CN-116024205-B - Method for improving activity of 2-deoxy-D-ribose 5-phosphate aldolase through immobilization

Abstract

The present invention provides a method for increasing the activity of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) by immobilization. The method can remarkably activate the aldolase activity of DERA, has simple operation, mild conditions, is simple and easy to operate, and can be applied to the preparation of statin chiral side chains.

Inventors

• CHEN YIJUN
• ZHANG MINGYUE
• CAI WUJIN

Assignees

• 中国药科大学

Dates

Publication Date

20260505

Application Date

20230223

Claims (6)

1. 1. A method for improving the activity of 2-deoxy-D-ribose 5-phosphate aldolase is characterized in that diatomite is added into a solution containing 2-deoxy-D-ribose 5-phosphate aldolase for adsorption and fixation for 1-4 hours, the protein concentration of the solution containing 2-deoxy-D-ribose 5-phosphate aldolase is 5-50 mg/ml, 75-200 mg of diatomite is added into each 1ml of solution, and the solution containing 2-deoxy-D-ribose 5-phosphate aldolase is a buffer solution with the pH value of 6.0-9.0.
2. 2. The method according to claim 1, characterized in that the pH of the buffer is 7.0.
3. 3. Method according to claim 1 or 2, characterized by comprising the steps of: (1) Collecting thalli of recombinant engineering strains containing 2-deoxy-D-ribose 5-phosphate aldolase genes obtained by fermentation culture, and re-suspending the thalli with buffer solution to obtain bacterial suspension; (2) After the thalli are crushed, centrifuging and collecting supernatant to obtain crude enzyme liquid; (3) Adding diatomite into the crude enzyme solution, and vibrating, adsorbing and fixing; (4) And after immobilization, centrifugally collecting precipitate, washing, and freeze-drying to obtain immobilized activated 2-deoxy-D-ribose 5-phosphate aldolase.
4. 4. An immobilized activated 2-deoxy-D-ribose 5-phosphate aldolase prepared by the method of any one of claims 1-3.
5. 5. Use of an immobilized activated 2-deoxy-D-ribose 5-phosphate aldolase according to claim 4 in the field of enzyme catalysis.
6. 6. Use according to claim 5, characterized in that the immobilized activated 2-deoxy-D-ribose 5-phosphate aldolase is used for the preparation of statin chiral side chains.

Description

Method for improving activity of 2-deoxy-D-ribose 5-phosphate aldolase through immobilization Technical Field The invention relates to the field of biocatalysis, in particular to a method for improving the activity of 2-deoxy-D-ribose 5-phosphate aldolase through immobilization. Background 2-Deoxy-D-ribose 5-phosphate aldolase (2-deoxy-D-ribose-5-phosphate aldolase, DERA, EC 4.1.2.4) is an acetaldehyde dependent class I aldolase capable of mediating stereoselective C-C bond formation between an acetaldehyde donor and various acceptor substrates, resulting in a new chiral center (J.Rouvinen, M.Andberg, appl Microbiol Biotechnol,2021,105 (16-17): 6215-6228). DERA is widely distributed in various bacterial, fungal, plant, mammalian cells and plays an important role in the metabolism of nucleic acids in organisms. The DERA-catalyzed aldol condensation reaction proceeds under mild conditions by formation of a Schiff base between the donor substrate and the amino group of the enzyme active site lysine residue, followed by stereoselective addition of the activated donor to the aldehyde acceptor via the enamine intermediate to form a plurality of chiral centers. Based on this reactive nature of DERA, it has been applied to the industrial production of a variety of chemicals, including pentaerythritol, deoxy sugars, fragrances, and complex drug molecules. The most interesting application of DERA to the synthesis of chiral side chains of hypolipidemic statins (atorvastatin and rosuvastatin) has significant advantages (W.Greenberg, A.Varvak.Proc NATL ACAD SCI U S A.2004,101 (16): 5788-93). However, in the practical use process, the enzyme is sensitive to the substrate and easy to inactivate, the substrate is inhibited, and the enzyme cannot be recycled. Therefore, the immobilization of DERA not only can improve the stability, but also can be recycled, and the industrial application value of DERA can be effectively improved. The enzyme immobilization means that free enzyme is bound in a certain space or on a carrier, and free flow of the free enzyme is limited, so that the free enzyme can play a catalytic role for a long time and can be recycled. Immobilization can increase the stability of the enzyme, the tolerance of the organic solvent, the recycling property, the storage stability and the like, and is an excellent method for improving the physicochemical properties of the enzyme. The immobilization method mainly comprises four steps of adsorption, crosslinking, embedding and covalent bonding. The materials used in the methods are easy to obtain and mature, but have various defects, such as difficult diffusion of enzyme substrates and products after embedding and fixing, easy enzyme inactivation caused by a covalent bonding method, poor mechanical properties of cross-linked enzyme, and the like. In the enzyme immobilization method, the adsorption method has the advantages of simple operation, low cost and unique advantages. The adsorption method is to fix enzyme molecules on a carrier through nonspecific physical adsorption, ion adsorption, biological specific adsorption, affinity adsorption, hydrophobic effect and the like generated between the carrier and the structures of the enzyme molecules. Although the specificity of enzyme immobilized by adsorption is not very high, the method is simple and convenient to operate, does not generate chemical reaction, does not change the natural structure of the enzyme, can prevent the function of the active site of the enzyme from being damaged, keeps the enzyme in a natural state, and is widely applied (T.Jesionowski; J.ZDarta, adaptation.2014, 20:801-821). Materials commonly used for the adsorption method to immobilize enzymes include silica, hydroxyapatite, diatomaceous earth, activated carbon, nanomaterials, and the like. Diatomite belongs to an opal structure in mineralogy and is an important nonmetallic mineral material. The diatom shell has unique diatom shell structure, and has the appearance of disc, straight chain, feather, needle and the like, and is characterized by strong adsorptivity, large specific surface area and high porosity, and can be applied to a plurality of industrial fields such as chemical industry, petroleum, building materials and the like. Among them, diatomite is the most widely used in preparing filter aid, adsorbent and carrier (Yuan Peng, wu Daqing. Mineral rock. 2000 (01): 101-104). In addition, the diatomite has the characteristics of acid resistance and heat resistance, excellent biocompatibility and no toxic or side effect on human bodies. Disclosure of Invention In the process of overcoming the problems of easy inactivation, difficult recovery, poor environmental tolerance and low recycling rate of the prior 2-deoxy-D-ribose 5-phosphate aldolase in practical application, the invention occasionally discovers a material and a method for improving the activity of the 2-deoxy-D-ribose 5-phosphate aldolase through immobilization when comparing