L-Serine, 5-hydroxyindole, peptone, and yeast extract were purchased from Alighting Biochemical Technology Co. Ltd. (Shanghai, China). Isopropyl-β-D-thiogalactoside, pyridoxal phosphate (PLP) were purchased from Sangon Biotech Co., Ltd. (Shanghai). Other chemicals were analytical reagents.
Error-prone PCR mutant library
According to the trpB sequence of E. coli k-12 tryptophan synthase (ID: 945768) and the MCS site of the pETDuet-1 vector published by the NCBI, two primers were designed using Primer Premier 5.0. NcoI and EcoRI were introduced at the 5' end of the upstream and downstream primers, respectively. The primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.
Random mutations were introduced into the trpBA gene using the Trans EasyTaq DNA polymerase kit. The PCR system and PCR procedure are described in the instructions.
Screening of tryptophan synthase
A single colony of mutant strain was selected from the petri dish and inoculated into a 96-well shallow orifice plate with 1mL LB liquid medium (35°C). Isopropyl-β-D-thiogalactoside and PLP were added to each well of 96-well shallow orifice plate at 18°C. The fermentation mutant strain was centrifuged after induction (4°C, 4000 r⋅min−1,10 min). L-Serine and 5-hydroxyindole were added to the 96-well shallow orifice plate (37°C, 30 min, 200 r⋅min−1). The absorbance value at 290 nm was measured by ELISA.
Purification of tryptophan synthase
The engineered bacteria of tryptophan synthase were crushed by ultrasonication (600 W, 10 min, 0 oC). The crude enzyme was centrifuged at 5000 r⋅min−1 for 10 min. The supernatant was collected to obtain the crude tryptophan synthase enzyme liquid. The supernatant of tryptophan synthase were purified by Ni-NTA column using the method reported in the literature .
Determination of enzymatic activity
Tryptophan synthase activity was determined by the L-5-hydroxytryptophan concentration. The enzyme reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 min. The conversion at this time point was 17%. The concentration of L-5-hydroxytryptophan was determined by an amino acid analyser (L-8900, Japan). Resin column was sulfonic acid type cationic resin column (4.6 mm×60.0 mm). Column temperature was 57.0 ℃. Reactor temperature was 130 ℃. The flow rate of pump A (elution solution) was 0.40 mL/min. The flow rate of B (ninhydrin solution) was 0.35 mL/min, and the injection volume was 20 µL. The detection wavelength was 570 nm and 440 nm.
Determination Of Kinetic Parameters
Determination of kinetics parameters was carried out using five L-serine concentrations (0.1 mmol/L, 0.2 mmol/L, 0.3 mmol/L, 0.4 mmol/L and 0.5 mmol/L). Kinetics parameters of tryptophan synthase were obtained through the Lineweaver-Burk methodology.
Preparation of L-5-hydroxytryptophan
The optimum reaction conditions for L-5-hydroxytryptophan synthesis catalysed by tryptophan synthase were studied. The effects of temperature, pH and substrate concentration on tryptophan synthase were investigated. L-5-Hydroxytryptophan was synthesized under the optimal reaction conditions. The optimal conditions and reaction volume for L-5-hydroxytryptophan preparation were L-serine 100 mmol/L, 35°C, pH 8.5 and 1000 ml. The reaction system (1000 ml) containing 10.5 g L-serine, 13.3 g 5-hydroxyindole, 0.01g pyridoxal phosphate and 0.1 g tryptophan synthase. L-5-hydroxytryptophan was analyzed with an FT-IR Spectrometer ( Gangdong Sci & Tech. Co., Ltd., Tianjing, China). All treatment of L-5-hydroxytryptophan synthesis catalysed by tryptophan synthase were repeated for three times. SPSS 22.0 was used to analysis of experimental data.