CN117660496A - Rhamnosyltransferase SGT822 and application thereof - Google Patents

Abstract

The invention discloses a rhamnosyl transferase SGT822 and application thereof. The applicant screens from Streptomyces sp.147326 to obtain rhamnosyl transferase SGT822, can glycosylate and modify the nosiheptide to generate rhamnosyl nosiheptide, greatly improves the water solubility of the nosiheptide, explains the modifiable property of NOS pyridine ring hydroxyl, provides a new site for the structural modification of NOS, and provides a new enzymatic element for the rhamnosylation modification of natural products.

Description

Rhamnosyltransferase SGT822 and application thereof

Technical Field

The invention relates to rhamnosyl transferase, in particular to rhamnosyl transferase SGT822 and application thereof.

Background

The thiopeptides antibiotic belongs to ribosomal synthesis and post-translational modification peptides (Ribosome synthesis andpost translational modifiedpeptides, ripps), a heterocyclic peptide antibiotic with high modification produced by a variety of bacteria. Consists of a central pyridine ring, a core macrocycle and a tail (Bagley MC, et al J. Chem Rev.2005Feb;105 (2): 685-714).

The structure of the nosiheptide is as follows

Nosiheptide (NOS) belongs to the e-series of sulfur peptide parent compounds, and was originally isolated from Streptomyces parvus, and exhibits excellent antibacterial activity against all methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus currently, and has been the mainstream of the study of sulfur peptide antibiotics due to its structural representativeness (Haste NM, et al J.Antiboot (Tokyo) 2012Dec;65 (12): 593-8.). Although NOS has super-strong antibacterial activity, the disadvantages of poor water solubility and no in vivo activity limit the development and clinical application of the patent medicine, and structural transformation to obtain analogues with various structures becomes one of the important means for improving the patentability of NOS. However, NOS is too complex in structure, extremely difficult to modify, few examples of successful modification exist, and modification of dehydroalanine side chains and side rings is concentrated.

In the structure of NOS, the hydroxyl group of the core pyridine ring has excellent reactivity, and is an ideal structural modification site. The water solubility of the rhamnoylated norcetin is enhanced by glycosylating the pyridine ring hydroxyl of the norcetin with a rhamnotransferase to form the rhamnoylated norcetin.

Disclosure of Invention

The invention aims to provide a rhamnosyl transferase SGT822 and application thereof, wherein the rhamnosyl transferase SGT822 can realize the regioselective glycosylation modification of NOS pyridine ring hydroxyl. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

1) The rhamnosyltransferase is SGT822 derived from Streptomyces sp.147326.

2) A rhamnosyl transferase SGT822, the nucleotide sequence of said rhamnosyl transferase SGT822 being as set forth in seq id NO:1, high efficiency catalyzing the formation of rhamnosylated norcetin from norcetin.

3) The rhamnosyltransferase SGT822 constructs a plasmid.

4) The rhamnosyltransferase SGT822 may be expressed in E.coli BL21 (DE 3) independently or in a co-expression, and the heterologous expression host is only an example, and the rhamnosyltransferase may be expressed in lactic acid bacteria, streptomyces, yeast, bacillus subtilis, etc., without limiting the scope of the present invention.

5) The present invention provides a rhamnosyl transferase which has the reactivity of catalyzing the conversion of a nosiheptide into a rhamnosylated nosiheptide.

The invention has the following advantages:

the invention discovers that the rhamnosyl transferase SGT822 from natural microorganism is a brand new enzyme, the structure of the enzyme is not reported in the literature, the enzyme has the function of directionally modifying the pyridine ring hydroxyl of the norcetylpeptides to form the rhamnosylated norcetylpeptides, namely, the two hydroxyl groups exist in NOS, the rhamnose can be modified by the regional selective glycosylation of the pyridine ring hydroxyl of NOS, and the 3-position light group of the other threonine is not affected.

Improves the water solubility of the norcetirin and provides a new method for improving the water solubility of the series of other compounds by utilizing the modification of the site.

Drawings

FIG. 1 is an HPLC assay of SGT822 to catalyze NOS;

FIG. 2 is a graph showing the effect of reaction pH on SGT 822;

FIG. 3 is a graph showing the effect of reaction temperature on SGT 822;

FIG. 4 is a graph showing the effect of temperature stability (A) and pH stability (B) on SGT 822;

FIG. 5 is a graph showing the effect of metal ions on SGT 822;

FIG. 6 is a graph of the Miq equation for SGT822 glycosylation modification of a northwest peptide to produce a rhamnosylated northwest peptide;

FIG. 7 is an elution profile of SGT822 over Superdex 200;

FIG. 8 is a schematic representation of pET22b-SGT822 plasmid.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

EXAMPLE 1 construction of pET22b-SGT822 expression plasmid

(a) Acquisition of the Gene of interest

The Streptomyces sp.147326 genome was obtained by a kit extraction method, specific primers were designed as shown in Table 1, DNA fragments of SGT822 target sequences were obtained by PCR amplification, and were recovered and purified by gel.

TABLE 1 SGT822 specific primers

(b) Cleavage of the fragment of interest and the vector

The target fragment and the vector were digested with restriction endonucleases, and the cleavage system is shown in Table 2. After mixing, the enzyme digestion system is incubated for 1h in a 37 ℃ incubator, enzyme digestion products are separated by 1% agarose gel electrophoresis, the electrophoresis voltage is 120V, the electrophoresis time is 30min, and the enzyme digestion fragments are recovered by using a gel recovery kit.

TABLE 2 enzyme digestion system

(c) Fragment of interest and vector ligation

The target fragment after cleavage and recovery was ligated to a linear vector, and the ligation system was shown in Table 3. After mixing, the system was placed in a 16 ℃ incubator and connected for 12h. A schematic representation of the pET22b-SGT822 plasmid is shown in FIG. 8.

TABLE 3 ligation System of fragments of interest with vector

EXAMPLE 2 protein expression and purification of pET22b-SGT822 plasmid

(a) Construction of expression strains

The pET22b-SGT822 plasmid was transformed into E.coli competent DH 5. Alpha. And sequenced to obtain the correct plasmid pET22b-SGT822. Transformation of pET22b-SGT822 plasmid into E.coli protein expression competent

BL21(DE3)。

(b) Inducible expression of rhamnosyl transferase SGT822

Selecting monoclonal strain, shake culturing in liquid LB culture medium at 37deg.C and 220rpm to OD ₆₀₀ ＝0.6，

IPTG was added at a final concentration of 0.5mM and induction was carried out at 20℃for 12 hours. After the induction is finished, the bacterial cells are collected,

the cells were resuspended in 50mM Tris-HCl and collected by centrifugation again.

(c) Purification of rhamnosyltransferase SGT822

5g of the cells were weighed and added to 50mL of Tris-HCl buffer solution to mix well. And crushing the cells by using an ultrasonic crusher, collecting the supernatant, and purifying by using nickel affinity chromatography to obtain the SGT822 rhamnosyl transferase.

Example 4 functional verification of rhamnosyltransferase SGT822

Verification of rhamnosyl transferase SGT822 function was performed according to the reaction system shown in Table 4 where NOS is acceptor and dTDP-L-rhamnose (dTR) is sugar donor. After reaction at 30℃for 6 hours at 100rpm, 500. Mu.L of methanol and 500. Mu.L of DMSO were added and mixed uniformly to terminate the reaction, the mixture was centrifuged at 12000rpm for 10 minutes, and then filtered through a 0.22 μm organic filter membrane, and the reaction mixture was subjected to HPLC detection using NOS-R as a positive control, the detection result being shown in FIG. 1.

TABLE 4 glycosyltransferase reaction System

EXAMPLE 5 rhamnosyltransferase SGT822 enzymatic Studies (a) enzymatic Property determination

1. Optimal reaction pH

The reaction system was set to a final concentration of 1mg/mL of NOS, 20. Mu.L of SGT822 obtained in example 3 and 100. Mu.L of dTR, three parallel experiments were performed for each sample, and after the completion of the reaction, HPLC detection was performed to examine the change in substrate conversion in the pH range of 6 to 11. As shown in FIG. 2, at a reaction pH of between 6 and 10, the yield increased with increasing pH, and at pH 10, the conversion reached a maximum of 39.1% with 50mM Glycine-NaOH as the buffer. In addition, the enzyme activity is affected by the difference of the buffer used at the same pH, and the enzyme activity is affected minimally by Tris.

2. Optimum reaction temperature

The change of the conversion rate of the substrate in the range of 25-70 ℃ is examined, and the reaction system is the same as above. As shown in FIG. 3, the reaction temperature is increased between 25 and 55 ℃, the yield is increased, the conversion rate reaches the highest at 55 ℃, 73.2%, the temperature is continuously increased, the yield is obviously reduced, and the yield is only 13.3% at 70 ℃.

3. Temperature stability and pH stability

As shown in FIG. 4, SGT822 has better stability at 4deg.C, there is no significant change in enzyme activity after 12 hours of standing, and stability at 30deg.C is poorer than that at 4deg.C. After 12h in 50mM Glycine-NaOH buffer, the enzyme activity was only lost 7%, indicating that it is suitable for use as a storage buffer for SGT822.

4. Influence of metal ions

EDTA and Mg with final concentration of 5mM are added ²⁺ 、Mn ²⁺ 、Zn ²⁺ 、Fe ³⁺ 、Cu ²⁺ 、Ca ²⁺ 、K ⁺ 、Na ⁺ Ni and ²⁺ the rest reaction systems are the same, the reaction is carried out at the optimal temperature and pH of the enzyme, and the relative ratio is calculated by taking the substrate conversion rate of the reaction of the control group without adding metal ions as a standardActivity. As shown in FIG. 5, mg ²⁺ The enzyme activity is increased by about 1.3 times, and the metal ion chelating agent EDTA reduces the enzyme activity by half. Other metal ions all have various degrees of inhibition on SGT822, mn ²⁺ 、Ca ²⁺ 、K ⁺ Na (sodium carbonate) ⁺ The influence on the enzyme activity is weak, and Cu ²⁺ Has the strongest inhibition effect, and can lead the enzyme activity to lose about 97 percent (b) the determination of the kinetic parameters of the SGT822 of the rhamnosyl transferase

The kinetic parameters of the individual substrates NOS were determined, the amount of immobilized dTR was 100. Mu.L, the final concentration of NOS was set between 0.0156 and 2mM, and the reaction was carried out at optimum temperature and pH for 45min. As shown in FIG. 6, the curve after nonlinear fitting has a maximum reaction rate Vmax of 1.465.+ -. 0.049. Mu.M/min, a Mie constant Km of 107.22.+ -. 13.38. Mu.M, and a catalytic constant Kcat of 0.033.+ -. 0.001min ^-1 Kcat/Km of 3.07×10 ^-3 min ^-1 μM ^-1 。

EXAMPLE 6 rhamnosyltransferase SGT822 active form

The gel chromatography is used to separate the monomer and oligomer with large molecular weight difference, and the protein with large molecular weight is washed out before the protein with small molecular weight. The elution results are shown in FIG. 7, where there are two peaks at 280nm, the first peak is multimeric and the second peak is monomeric, by the control protein standard curve. The activity was verified after collecting the monomer and the multimer separately, and the results showed that the monomer was active and the multimer was inactive. Embodiment 7 rhamnosyltransferase SGT822 catalytic residue positioning

The conserved residues His13 and Asp308 were selected as mutation sites, which were mutated to alanine using a site-directed mutagenesis kit, the primers required for this procedure are shown in Table 5 (bolded bases are mutation sites), and the PCR procedure is shown in Table 6. As shown in the following formula, his13 and Asp308 are not necessarily catalytic, and act as catalytic diads.

TABLE 5 primers for alanine mutations

TABLE 6 PCR amplification procedure

Claims (5)

1. The rhamnosyl transferase SGT822 is characterized in that the nucleotide sequence of the rhamnosyl transferase SGT822 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO. 2.

2. A recombinant expression vector comprising the nucleotide sequence of rhamnosyltransferase SGT822 of claim 1.

3. An engineered host cell, wherein the recombinant bacterium or engineered host cell line comprises the recombinant expression vector of claim 2.

4. The engineered host cell of claim 3, wherein the host cell is e.

5. Use of a rhamnosyltransferase SGT822 according to claim 1 or a host cell according to claim 3 or 4 for the synthesis of rhamnosylated norcetin from glycosylation modified norcetin.