A mutant of an aromatic prenyltransferase derived from bacillus subtilis

Abstract

The application provides a kind of aromatic isopentenyl transferase mutant, which is based on the aromatic isopentenyl transferase gene MenA derived from Bacillus subtilis ( Bacillus subtilis ) and six aromatic isopentenyl transferase mutants MenA-1 to MenA-6 are obtained by site-directed mutagenesis PCR technology, and the recombinant bacteria Bacillus subtilis expressing the above mutants are constructed, respectively. The yield of heptamethylnaphthoquinone fermented by the recombinant bacteria is 64.49 mg / L, 69.30 mg / L, 83.86 mg / L, 105.84 mg / L, 119.23 mg / L and 125.45 mg / L, respectively, which is 133%, 143%, 173%, 219%, 246% and 259% of the yield of heptamethylnaphthoquinone fermented by the Bacillus subtilis expressing the wild-type aromatic isopentenyl transferase MenA.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and protein engineering, and specifically relates to an aromatic isopentenyltransferase mutant protein. Background Technology

[0002] Aromatic isoprene transferases (EC 2.5.1.74) are complex, biologically active substances formed by the alkylation of aromatic compounds as substrates and isoprene compounds as side chains, creating C-C bonds. Currently, methods for preparing aromatic isoprene compounds include chemical and biosynthetic methods, with enzymatic biosynthesis being a major research direction. This involves cloning relevant genes from naturally occurring organisms capable of synthesizing isoprene aromatic compounds, expressing them exogenously, and constructing engineered microorganisms and mutants of aromatic isoprene transferases to achieve targeted isopreneylation of aromatic compounds. Aromatic isoprene transferases have been found in bacteria, fungi, and plants, and they participate in the synthesis of secondary metabolites such as menadione, ubiquinone, and tocopherol. Aromatic isopentenyltransferases are mainly classified into two types: membrane-bound aromatic isopentenyltransferases and soluble proteins lacking the isopentenyl binding region (N / D)DxxD. Microbial isopentenyltransferases of aromatic compounds exhibit broad substrate selectivity. Researching the protein structure and identifying the enzyme function of aromatic isopentenyltransferases provides novel methods for the synthesis of isopentenyl aromatic compounds.

[0003] Site-directed mutagenesis refers to the introduction of desired changes (usually changes indicating a favorable direction) into DNA fragments encoding a target protein (which can be in the genome or plasmids) using methods such as polymerase chain reaction (PCR). These changes include base addition, deletion, and point mutations. Site-directed mutagenesis can rapidly and efficiently improve the traits and characterization of the target protein expressed by DNA, making it a very useful tool in gene research. In vitro site-directed mutagenesis is an important experimental technique in various fields of biology and medicine, a convenient method for modifying and optimizing genes, and a powerful tool for studying the complex relationship between protein structure and function. By selectively altering, deleting, or inserting specific bases in a known gene, the corresponding amino acid sequence and protein structure can be changed. Studying the expression products of mutated genes helps us understand the relationship between protein structure and function and explore protein structure / domains. The potential applications of site-directed mutagenesis are wide-ranging, such as studying the structure of protein-protein interaction sites, modifying the different activities or kinetic properties of enzymes, improving protein stability and activity, studying protein crystal structures, and gene therapy. Site-directed mutagenesis of enzymes has opened up new avenues for understanding the structure and function of enzymes, achieving great success in fields such as industry, agriculture, food processing, and the environment. Summary of the Invention

[0004] This invention provides an aromatic isopentenyl transferase mutant with significantly enhanced enzyme activity to address the problems of existing technologies. The mutant is derived from Bacillus subtilis (…). Bacillus subtilis The aromatic isopentenyltransferase gene MenA was obtained using site-directed mutagenesis and expressed in Bacillus subtilis.

[0005] In one aspect, the present invention provides an aromatic isopentenyltransferase mutant, wherein the 67th amino acid of the aromatic isopentenyltransferase MenA with the amino acid sequence SEQ ID NO:1 is changed from Gln to Tyr, and the amino acid sequence of the mutant is SEQ ID NO:2.

[0006] In one aspect, the present invention provides an aromatic isopentenyltransferase mutant in which the 67th amino acid of the wild-type aromatic isopentenyltransferase MenA is changed from Gln to Tyr, and the mutated amino acid sequence is shown in SEQ ID NO:2.

[0007] Or, in the amino acid sequence of the aromatic isopentenyltransferase MenA relative to the wild type, the 22nd amino acid is changed from Leu to Phe, and the mutated amino acid sequence is shown in SEQ ID NO:3;

[0008] Or, in the amino acid sequence of the aromatic isopentenyltransferase MenA relative to the wild type, the 102nd amino acid is changed from Pro to Leu, and the mutated amino acid sequence is shown in SEQ ID NO:4;

[0009] Or, in the amino acid sequence of the aromatic isopentenyltransferase MenA relative to the wild type, the 185th amino acid is changed from Gln to Tyr, and the mutated amino acid sequence is shown in SEQ ID NO:5;

[0010] Or, in the amino acid sequence of the wild-type aromatic isopentenyltransferase MenA, amino acid 268 is changed from Val to Phe, and the mutated amino acid sequence is shown in SEQ ID NO:6.

[0011] Or, in the amino acid sequence of the wild-type aromatic isopentenyltransferase MenA, the 60th amino acid is changed from Leu to Ile, and the mutated amino acid sequence is shown in SEQ ID NO:7.

[0012] Furthermore, the present invention provides a gene encoding an aromatic isopentenyltransferase MenA mutant, which encodes the aforementioned aromatic isopentenyltransferase MenA mutant.

[0013] Furthermore, the present invention provides a recombinant expression vector carrying a gene encoding the MenA mutant of the aromatic isopentenyltransferase; preferably, its starting expression vector PDG1730 is generated by the promoter P glv Or P cspd Or P cspB The integration site is either amyE, ldh, or aprE, and the recombinant expression vector preferably uses promoter P. glv Integration site amyE.

[0014] The present invention also provides a recombinant host cell, wherein the recombinant host cell is a host cell transformed / transfected with the recombinant expression vector.

[0015] Preferably, the host cell is Bacillus subtilis (B. subtilis). Bacillus subtilis ).

[0016] The present invention further provides the application of the aforementioned aromatic isopentenyltransferase MenA mutant or its encoding gene in the preparation of heptaene-menaquinone.

[0017] The present invention also provides a method for preparing heptaenemenaquinone, wherein recombinant host cells as described in claim 3 or 4 are cultured to produce heptaenemenaquinone. Preferably, the method further includes the steps of collecting and purifying heptaenemenaquinone.

[0018] Specifically, the fermentation medium used during cultivation consisted of: 3% glycerol, 6% soybean peptone, 0.5% yeast extract, 0.3% K2HPO4, 0.5% MgSO4·7H2O, 0.1% L-Tryptophan, and 3% maltose.

[0019] Preferably, the culture conditions are 40 ℃ and 200 rpm shaking culture for 96 h.

[0020] More preferably, under light-protected conditions, the collection is carried out by adding 4 times the volume of an extract of isopropanol:n-hexane = 1:2 to the fermentation broth, shaking for more than 15 minutes; then allowing it to stand, and after separation, the supernatant is collected.

[0021] This invention is based on the aromatic isopentenyltransferase MenA derived from Bacillus subtilis. Six aromatic isopentenyltransferase mutants, MenA-1, MenA-2, MenA-3, MenA-4, MenA-5, and MenA-6, were obtained through site-directed mutagenesis. These mutants were then fermented in Bacillus subtilis, yielding heptaene-menaquinone yields of 64.49 mg / L, 69.30 mg / L, 83.86 mg / L, 105.84 mg / L, 119.23 mg / L, and 125.45 mg / L, respectively. These yields represent 133%, 143%, 173%, 219%, 246%, and 259% of the heptaene-menaquinone yield of Bacillus subtilis expressing the wild-type aromatic isopentenyltransferase MenA. Attached Figure Description

[0022] Figure 1 Plasmid map of aromatic isopentenyltransferase expression vector;

[0023] Figure 2 Bar graph showing the yield of heptaene-naphthoquinone in the Aromatic Isopentenyltransferase mutant Bacillus subtilis strain. Detailed Implementation

[0024] The method of the present invention will be further illustrated below with reference to examples. Experimental methods not specified in the examples can generally be performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual* by J. Sambrook et al., or according to the manufacturer's recommendations. Those skilled in the art can better understand and master the present invention with the help of these examples. However, the methods for implementing the present invention should not be limited to the specific method steps described in the embodiments of the present invention.

[0025] The terminology and related assay methods involved in this invention are explained as follows: Aromatic isopentenyltransferase mutants are identified by the term "amino acid replaced at the original amino acid position," indicating the mutated amino acid in the aromatic isopentenyltransferase mutant. For example, Gln185Tyr indicates that the amino acid at position 185 has been replaced by Tyr from the parental aromatic isopentenyltransferase (Gln), and the position number corresponds to the number in SEQ ID NO:1 of the appendix sequence listing. For example, Pro102Leu indicates that the amino acid at position 102 has been mutated.

[0026] Example 1: Construction of the Aromatic Isopentenyltransferase MenA Expression Vector and Recombinant Strains

[0027] Using PDG1730 plasmid as a template, the vector DNA sequence was amplified by PCR to obtain the vector backbone sequence to be cloned. Using Bacillus subtilis genomic DNA as a template, the complete MenA gene was amplified by PCR to obtain the aromatic isopentenyltransferase MenA sequence, whose encoded amino acid sequence is SEQ ID NO: 1. The PCR amplification conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles, 72℃ for 5 min, and incubation at 4℃. The PCR amplification products were recovered using the EZNA Gel Extraction Kit. The recovered enzyme gene sequence and vector backbone sequence were analyzed using Clon Express. ® The recombinant plasmid was ligated using the OneStep Cloning Kit to assemble the vector and fragment. The assembly system was as follows: 4 μL of 5×CEⅡBuffer, 2 μL of Exnase Ⅱ, gene fragment (MenA fragment) (0.04× number of insert bases) ng / concentration (ng / μL), vector backbone fragment (PDG1730) (0.02× number of cloning vector bases) ng / concentration (ng / μL), and ddH2O to a final volume of 20 μL. After adding the appropriate components as described above, the mixture was incubated in a 37 ℃ water bath for 30 min to complete the assembly. The assembly product was then transformed into E. coli competent cells DH5α. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDG-MenA. The plasmid map is shown in the attached image. Figure 1 As shown. The plasmid PDG-MenA was transformed... Bacillus subtilis 168 The host bacterium was used to screen for positive transformants, resulting in a recombinant strain expressing the wild-type aromatic isopentenyltransferase MenA, which was named Bacillus subtilis MenA. Bacillus subtilis MenA).

[0028] The above-mentioned Bacillus subtilis ( Bacillus subtilis )168 was transformed using the competent state method. BS1 competent states were prepared via a two-step Spizizen method, as described in the article (Spizizen J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate[J].Proc Natl Acad Sci USA, 1958, 44: 1072-1075.). The specific transformation process is as follows: freshly activated... Bacillus subtilis 168 μL of LB agar (1% tryptone, 0.5% yeast extract, 1% NaCl) was inoculated into 5 mL GMⅠ (GMI) plates. The GMI solution was prepared as follows: 0.5 mL 10× Spizizen salt solution, 0.1 mL 2% acid-hydrolyzed casein, 0.1 mL 40% glucose, 0.1 mL 5% yeast extract, 0.005 mL 20% MgSO4·7H2O, 0.05 mL 0.5% L-Tryptophan, and 3.145 mL ddH2O. The 10× Spizizen salt solution was prepared as follows: 18.3 g / L K2HPO4, 6 g / L KH2PO4, 2 g / L (NH4)2SO4, 1.2 g / L trisodium citrate, and ddH2O. The solution was dissolved sequentially in distilled water. The plates were incubated overnight at 37 °C with shaking at 220 rpm. The next day, 0.5 mL of the solution was transferred to a freshly prepared 4.5 mL plate. Incubate in 4.25 mL of sterile GMⅠ medium at 37°C and 220 rpm for 4.5 h. Then, transfer 0.75 mL of the culture medium from the previous step to 4.25 mL of freshly prepared sterile GMⅡ medium (GMⅡ is prepared as follows: 0.5 mL of 10×Spizizen salt solution, 0.05 mL of 2% acid-hydrolyzed casein, 0.1 mL of 40% glucose, 0.035 mL of 20% MgSO4·7H2O, 0.005 mL of 0.5% L-Tryptophan, and 3.310 mL of ddH2O). Incubate at 37°C and 220 rpm for 90 min, then centrifuge at 5000 g for 10 min to collect the bacterial cells. Gently resuspend the bacterial cells in 10 mL of the original culture supernatant; these are the competent cells. Add an appropriate amount of DNA to 1 mL of competent cells and incubate at 37°C and 220 rpm with shaking for 1.5 h, then centrifuge and plate. Finally, incubate at 37°C... Incubate overnight at ℃ with the inverted incubator, and check and verify the transformants the next day.

[0029] Example 2: Construction of the MenA mutant of aromatic isopentenyltransferase using point mutagenesis technology

[0030] Emergent analysis of the MenA gene, an aromatic isopentenyltransferase derived from Bacillus subtilis, was performed to compare its amino acid sequence with that of other aromatic isopentenyltransferases with high homology, in order to identify potential amino acid mutation sites that could improve its enzymatic properties. After a series of ClustalX sequence alignments, amino acid residues at positions 67, 22, 102, 185, 268, and 60 were selected for mutation.

[0031] Nucleotide mutations were introduced into the MenA gene, an aromatic isopentenyl transferase derived from Bacillus subtilis, using point mutagenesis technology. The amplification system for the Gln67Tyr site mutation PCR was as follows: 2×PimerStarMax 25 μL, upper primer (10 μM) 2.5 μL, lower primer (10 μM) 2.5 μL, template DNA PDG-MenA plasmid (100 ng / μL) 2 μL, ddH2O 18 μL, upstream mutation primer 5'-atcgtttgtgtgggtgctgctcggaaccgttttg-3', downstream mutation primer 5'-cgagcagcacccacacaaacgatgcggttaacgtatgag-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; incubation at 4℃. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDG-MenA-1.

[0032] The amplification system for the Ser34Thr site mutation PCR was as follows: 25 μL of 2×PimerStarMax, 2.5 μL of upper primer (10 μM), 2.5 μL of lower primer (10 μM), 2 μL of template DNA PDG-MenA plasmid (100 ng / μL), 18 μL of ddH2O, upstream mutation primer 5'-gtttttcatttatacagataagatcaatatgcagagcattttgatttcc-3', and downstream mutation primer 5'-gatcttatctgtataaatgaaaaacgaaatcagcaaacaccg-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDG-MenA-2.

[0033] The amplification system for the Leu22Phe site mutation PCR was as follows: 2×PimerStarMax 25 μL, upper primer (10 μM) 2.5 μL, lower primer (10 μM) 2.5 μL, template DNA PDG-MenA plasmid (100 ng / μL) 2 μL, ddH2O 18 μL, upstream mutation primer 5'-gtttttcatttatacagataagatcaatatgcagagcattttgatttcc-3', downstream mutation primer 5'-gatcttatctgtataaatgaaaaacgaaatcagcaaacaccg-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; 4℃ infinity. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDG-MenA-3.

[0034] The amplification system for the Pro102Leu site mutation PCR was as follows: 25 μL of 2×PimerStarMax, 2.5 μL of upper primer (10 μM), 2.5 μL of lower primer (10 μM), 2 μL of template DNA PDG-MenA plasmid (100 ng / μL), 18 μL of ddH2O, upstream mutation primer 5'-gtttttcatttatacagataagatcaatatgcagagcattttgatttcc-3', and downstream mutation primer 5'-gatcttatctgtataaatgaaaaacgaaatcagcaaacaccg-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDG-MenA-4.

[0035] The amplification system for the Gln185Tyr site mutation PCR was as follows: 25 μL of 2×PimerStarMax, 2.5 μL of upper primer (10 μM), 2.5 μL of lower primer (10 μM), 2 μL of template DNA PDG-MenA plasmid (100 ng / μL), 18 μL of ddH2O, upstream mutation primer 5'-gtttttcatttatacagataagatcaatatgcagagcattttgatttcc-3', and downstream mutation primer 5'-gatcttatctgtataaatgaaaaacgaaatcagcaaacaccg-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDG-MenA-5.

[0036] The amplification system for the Leu60Ile site mutation PCR was as follows: 25 μL of 2×PimerStarMax, 2.5 μL of upper primer (10 μM), 2.5 μL of lower primer (10 μM), 2 μL of template DNA PDG-MenA plasmid (100 ng / μL), 18 μL of ddH2O, upstream mutation primer 5'-cggattcctgatttcgatcggattattgatcagctat-3', and downstream mutation primer 5'-aatccgatcgaaatcaggaatccgaaaaatgtatttgt-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDG-MenA-6.

[0037] Transform plasmids PDG-MenA-1, PDG-MenA-2, PDG-MenA-3, PDG-MenA-4, PDG-MenA-5 and PDG-MenA-6 Bacillus subtilis 168 host bacteria were screened for positive transformants to obtain recombinant strains expressing the wild-type aromatic isopentenyltransferase MenA mutant, which were named Bacillus subtilis MenA-1. Bacillus subtilis MenA-1), MenA-2 Bacillus subtilis MenA-2), MenA-3 Bacillus subtilis MenA-3), MenA-4 Bacillus subtilis MenA-4), MenA-5 Bacillus subtilis MenA-5) and MenA-6( Bacillus subtilis MenA-6).

[0038] Example 3 Screening of MenA mutants of aromatic isopentenyltransferases

[0039] 3.1 Shake-flask fermentation

[0040] The six aromatic isopentenyltransferase mutant recombinant strains (MenA-1, MenA-2, MenA-3, MenA-4, MenA-5, and MenA-6) and the control strain Bacillus subtilis MenA were inoculated into 5 mL of seed culture medium (0.5% yeast extract, 1% tryptone, 1% NaCl, 25 μg / mL kanamycin) and cultured at 37 ℃ with shaking at 200 rpm for 12 h. Then, the seed culture was inoculated into 30 mL of fermentation medium (3% glycerol, 6% soybean peptone, 0.5% yeast extract, 0.3% K2HPO4, 0.5% MgSO4·7H2O, 0.1% L-Tryptophan, 3% maltose) at an inoculation rate of OD600=0.1 and cultured at 40 ℃ with shaking at 200 rpm for 96 h.

[0041] 3.2 Collect bacterial cells and detect heptaene-naphthoquinone production

[0042] Add 4 times the volume of extraction buffer (isopropanol: n-hexane = 1:2) to the fermentation broth, shake for 20 min, and perform all operations in the dark. After standing and separating the layers, aspirate the supernatant into a centrifuge tube. Add the chromatographic detection solution to the centrifuge tube after vacuum freeze concentration for reconstitution, and filter the dissolved solution through a 0.22 μm organic filter membrane to obtain the sample detection solution. The yield of heptaene-menaquinone was detected by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). The detection conditions were as follows: Waters XBridge @ BEH C18 column (2.5 µm, 3.0 × 100 mm), column temperature 40 ℃, mobile phase 100% pure methanol, flow rate 0.8 mL / min, detection wavelength 270 nm, and single injection volume 3 µL. Meanwhile, before the production of heptaene-menaquinone, five standard samples with different concentration gradients were prepared. The production of heptaene-menaquinone in the fermentation broth was measured simultaneously with the standard samples and the fermentation samples. The production of heptaene-menaquinone in the fermentation broth was obtained by calculating the results using the standard curve.

[0043] Specific results: The yield of heptaene-menaquinone in Bacillus subtilis expressing the wild-type aromatic isopentenyltransferase MenA was 48.35 mg / L. The yields of heptaene-menaquinone after fermentation of the aromatic isopentenyltransferase mutants MenA-1 to MenA-6 were 64.49 mg / L, 69.30 mg / L, 83.86 mg / L, 105.84 mg / L, 119.23 mg / L, and 125.45 mg / L, respectively. These represent 133%, 143%, 173%, 219%, 246%, and 259% of the heptaene-menaquinone yield of Bacillus subtilis expressing the wild-type aromatic isopentenyltransferase MenA, respectively.

Claims

1. An aromatic isopentenyltransferase MenA mutant, characterized in that, The amino acid sequence of MenA, an aromatic isopentenyltransferase, has its 60th amino acid changed from Leu to Ile. The mutated amino acid sequence is encoded by the nucleotide sequence shown in SEQ ID NO:

7.

2. A gene encoding an aromatic isopentenyltransferase MenA mutant, wherein the gene encodes the aromatic isopentenyltransferase MenA mutant as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector carries a gene encoding the MenA mutant of the aromatic isopentenyltransferase described in claim 1.

4. The recombinant expression vector as described in claim 3, characterized in that, Its starting expression vector is PDG1730, and the gene is generated by the promoter P. glv Or P cspd Or P cspB The initiation integration site is either amyE, ldh, or aprE.

5. A recombinant host cell, characterized in that, The recombinant host cell is a host cell transformed / transfected with the recombinant expression vector as described in claim 3 or 4.

6. The recombinant host cell as described in claim 5, characterized in that, The host cell is Bacillus subtilis ( Bacillus subtilis ).

7. The use of the MenA mutant of the aromatic isopentenyltransferase as described in claim 1 or its encoding gene in the preparation of heptaene-menaquinone.

8. A method for preparing heptaene-menaquinone, characterized in that, Culturing the recombinant host cells as described in claim 5 or 6 to produce heptaene-menaquinone.

9. The method as described in claim 8, characterized in that, It also includes the steps of collecting and purifying heptaene-menaquinone.

10. The method as described in claim 8, characterized in that, The fermentation medium used during cultivation consisted of: 3% glycerol, 6% soybean peptone, 0.5% yeast extract, 0.3% K2HPO4, 0.5% MgSO4·7H2O, 0.1% L-Tryptophan, and 3% maltose.

11. The method as described in claim 8, characterized in that, The culture conditions were 40 ℃ and 200 rpm shaking culture for 96 h.

12. The method as described in claim 9, characterized in that, Under light-protected conditions, the collection is carried out by adding 4 times the volume of an extract of isopropanol:n-hexane = 1:2 to the fermentation broth, shaking for more than 15 minutes; then letting it stand, separating the layers, and then collecting the supernatant.

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