CN-121975768-A - Recombinant macrolide as well as preparation method and application thereof

Abstract

The scheme provides a recombinant macrolide and a preparation method and application thereof, wherein the amino acid sequence of the recombinant macrolide is shown as SEQ ID NO.1, and is obtained by site-directed mutagenesis of erythromycin esterase family protein sequences derived from enterobacter cholerae, wherein the mutated sites are 44 th glutamic acid mutated to asparagine, 55 th tyrosine mutated to proline, 153 th proline mutated to alanine and 219 th serine mutated to glutamic acid, and the recombinant macrolide is prepared efficiently, stably, easily and massively and is more suitable for specific macrolide with environmental requirements, so that macrolide antibiotic pollution is removed, and the recombinant macrolide has good economic benefit and practical value.

Inventors

• MA PENGFEI
• LI JIA
• XU LEI
• WANG YUNGUANG
• YE XIANGYU
• SHEN ZHILIN

Assignees

• 浙江泰林生命科学有限公司

Dates

Publication Date

20260505

Application Date

20260330

Priority Date

20251113

Claims (13)

1. 1. The recombinant macrolide is characterized in that the amino acid sequence of the recombinant macrolide is shown as SEQ ID NO. 1.
2. 2. The recombinant macrolide as claimed in claim 1, wherein the recombinant macrolide is derived from erythromycin esterase family proteins of E.cholerae and is obtained by site-directed mutagenesis at the 44 th glutamic acid to asparagine, 55 th tyrosine to proline, 153 th proline to alanine and 219 th serine to glutamic acid.
3. 3. A polynucleotide for encoding the recombinant macrolide enzyme of claim 1, which has the nucleotide sequence shown in SEQ ID No. 2.
4. 4. A recombinant vector comprising the polynucleotide for encoding a recombinant macrolide according to claim 3.
5. 5. An engineered strain comprising the recombinant vector of claim 4.
6. 6. The engineering strain according to claim 5, wherein the engineering strain is obtained by a chemical conversion method, the recombinant vector of claim 4 is transformed into competent cells of escherichia coli BL21, and the engineering strain with high expression of recombinant macrolide is obtained through colony PCR verification and induced expression screening, wherein primer sequences verified by colony PCR are shown as SEQ ID NO.3 and SEQ ID NO. 4.
7. 7. A process for the preparation of a recombinant macrolide comprising the steps of: s1, carrying out activation culture on the engineering strain of claim 5 to obtain seed solution; s2, inoculating the seed solution into a fermentation medium for culture to obtain a fermentation liquid, and inducing the fermentation liquid by adopting an inducer at low temperature to obtain thalli for expressing the recombinant macrolide; s3, collecting thalli, and then crushing to obtain recombinant macrolide crude enzyme liquid; S4, separating and purifying the crude enzyme solution of the recombinant macrolide to obtain the recombinant macrolide.
8. 8. The method for producing a recombinant macrolide as claimed in claim 7, wherein the inducer for inducing the expression of the macrolide is isopropyl- β -D-thiogalactoside and the crushing means is homogenizer crushing.
9. 9. The method for producing a recombinant macrolide as defined in claim 7, wherein the separation and purification are carried out by subjecting the crude enzyme solution of the recombinant macrolide to ultrafiltration clarification and anion exchange chromatography in this order.
10. 10. A recombinant macrolide protection agent comprising the recombinant macrolide of claim 1.
11. 11. Use of a recombinant macrolide as claimed in claim 1-2 for degrading macrolide antibiotics.
12. 12. Use according to claim 11 as a neutralising agent in the aseptic inspection of macrolide antibiotic drugs.
13. 13. Use according to claim 11, as a treatment of wastewater or environmental samples containing macrolide antibiotics.

Description

Recombinant macrolide as well as preparation method and application thereof Technical Field The invention relates to the field of enzyme products, in particular to a recombinant macrolide as well as a preparation method and application thereof. Background Macrolide antibiotics (such as erythromycin, azithromycin, clarithromycin and the like) are clinically widely used antibiotics due to their broad-spectrum antibacterial activity, and most of them are basic lipophilic compounds having macrolide groups in their molecular structures. However, its widespread use also presents two important technical challenges: On one hand, the sterility test of medicines is interfered, and the sterility test is a core link for guaranteeing the medication safety of products in the production and quality control flow of medicines (especially injection). The sterility of the product is judged by observing the growth condition of microorganisms in a culture medium by using a sterility detection method (such as a film filtration method and a direct inoculation method) specified by the current pharmacopoeia. At present, the bacteria-free inspection and microorganism limit inspection of macrolide antibiotics mainly adopt a film filtration method, but the method has obvious defects that on one hand, ethanol and other components which can influence the activity of microorganisms need to be added into flushing liquid, the flushing pressure needs to be more than 0.3bar, the operation is easy to cause the falling of microorganisms, on the other hand, the method has complex applicability verification flow and poor reproducibility of verification results. More importantly, the existing neutralization method lacks a specific neutralizer aiming at macrolide antibiotics, and has weak neutralization pertinence or limited effect on the antibiotics (particularly lactone rings in the molecular structure). And if the amount of the common broad-spectrum neutralizer (such as lecithin-Tween 80) is too large, the microbial growth can be inhibited. If a trace amount of macrolide antibiotics with activity remain in the sample to be detected, the residual antibacterial activity of the macrolide antibiotics can still obviously inhibit or even completely prevent the growth of potential pollution microorganisms in the detection culture medium even through conventional treatments such as dilution, addition of a general neutralizer and the like, so that a false negative result appears in the sterile detection, and a great risk is brought to clinical medication safety. On the other hand, the large-scale antibiotics exist in a large amount in production wastewater, cultivation wastewater, medical wastewater and expired medicines, and after the residual antibiotics enter the environment, the residual antibiotics can directly generate ecological toxicity to destroy aquatic ecosystems, and more importantly, the residual antibiotics are used as strong selective pressure to continuously promote the generation and transmission of Antibiotic Resistance Genes (ARGs) and Antibiotic Resistant Bacteria (ARBs) in the environment, so that public health safety is seriously threatened. At present, the method for removing macrolide antibiotics still has obvious defects, and particularly in the aspect of the treatment of the pollution of the antibiotics in the environment, no efficient and feasible removing technology is found yet. The method has the advantages of low removal efficiency, unstable effect, strong promotion of propagation of antibiotic resistance genes and resistant bacteria possibly due to selective enrichment, risk of resistance gene diffusion, poor selectivity, limited adsorption capacity, high synthesis cost, large-scale preparation difficulty, poor environmental stability (such as easy dissolution in water), poor recycling performance and the like of the traditional biological treatment method, and the defects of high operation cost, easy substrate interference and poor selectivity of the advanced oxidation process. The esterase capable of efficiently hydrolyzing the macrolide antibiotics provides a new idea for the application of the esterase in the aspect of environmental removal of macrolide antibiotics pollution. Macrolides theoretically have the potential to hydrolyze macrolide internal ester bonds, irreversibly destroying their antibacterial activity. However, there is still a lack of mature preparation technology in the industry, and it is difficult to obtain specific macrolides that are efficient, stable and easy to prepare on a large scale. The main reasons for this dilemma are that although macrolides are widely present in prokaryotes and eukaryotes, the enzyme production process of natural strains is complex and the yield is extremely low, and meanwhile, most of natural strains capable of producing the enzyme are pathogenic bacteria (such as enterobacter cholerae) and cannot be produced by a large-scale fermentation mode. The two factors limit the