LEH characteristics and screeing mutant library
Based on the mutant library constructed with the vector pBAD/Myc-His A[12], all mutants were transformed, cultured and purified to verify the enantioselectivity (Table 1). Since mutant BE3 had significant (S) - enantioselectivity and mutant BG5 had (R) – enantioselectivity in our experiment, they were selected for the subsequent test.
Table 1 Screening and verification of LEH mutant library
Mutants
|
Mutation site
|
ee (%)
|
C (%)
|
AG9
|
T76K/T85V/N92K/Y96F/E124D/ I5C/E84C/G89C/S91C
|
10.59±0.29 (R,R)
|
99.15
|
AH6
|
T76K/T85V/N92K/Y96F/E124D/
I5C/A17C/E84C/N92C
|
16.68±0.25 (R,R)
|
99.99
|
BE3
|
M32L/L74F/I80F/L103I/I116V/F139L
|
86.44±0.78 (S,S)
|
22.06
|
BE5
|
M78L/I80F/I116V/F139W
|
14.05±0.67 (S,S)
|
91.25
|
BE6
|
M32A/L35W/M78I/I80A/V83A/I116V
|
34.86±6.49 (S,S)
|
10.99
|
BE7
|
M32L/I80W/L103I/I116V/F139W
|
6.67±5.77 (R,R)
|
17.34
|
BE9
|
M32L/L35M/L74I/M78I/I80F/V83I/ L103V/I116V/F139L
|
83.30±8.53( S,S)
|
8.57
|
BF2
|
M32L/M78L/I80F/I116V/F139W
|
0.61 (S,S)
|
61.17
|
BF3
|
M78W/F139W
|
41.56±1.27 (R,R)
|
32.15
|
BF4
|
M32A/L35M/M78L/I80W/L103V/ I116V/F139W
|
1.79±0.21 (R,R)
|
19.74
|
BF5
|
M32L/L35M/L74I/M78V/I80F/V83G/ L103V/F139L
|
49.66±14.27 (R,R)
|
0.29
|
BF6
|
L74I/I80F/I116V/F139W
|
5.87±2.71( R,R)
|
99.04
|
BF7
|
M32L/L35M/M78L/I80F/L103I/I116V/F139L
|
60.11±2.11 (S,S)
|
79.84
|
BF8
|
M32L/L35M/M78L/I80W/V83I/ L103I/I116V/F139W
|
11.28( S,S)
|
10.62
|
BF9
|
M32L/L35W/L103I/I116V/F139W
|
9.31±0.46 (S,S)
|
64.32
|
BG1
|
M32L/L74F/M78A/I80F/L103I/ I116V/F139L
|
73.63±2.27 (S,S)
|
64.43
|
BG2
|
M32L/M78I/I80W/L103I/I116V/F139L
|
60.58±4.97 (S,S)
|
13.24
|
BG3
|
M32L/L35W/M78I/I80A/L103I/I116V/F139L
|
25.51±5.89 (R,R)
|
37.48
|
BG5
|
M32L/L74I/I80V/L103F/F139L
|
79.41±0.92 (R,R)
|
62.37
|
BG6
|
M32L/L74I/I80V/L103F/F139W
|
70.89±1.58 (R,R)
|
73.90
|
BG7
|
M32L/L74I/L103F/F139W
|
60.40±1.20 (R,R)
|
58.34
|
BG8
|
M32L/M78G/L103F/F139M
|
30.35±0.28 (R,R)
|
84.36
|
BG9
|
M32L/M78G/L103F/I116V/F139L
|
54.63±1.80 (S,S)
|
97.61
|
BH2
|
M32A/M78G/L103F/F139L
|
18.46±0.91 (R,R)
|
97.85
|
Note: for the substrate CHO; C%: conversion rate
LEH catalyzes the hydrolysis of cyclohexane oxide to vicinal diol cyclohexane-1,2-diol (Fig. 3). The substrate CHO was molecular docked with BE3 and BG5. The docking grids of BE3 was set to 46×54×64 Å, and the docking grids of BG5 was set to 54×52×40 Å. For the docking results, CHO was located on the surface of the protein to the catalyst. BG5 has a larger cavity than BE3, which can reduce steric hindrance and is more conducive for CHO to enter the ligand channel (Fig.4).
Construction of pHY-p43-LEH expression vector
Mutants BE3 and BG5 genes were amplified from plasmid pBAD-LEH mutants library. The double enzyme digestion products of target fragments BE3, BG5 and vector pHY-p43 were successfully recovered (Fig. 5A). After connection and transformation, some single colony was selected by bacteria liquid PCR, then the transformed plasmids were extracted for plasmid PCR and double enzyme digestion (Fig. 5B-C).
LEH heterologous expression in E. coli Top10 and B. subtilis WB800
Plasmids pHY-p43-BE3 and pHY-p43-BG5 transformed into E. coli TOP10 were cultured over 12 h, and proteins were purified by 6×His affinity chromatography. SDS-PAGE detection showed that pHY-p43 /TOP10 system expressed LEH successfully (Fig. 6A), and the sequencing results also showed that the target fragments were correctly connected.
Meanwhile, pHY-p43-BE3 and pHY-p43-BG5 were transformed into B. subtilis WB800 by electrotransformation. From the SDS-PAGE results, the expression in B. subtilis WB800 was lower than the expression in E. coli, therefore the latter strain was considered for the optimized system (Fig. 6B).
Enzymatic characteristics of mutants BE3 and BG5 in different systems
For the protein expression of pBAD/Myc-HisA–LEH system needs to add a certain concentration of inducer and control the corresponding induction temperature and time, also the culture time is more than 20 h. In order to make an efficient and economic procedure, the expression system of LEH was considered to be optimized. The constitutive vector pHY-p43-LEH containing strong promoter p43 was our selection for promoting the expression,pHY-p43 system did not need inducer, and the amount of protein needed in the experiment could be obtained by conventional temperature 37℃ and LB medium for cultured more than 12 h.
In our experiment, LEH mutants were designed to catalyze substrate cyclohexene oxide (CHO). During the gas chromatography, retention times of cycloheptene oxide ca. 9.3min; acetonitrile ca. 8.8 min; (S,S)-cycloheptane diol ca. 13.7 min; (R,R)-cycloheptane diol ca. 14.0 min; IS ca. 16.3 min. Measure peak area's and the internal standard peak area's and a calibration curve, calculate concentrations (Table 2).
Table 2 The substrate conversion and enantiomeric excess of mutants BE3 and BG5
Mutant
|
CHO conversion (%)
|
CHO ee(%)
|
pBAD-BE3
|
46.212
|
-56.987
|
pBAD-BG5
|
33.477
|
40.886
|
pHY-P43-BE3
|
37.71
|
-71.57
|
pHY-P43-BG5
|
39.93
|
69.55
|
The mutants under the expression system were purified from E.coli TOP10, and the LEH expressed by different expression systems was determined to be affected by pH, temperature and reaction time In the experiment, the 1M substrate stock solution was diluted with 50 mM potassium phosphate buffer to 50 mM, and 990 μL substrate dilution and 10 μL LEH purified enzyme solution were incubated in a 5 mL glass bottle at 30°C for 90 min. First, the optimal reaction temperature was determined, and the temperature was set at 30 to 85℃. It was found that the same mutant had the same optimal temperature in different expression systems The optimal reaction temperatures for pBAD-BE3 and pHY-p43-BE3 are both at 65°C, and the optimal reaction temperatures for pBAD-BG5 and pHY-p43-BG5 are both at 55°C (Fig. 7). Then the optimal pH was determined and configure the substrate diluted potassium phosphate buffer at 5.8, 6.2, 6.6, 7, 7.4, 7.8, 8. For pHY-p43-BE3 and pBAD-BG5, the catalysis was at the highest level at pH 6.2, and while the optimal pH of pBAD-BE3 is 5.8, pHY-P43-BG5 is 7.4 (Fig. 8).
Finally, the change of the different reaction time of each mutant was determined, and it basically belonged to a steady ascending state, and there were no significant changes (Fig. 9). Therefore, the reaction time of the subsequent experiment kept for 90min. The enzyme-catalyzed reaction was carried out under the above-mentioned single-factor optimal conditions, and it was found that the substrate conversion rate was significantly increased, and the conversion rate of pBAD-BE3 on CHO reached 100%, but after the conversion rate increased, the ee value decreased (Fig. 10). It was speculated that the optimal temperature and pH of single factor had certain influence on the conformation of LEH, and the formation of chiral products was also affected by the increase of catalytic efficiency.
Kinetic analysis of LEH mutants expressed in E. coli Top10
The kinetic parameters of mutant BE3 and BG5 were determined using different concentrations of substrate CHO from 1 mM to 70 mM, and the reaction system was 990 μL substrate dilution plus 10 μL LEH solution in 5 mL incubater at 30°C for 90 min, in which the substrate concentration was unique variable. The Km value and Vmax value are calculated by the Michaelis-Menten equation in the software Originpro 2021. From the table 3, that the mutants of the p43 system have stronger substrate affinity and better catalytic efficiency than the pBAD system. Therefore, the p43 system was selected as the optimized plasmid expression system.
Table 3 The kinetic parameters of mutants BE3 and BG5
Mutant
|
Enzyme concentration(μmol/L)
|
Km(mM)
|
Vmax
(umol/min·mg)
|
Kcat(s-1)
|
Kcat/ Km
(mM-1s-1)
|
pBAD-BE3
|
3.557
|
20.425 ± 5.870
|
19.149
|
0.822
|
0.040
|
pBAD-BG5
|
2.548
|
10.930 ± 0.403
|
5.212
|
0.313
|
0.029
|
P43-BE3
|
1.962
|
9.797 ±1.640
|
5.670
|
0.546
|
0.056
|
P43-BG5
|
1.188
|
9.08 ± 4.11
|
4.735
|
0.369
|
0.045
|
Note: for the subatrate cyclohexane oxide