Method for preparing r-hydroxynitrile lyase and use thereof

Abstract

Methods for synthesizing and using R-hydroxynitrile lyase derived from mango. Specifically, a variety of mutant cyanohydrin lyases were obtained by mutating a wild-type hydroxynitrile lyase gene, adding an enzyme cleavage site thereto, preparing a recombinant plasmid by inserting the mutated wild-type hydroxynitrile lyase gene into a vector, introducing the recombinant plasmid into a strain of bacteria, and secreting and expressing the R-hydroxynitrile lyase in the strain. The method Is safe, simple and easy to operate.

Claims

1. A method for preparing R-hydroxynitrile lyase, comprising: mutating R-hydroxynitrile lyase wild-type gene, comprising: mutating the wild-type gene sequence of R-hydroxynitrile lyase set forth in SEQ ID NO. 1 to obtain a hydroxynitrile lyase mutant gene having the gene sequence set forth in SEQ ID NO. 2, wherein: bases ATC at positions 325-327 of SEQ ID NO. 1 are substituted for bases ATG, bases AAT at positions 328-330 of SEQ ID NO. 1 are substituted for bases AGT, bases ATT at positions 961-963 of SEQ ID NO. 1 are substituted for bases ACT, bases CCA at positions 1060-1062 of SEQ ID NO. 1 are substituted for bases ATA, and bases TCT at positions 1063-1065 of SEQ ID NO. 1 are substituted for bases TTA; adding an enzyme cleavage site, comprising: adding a double digestion site to the hydroxynitrile lyase mutant gene, wherein the double digestion site is Ndel/Hindllll; preparing a recombinant plasmid, comprising: inserting the hydroxynitrile lyase mutant gene into an expression vector to obtain a recombinant plasmid, wherein the expression vector is pET26b(+), N-terminus of the expression vector is a signal peptide pelB leader having the gene sequence set forth in SEQ ID NO. 5; introducing the recombinant plasmid into a strain, comprising: introducing the recombinant plasmid with the hydroxynitrile lyase mutant gene into the strain to obtain a recombinant expression strain, wherein the strain is E. coli BL21 (DE3); and expressing and secreting the R-hydroxynitrile lyase in the strain, comprising: culturing the recombinant expression strain, and inducing the recombinant expression strain to express the enzyme of R-hydroxynitrile lyase in a culture medium and collecting a resulting enzyme liquid.

2. The method according to claim 1, wherein the mutating is conducted by error-prone PCR.

3. The method according to claim 1, wherein the culture medium is LB culture medium.

4. The method according to claim 1, wherein the method further comprises a step of performing induction culturing when expressing and secreting the R-hydroxynitrile lyase in the strain, comprising adding 0.2 mM IPTG to the culture medium when OD600 of the culture medium is 1.0, keeping a culture temperature at 30 C., and inducing expression for 4-5 hours.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

(1) In order to make the above objectives, features and advantages of the present disclosure more apparent and easy to understand, the specific embodiments of the present disclosure will be described in detail below with reference to the embodiments of the present disclosure.

(2) In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present disclosure can also be implemented in other ways different from those described herein, and those skilled in the art can make such modifications without departing from the connotation of the present disclosure. Therefore, the present disclosure is not limited by the specific embodiments disclosed below.

(3) Moreover, reference herein to one embodiment or an embodiment refers to a particular feature, structure, or characteristic that may be included in at least one implementation mode of the present disclosure. The appearances of in one embodiment in various places in this specification are not intend to refer to the same embodiment, nor are they separate or selectively mutually exclusive from other embodiments.

(4) In the examples of the present disclosure, R-selective hydroxynitrile lyase derived from mango (Mangifera indica) was used.

(5) Both methyl tert-butyl ether and liquid hydrocyanic acid used in the present disclosure were of analytical grade, and other raw materials and reagents were commercially available unless otherwise specified.

Example 1

(6) The specific principle for the design of the present disclosure is to obtain a variety of mutant hydroxynitrile lyase by constructing random mutation and site-saturation mutagenesis libraries and high-throughput screening.

(7) The source of the R-hydroxynitrile lyase mutant is wild-type hydroxynitrile lyase. The specific synthesis method was as follows: restriction sites NdeI and HindIII were added to the hydroxynitrile lyase wild gene set forth in SEQ ID NO. 1 or the hydroxynitrile lyase mutant gene set forth in SEQ ID NO. 2, and inserted into the expression vector pET26b (+) after DNA digestion to obtain a recombinant plasmid, and the recombinant plasmid was transformed into E. coli BL21(DE3) to construct a recombinant expression strain. After the above recombinant strains were cultured in LB culture medium to OD600=1.0, 0.2 mM IPTG was added and cultured at 30 C. for 4 to 5 hours. The wild-type or mutant cells were collected by centrifugation, and the crude enzyme solution was obtained after the cells were lysed by sonication.

Example 2

(8) The method for preparing wild-type hydroxynitrile lyase mutant was conducted as follows.

(9) The hydroxynitrile lyase wild-type gene sequence of mango (Mangifera indica) is set forth in SEQ ID NO. 1, and the random mutations were introduced by error-prone PCR and/or the site-saturation mutagenesis was introduced based on docking between the enzyme protein structure and the mimetic substrate. Then high-throughput screening was performed to obtain the wild-type mutant of hydroxynitrile lyase of the present disclosure, which had a sequence set forth in SEQ ID NO. 2.

(10) The protein sequence of the wild-type hydroxynitrile lyase of mango (Mangifera indica) after transcription and translation is shown in SEQ ID NO. 3. The protein sequence of the wild-type hydroxynitrile lyase mutant is shown in SEQ ID NO. 4. Compared with the wild-type hydroxynitrile lyase, the wild-type hydroxynitrile lyase mutant had the difference lied in that isoleucine at position 109 was mutated to methionine, asparagine at position 110 was mutated to alanine or serine, and isoleucine at position 321 was mutated to threonine or alanine, proline at position 354 was mutated to isoleucine, and serine at position 355 was mutated to leucine. The sequence (SEQ ID NO. 4) indicated a possible protein sequence. However, other protein sequences that were possibly mutated had the same property.

(11) Except for the sequence as shown in SEQ ID No.2, there was a possibility that the residues of R-hydroxynitrile lyase mutant gene at positions 325-327 were altered to ATG, residues at positions 328-330 were altered to GCT or AGT, residues at positions 961-963 were altered to ACT or GCT, residues at positions 1060-1062 were altered to ATA, and residues at positions 1063-1065 were altered to TTA or TTG. There were also included any case where alterations occurred at any above-mentioned sites.

Example 3

(12) The steps of hydroxynitrile lyase high-throughput screening were conducted as follows: 130 L of 100 mM potassium phosphate-citric acid buffer (pH=5.0), 20 L of diluted hydroxynitrile lyase solution were added to the 96-well plate in sequence at 25 C., and finally 50 L of the substrate solution of phenylethyl cyanohydrin or o-methyl phenylethyl cyanohydrin was added. Change in absorbance at 280 nm within 5 minutes was read using a microplate reader, and the change in absorbance represented the level of enzyme activity.

(13) Substrate solution: 100 mM pH=3.5 potassium phosphate-citric acid buffer was used to prepare a buffer having a concentration of 8 microliters per milliliter of buffer.

Example 4

(14) Application of R-hydroxynitrile lyase: R-hydroxynitrile lyase was used in a method for synthesizing R-phenylethyl cyanohydrin and R-o-methyl phenylethyl cyanohydrin. And the method was conducted as follows.

(15) 15 mmol of substrate was dissolved in 2.1 mL of methyl tert-butyl ether (MTBE) to obtain a MTBE solution of the substrate. The resulting hydroxynitrile lyase was diluted in 10 mM phosphate-citrate buffer to make a total volume of 3.7 mL, and the pH was adjusted to 3.4. The MTBE solution of the substrate and the enzyme solution were fully mixed, and the temperature of the system was lowered to 10 C. After stirring, an emulsion was formed, and 1.2 ml of liquid hydrocyanic acid was added to start the reaction.

(16) Sampling were conducted at different time points, a column was derivatized with acetic anhydride in the presence of pyridine and dichloromethane, and analyzed by GC on a cyclodextrin column (CP-Chirasil-Dex CB) to determine the substrate conversion and enantiomers value.

(17) After the conversion of the substrate was completed, MTBE was added in 1 volume of the aqueous phase, extracted three times, the organic phases were combined and dehydrated with anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the product. Table 1 shows the relationship between the product yield, product chirality value and the amount of hydroxynitrile lyase added when 15 mmol of substrate was added.

(18) TABLE-US-00001 TABLE 1 Relationship between product yield, product chirality value and the amount of hydroxynitrile lyase added Addition Product amount of Product chirality cyanohydrin yield value Substrate name (mg) (%) (e.e) phenylethyl cyanohydrin 0 92.33 75.32 phenylethyl cyanohydrin 1.5 99.55 99.63 phenylethyl cyanohydrin 3.0 99.87 99.92 o-methyl phenylethyl 0 91.08 84.77 cyanohydrin o-methyl phenylethyl 1.5 99.77 99.57 cyanohydrin o-methyl phenylethyl 3.0 99.82 99.79 cyanohydrin

(19) It can be concluded from Table 1 that both of the yields of the synthesized R-phenylethyl cyanohydrin and R-o-methyl phenylethyl cyanohydrin were 99.5%, and the chirality values (ee values) are both 99.5%.

(20) The present disclosure provides a new method for preparing and use of R-hydroxynitrile lyase derived from mango. Particularly, the present disclosure provides a method in which a variety of mutant hydroxynitrile lyase are obtained through the construction of random mutation and site-saturation mutagenesis libraries, as well as high-throughput screening, the method is safe, simple and easy to operate.

(21) It should be noted that the above embodiments are merely used to describe the technical solutions of the present disclosure and not intend to limit technical solutions of the present disclosure. Although the present disclosure has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that modifications or equivalent substitutions of the technical solutions of the present disclosure can be made without departing from the spirit and scope of the technical solutions of the present disclosure, and those modifications or equivalent substitutions should be deem as falling within the scope of the claims of the present disclosure.