is located at the entrance of the active site, and mutation of it can extend the length of the side chain at this site, increase the steric hindrance of this site, narrow the entrance of the active site, and increase the stability of lactose binding in the active site.

[0074] Design degenerate primers: 5'-3' direction:

[0075] PF1-AAACGCATTTATNNKGCGAGCCTGGAAA,

[0076] PR1-GCTCCGCMNNATAAATGCGTTTCACTTTC.

[0077] Amplify in a PCR instrument according to the following procedure:

[0078] Primer PF1 (10 μM) 1μL Primer PR1 (10 μM) 1μL template(40ng) 0.5μL 2x phanta max master mix 10μL <![CDATA[ddH2O]]> Up to 20μL

[0079] PCR machine procedure:

[0080]

[0081] After PCR, the machine was removed and the PCR products were run on gel electrophoresis. The PCR-positive products were purified by gel recovery. 4 μL of the homologous recombination product was transferred into 30 μL DH5α competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, ice-bathed for 2-3 minutes, and then 500 μL of non-antibiotic LB medium was added. The culture was shaken for 45 minutes, and the whole was centrifuged and spread on a solid LB plate containing 50 μg / ml kanamycin. The cells were cultured in a 37°C incubator overnight for 12-16 hours. The bacteria on the plate were washed off with non-resistant LB medium and the mixed plasmid was extracted with a kit. 5 μL of the mixed plasmid was transferred into 100 μL BL21 (D3) competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, and ice-bathed for 2-3 minutes. Then, 500 μL of antibiotic-free LB medium was added, and after shaking culture for 1 hour, 150 μL was spread on a solid LB plate containing 50 μg / ml kanamycin, and cultured in a 37°C incubator overnight for 12-16 hours.

[0082] A nitrocellulose membrane (0.45 μm) with a diameter of 80 cm was placed on a plate with a moderate colony density and marked. Then the side with the colonies printed on it was pasted onto an induction plate containing 50 μg / ml kanamycin and 0.5 mM IPTG and induced expression at 30°C for 8 h.

[0083] 3. Preparation of color plate:

[0084] Add 20mM cytidine monophosphate-sialic acid (CMP-SA), 24mM lactose, 0.4mM bromothymol blue solution (BTB) to the 50mM pH=8 Tris-HCl system, and finally add 0.8% agarose powder. Mix the color-developing solution evenly and boil it to dissolve the agarose. After cooling to about 50°C, quickly pour the plate. After the plate is completely solidified, stick the nitrocellulose membrane with the induced bacteria on the color-developing plate (the side containing the colonies faces up). Observe the color development results after reacting at 28°C for 2h. Figure 4 The overall color was green, and there was no superior mutant.

[0085] Example 3

[0086] 1. Same as Example 2

[0087] Second, by constructing a correlation network with the catalytic residues, we selected the site T196 with a higher impact, performed saturation mutation, and screened the mutation types that positively affected the catalytic residues and improved the catalytic activity.

[0088] Design degenerate primers, 5'-3' direction:

[0089] PF2-GATGAACATTATNNKATTTTTAAAGGCCTGA,

[0090] PR2-CTTTAAAAAATMNNATAATGTTCATCGCTCG.

[0091] Amplify in a PCR instrument according to the following procedure:

[0092] Primer PF2 (10 μM) 1μL Primer PR2 (10 μM) 1μL template(40ng) 0.5μL 2x phanta max master mix 10μL <![CDATA[ddH2O]]> Up to 20μL

[0093] PCR machine procedure:

[0094]

[0095] After PCR, the machine was removed and the PCR products were run on gel electrophoresis. The PCR-positive products were purified by gel recovery. 4 μL of the homologous recombination product was transferred into 30 μL DH5α competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, ice-bathed for 2-3 minutes, and then 500 μL of non-antibiotic LB medium was added. The culture was shaken for 45 minutes, and the whole was centrifuged and spread on a solid LB plate containing 50 μg / ml kanamycin. The cells were cultured in a 37°C incubator overnight for 12-16 hours. The bacteria on the plate were washed off with non-resistant LB medium and the mixed plasmid was extracted with a kit. 5 μL of the mixed plasmid was transferred into 100 μL BL21 (D3) competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, and ice-bathed for 2-3 minutes. Then, 500 μL of antibiotic-free LB medium was added, and after shaking culture for 1 hour, 150 μL was spread on a solid LB plate containing 50 μg / ml kanamycin, and cultured in a 37°C incubator overnight for 12-16 hours.

[0096] A nitrocellulose membrane with a diameter of 80 cm was placed on a plate with a moderate colony density and marked. Then the side with the colonies printed on it was pasted onto an induction plate containing 50 μg / ml kanamycin and 0.5 mM IPTG and induced for expression at 30°C for 8 h.

[0097] 3. Preparation of color plate:

[0098] Add 20mM cytidine monophosphate-sialic acid (CMP-SA), 24mM lactose, 0.4mM bromothymol blue solution (BTB) to the 50mM pH = 8 Tris-HCl system, and finally add 0.8% agarose powder. Mix the color-developing solution evenly and boil it to dissolve the agarose. When it cools to about 50°C, quickly pour it into a plate. After the plate is completely solidified, stick the nitrocellulose membrane with the induced bacteria on the color-developing plate (the side containing the colonies faces up). Observe the color development results after reacting at 28°C for 2h. Figure 5 The overall color was green, and there was no superior mutant.

[0099] Example 4

[0100] 1. Same as Example 2

[0101] Second, the enzyme design module in Rosetta was used to redesign the shape of the active site according to the ligand. In the constructed mutant, the F314 mutation increased the cavity volume of the active site. After the mutant was re-docked with lactose, the docking results showed that the binding position of lactose was deeper into the bottom of the active site, which was more conducive to the stable binding of lactose and SL09 enzyme.

[0102] Design degenerate primers: 5'-3' direction:

[0103] PF3-TGAAAGATNNKCCGAACGTGCATGTG,

[0104] PR3-ACGTTCGGMNNATCTTTCATGGTCAGCG.

[0105] Amplify in a PCR instrument according to the following procedure:

[0106] Primer PF3 (10 μM) 1μL Primer PR3 (10 μM) 1μL template(40ng) 0.5μL 2x phanta max master mix 10μL <![CDATA[ddH2O]]> Up to 20μL

[0107] PCR machine procedure:

[0108]

[0109] After PCR, the machine was removed and the PCR products were run on gel electrophoresis. The PCR-positive products were purified by gel recovery. 4 μL of the homologous recombination product was transferred into 30 μL DH5α competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, ice-bathed for 2-3 minutes, and then 500 μL of non-antibiotic LB medium was added. The culture was shaken for 45 minutes, and the whole was centrifuged and spread on a solid LB plate containing 50 μg / ml kanamycin. The cells were cultured in a 37°C incubator overnight for 12-16 hours. The bacteria on the plate were washed off with non-resistant LB medium and the mixed plasmid was extracted with a kit. 5 μL of the mixed plasmid was transferred into 100 μL BL21 (D3) competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, and ice-bathed for 2-3 minutes. Then, 500 μL of antibiotic-free LB medium was added, and after shaking culture for 1 hour, 150 μL was spread on a solid LB plate containing 50 μg / ml kanamycin, and cultured in a 37°C incubator overnight for 12-16 hours.

[0110] A nitrocellulose membrane with a diameter of 80 cm was placed on a plate with a moderate colony density and marked. Then the side with the colonies printed on it was pasted onto an induction plate containing 50 μg / ml kanamycin and 0.5 mM IPTG and induced for expression at 30°C for 8 h.

[0111] 3. Preparation of color plate:

[0112] Add 20mM cytidine monophosphate-sialic acid (CMP-SA), 24mM lactose, 0.4mM bromothymol blue solution (BTB) to the 50mM pH = 8 Tris-HCl system, and finally add 0.8% agarose powder. Mix the color-developing solution evenly and boil it to dissolve the agarose. When it cools to about 50°C, quickly pour it into a plate. After the plate is completely solidified, stick the nitrocellulose membrane with the induced bacteria on the color-developing plate (the side containing the colonies faces up). Observe the color development results after reacting at 28°C for 2h. Figure 6 Among them, the single colony with yellower color is the mutant with higher 3-SL production.

[0113] Example 5

[0114] Compare the position of the yellowish single colony with the original plate (after covering the bacteria with a membrane, culture at 37°C for 4 hours and then store in a 4°C refrigerator), and pick the single colony from the original plate and the control (unmutated strain) in advance and culture them in 96 deep-well plates. Use a toothpick to inoculate the picked single colony into 600μL LB medium containing 50μg / ml kanamycin, 37°C, 220r. When OD600=2, take 20μL of bacterial solution and add it to LB medium containing 50μg / ml kanamycin and 0.3mM inducer IPTG, and culture it overnight at 37°C and 220r. After the culture is completed, centrifuge and put the 96 deep-well plate containing the bacteria into the refrigerator at -20°C for freezing.

[0115] Take out the cultured 96-deep-well plate containing only bacteria from the -20°C refrigerator, add 100 μL of lysis buffer to each well, blow thoroughly to suspend the bacteria and then break them with lysis buffer at 25°C, 800 rpm, and 2 h.

[0116] Lysis buffer formulation concentration Dosage Lysozyme / 20mg Tris-HCl 10mM, pH 8.0 19.8mL DNase I 2000U / mL 2.5μL <![CDATA[MgCl2]]> 1M 0.2mL

[0117] 30mM CMP-SA was mixed with 36mM lactose and 50mM Tris, and 600μL was added to a new 96-deep-well plate. Then 50μL of each mutant containing lysis solution was added to each well. The reaction was carried out at 28℃ and 220r for 1h. After the reaction, 100μL of the sample was added to 60% acetonitrile for quenching and liquid phase verification. After that, the point with higher 3-SL production detected by liquid phase was selected from the original plate for bacterial culture and sent for sequencing.

[0118] Among them, the strain with F314 mutated to Y314 produced the highest yield of 3-SL ( Figures 7-8 ), the yield increased by 1.46 times compared with the control group. This proves that the plate high-throughput screening method of the present invention is efficient and rapid, and the results are reliable.

[0119] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A reagent for screening 3-sialyltransferase mutants, comprising: buffer, cytidine monophosphate-sialic acid, lactose, and pH indicator; The pH value of the buffer solution is 7.5 to 8; The color change range of the pH indicator includes 6.0 to 7.

6.

2. The reagent according to claim 1, characterized in that The buffer is Tris-HCl, phosphate buffer or TM buffer; The pH indicator is bromothymol blue, neutral red or catechol violet.

3. The reagent according to claim 1 or 2, characterized in that It includes: 30-50 mM Tris-HCl buffer, 15-25 mM cytidine monophosphate-sialic acid, 20-30 mM lactose and 0.1-1 mM bromothymol blue.

4. The reagent according to claim 3, characterized in that It includes: 50 mM Tris-HCl buffer, 24 mM lactose, 20 mM cytidine monophosphate-sialic acid, 0.4 mM bromothymol blue and 0.8% agarose.

5. The reagent according to any one of claims 1 to 4, characterized in that Also included are nitrocellulose membranes.

6. A method for screening 3-sialyltransferase mutants, comprising contacting a colony with the reagent according to any one of claims 1 to 5, and determining the activity of the 3-sialyltransferase based on color changes.

7. The method according to claim 6, characterized in that: Specifically include: Step 1), applying the bacterial solution on a solid culture medium for cultivation; Step 2), printing the bacterial colony on the nitrocellulose membrane, and then applying the membrane with the bacterial colony printed on it upward on the solid culture medium for induction culture; Step 3), stick the nitrocellulose membrane containing the colonies after the culture in step 2) on the reagent according to any one of claims 1 to 6 with the side facing upward, and observe the color change after the reaction.

8. The method according to claim 7, characterized in that The bacterial solution is Escherichia coli transformed with a mutant fragment.

9. The method according to claim 7, characterized in that: The solid culture medium of step 1) is LB solid culture medium containing 50 μg / ml kanamycin. Step 2) The solid culture medium for the culture is LB solid culture medium containing 50 μg / ml kanamycin and 0.5 mM IPTG, and the culture conditions include induction culture at 30° C. for 8 h; Step 3) The reaction conditions include standing the reaction at 28°C for 2 hours.

10. The method according to claim 8, characterized in that The activity of 3-sialyltransferase is determined based on the color change: if the colonies are blue or green, the activity of 3-sialyltransferase is weak; if the colonies turn yellow, the activity of 3-sialyltransferase is strong.

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