Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia Coli

microorganisms

Article Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

Lisheng Xu 1,*, Fangkai Han 1, Zeng Dong 1 and Zhaojun Wei 2 1 Department of Life and Food Science, Suzhou University, Suzhou 234000, China; [email protected] (F.H.); [email protected] (Z.D.) 2 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China; [email protected] * Correspondence: [email protected]; Tel.: +86-557-287-1681

Received: 23 March 2020; Accepted: 3 April 2020; Published: 5 April 2020

Abstract: To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 1 1 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM s , which was 4.8 times higher than that − · − of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 ◦C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.

Keywords: tryptophan synthase; thermostability; L-tryptophan; directed evolution

1. Introduction Tryptophan is a precursor of serotonin, an important neurotransmitter, and an essential amino acid in the human body. Tryptophan synthetase (EC 4.2.1.20) is a heterotetramer with an aaββ subunit structure in Escherichia coli. The enzyme can synthesize L-tryptophan with indole and L-serine as substrates. In L-tryptophan metabolism, tryptophan synthase is one of the important enzymes that affect the yield of L-tryptophan. The trp B and trp A genes (or trp BA genes) coexist in the tryptophan operon of the E. coli genome. The function of the a subunit is to decompose indole-3-glycerol phosphate into indole and glyceraldehyde-3-phosphate, while the function of the β subunit is to synthesize L-tryptophan [1–3]. The substrate spectra of tryptophan synthase derived from Salmonella, including fluorine, chlorine, bromine, and iodine-substituted indole derivatives, have been reported in the literature. This enzyme can synthesize L-tryptophan derivatives [4]. The active sites of tryptophan synthase in Pyrococcus furiosus mainly include Asp300, Glu104, Lys82, and Ala106, among which hydrogen bond formation between substrate complex E(Aex1) and Asp300 plays an important role in the catalytic process. The tryptophan synthase from Pyrococcus furiosus was modified via directed evolution, and the ability of the enzyme to synthesize 5-bromo-L-tryptophan and 6-hydroxy-L-tryptophan was promoted by modification of the tryptophan synthase β subunit [5]. S-phenyl-L-cysteine and

Microorganisms 2020, 8, 519; doi:10.3390/microorganisms8040519 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 519 2 of 11

L-2-methyltryptophan were synthesized by tryptophan synthase [6–8]. However, modification of the tryptophan synthase derived from E. coli K-12 has not been reported. In recent years, the molecular modification was applied to enzyme catalysis [9–12]. Useful compounds such as 5-aminolevulinic acid, ginsenoside, and L-theanine can be synthesized from microorganisms [13–15]. Frances Arnold proposed the concept of directed evolution of enzymes in the 1990s. Directed evolution greatly broadens the biocatalytic potential of an enzyme by modifying its corresponding gene sequence, enabling it to possess a wide range of substrate universality and even catalyze entirely new chemical reactions. Through the directed evolution of the cytochrome P411 enzyme, dicyclobutane, and cyclopropylene were efficiently synthesized [16]. The thermal stability, spatial selectivity, and activity of epoxide hydrolase were optimized by iterative saturation mutation and triplet codon saturation mutation strategies [17]. An efficient protein engineering strategy based on the fusion of directed evolution and rational design was used to obtain lipase mutant strains with high stereoselectivity, and the strategy required minimal screening. Crystal structure analysis and molecular dynamics simulation of the nonaqueous phase were used to explain the specific stereoselectivity and the molecular mechanism of the four mutant strains, respectively [18]. Directed evolution was used to modify threonine aldolase to improve the synergistic effect of its amino acid residues [19]. High stereoselective mutations in the R and S isomers of a-2-aryl propionate were obtained by iterative saturation mutation directed evolution of its active sites. Nearly 1000 mutant strains of Candida antarctica lipase B were selected for high-throughput screening [20]. By mutating transaminase, its biocatalytic activity increased by 25,000 times, and the modified transaminase could be used in the green production of the hypoglycemic drug citastatin [21]. The biosynthesis pathway of tryptophan in Escherichia coli mainly includes the central metabolic pathway, a common pathway, and a branch pathway. In the central metabolic pathway, the ppsA and tktA genes play key roles in the generation of phosphoenolpyruvate, precursors of tryptophan biosynthesis. In the common pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes the condensation of E4P and phoshoenolpyruvate to produce DAHP as a rate-limiting step. In the branching pathway, anthranilic acid synthetase is the key enzyme in tryptophan biosynthesis [22]. The molecular kinetics of the catalysis of tryptophan synthetase have been studied. The structure and reaction mechanism of tryptophan synthetase from Salmonella typhimurium have been analysed by NMR [23,24]. Excellent thermostability and catalytic activity of tryptophan synthase were needed in industrial production. In this study, tryptophan synthase derived from Escherichia coli K-12 was modified by directed evolution and rational design to improve its activity and thermostability. L-tryptophan was synthesized by modified tryptophan synthetase.

2. Materials and Methods

2.1. Chemicals, Strains and Culture Conditions The plasmid extraction kit, DNA polymerase, indole, and isopropyl-beta-D-thiogalactopyranoside (IPTG) were purchased from Sangon Biotech (Shanghai, China) Co., Ltd. E. coli BL21(DE3) and the expression plasmid pet-28a (+) were preserved in our laboratory.

2.2. Mutation Site Reetz et al. successfully improved the stability of Bacillus subtilis lipase based on B-FITTER [18]. In this study, B-FITTER software was used to analyze the temperature factors of 397 amino acid residues of tryptophan synthase trpB (PDB: 2dh6), and the temperature factors were obtained. The temperature factors of the amino acid residues within the entrance range of the substrate channel were analyzed by a Swiss-model online simulation of the three-dimensional structure. According to the temperature factor, the key amino acid residues related to substrate binding were screened out. Microorganisms 2020, 8, 519 3 of 11

2.3. Site-Saturated Mutation Library pET-28a (+) was used as the template plasmid for site-specific saturation mutation of 395 amino acids. The primers used for mutagenesis are presented in Table1. The PCR reaction system included 10 × PCR buffer solution, 2.5 mmol L 1 dNTP mixture, 15 pmol L 1 primers, pET28a(+)-trp B, and 5 U L 1 · − · − · − DNA polymerase (PCR reaction system 50 µL). The PCR reaction conditions included denaturation at 95 ◦C for 4 min, followed by 20 cycles of 95 ◦C for 30 s, 55 ◦C for 1 min, and 72 ◦C for 16 min, and finally, 72 ◦C for 5 min. The PCR products were enzymatically digested at 37 ◦C for 4 h to eliminate the parent template, and the enzymatic hydrolysis products were transformed into E. coli BL21(DE3) competent cells by the calcium chloride method. The transformation liquid was coated with agar medium containing 50 µg L 1 kanamycin to obtain the site-saturated mutant library. · − Table 1. Primers used in this study.

Primer Sequence (50 - 30)

G395S For (50-AACTTTCGTGCTNNNCTTTAGACT-30) G395S Rev (50-TTTTGCTGGTACANNNAAATCCTTC-30)

2.4. Error-Prone PCR Mutant Library With pET-28a (+)-trpB as the template, the trans-EasyTaq DNA Polymerase kit was used to introduce random mutations in the full-length trpB gene. Recombinant mutant plasmids were constructed. The genes were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. The target gene was constructed between the BamH I and Hind III cleavage sites of the pET-28a (+) vector. Successfully transformed clones were inoculated in 5 mL LB liquid medium (50 µg mL 1 Kan), were · − cultured at 37 C and 200 r min 1 for 8 h and were then transferred to 50 mL LB liquid medium ◦ · − (50 µg mL 1 Kan). IPTG was added at 18 C for induction. The mutant library was obtained by · − ◦ transforming E. coli competent cells.

2.5. Screening of Mutated Enzymes LB liquid medium (200 mL) was added to each well of the 96-well plate (50 µg mL 1 Kan). A single · − colony was selected from the plate with a sterilized toothpick and inoculated into the 96 shallow oriﬁce plate (37 C, 200 r min 1, 3 h). IPTG was added at 18 C for induction. The fermentation broth was ◦ · − ◦ centrifuged (4 C, 4000 r min 1,10 min). The cells were washed with PBS buﬀer and were then added ◦ · − to the reaction plate according to the corresponding position. L-Serine (10 mmol L 1) was added to · − the reaction plate (37 C, 30 min, 200 r min 1). After the reaction, the reactant was centrifuged (4 C, ◦ · − ◦ 4000 r min 1,10 min). The absorbance value at 290 nm was determined by ELIASA, and the mutant · − strain with an absorbance value that increased relative to the absorbance value of TrpB was selected for re-screening.

2.6. Protein Puriﬁcation To purify tryptophan synthase, the supernatant was applied to a Ni-NTA column based on the methods described in the literature [25]. The quality of puriﬁed tryptophan synthase was analyzed by SDS-PAGE.

2.7. Synthesis and Identiﬁcation of L-tryptophan L-tryptophan (Alighting reagent Co., Ltd., Shang hai, China) was synthesized by tryptophan synthase from L-serine and indole (50 mmol/L L-serine, 50 mmol/L indole, 40 ◦C, pH 8, 12 h). L-tryptophan was analyzed with an FT-IR Spectrometer ( Gangdong Sci & Tech. Co., Ltd., Tianjing, Microorganisms 2020, 8, 519 4 of 11

China), Nuclear Magnetic Resonance Spectrometer (Bruker Avance 500 MHz, Switzerland), and Liquid Chromatography-Mass Spectrometer (Agilent, Palo alto, California, USA). SPSS 22.0 (SPSS, Chicago, Illinois, USA) was used to calculate the standard error and the signiﬁcance of diﬀerences.

3. Results

3.1. Selection of Mutation Sites B-FITTER software was used to analyze 397 amino acid residue sites of tryptophan synthase and obtain their temperature factor values. Then, the temperature factor values of the amino acids at the entrance of the substrate channel were sorted, and the top 20 amino acid residue sites were identiﬁed (Table2). Among these 20 amino acid residues, we selected glycine 395 (G395), as it was the site with the largest temperature factor value for molecular modiﬁcation.

Table 2. B-factor values of some of the amino acid residues of tryptophan synthase.

Rank Residue Sequence Number Amino Acid B-Factor Value 1 395 Gly 95.24 2 394 Arg 93.55 3 382 Lys 93.34 4 393 Ala 92.39 5 392 Lys 89.52 6 383 Asp 88.7 7 313 His 88.7 8 257 Pro 87.61 9 329 Asp 86.18 10 233 Gly 86.17 11 191 Ala 86.06 12 234 Gly 85.96 13 18 Pro 85.83 14 258 Gly 85.79 15 388 His 85.68 16 17 Val 85.64 17 378 Gly 85.18 18 190 Thr 85.01 19 256 Glu 83.42 20 232 Gly 82.93

3.2. Mutation Library Screening A site-saturated mutant library consisting of 300 mutants was constructed, and a mutant enzyme with signiﬁcantly improved thermal stability was obtained by initial screening and rescreening. DNA sequencing revealed that the amino acid residue at position 395 of the mutant enzyme was mutated from glycine to serine (the codon was mutated from GGT to AGT).

3.3. Optimum Temperature of the Mutant Enzyme In the same reaction system, the activities of the mutant enzyme and the parent enzyme at diﬀerent temperatures (the absorbance increment caused by the formation of the product L-tryptophan was taken as the indicator) were investigated. The results showed that the optimal reaction temperature of the parent enzyme was 35 ◦C, while the optimal reaction temperature of the mutant enzyme was approximately 40 ◦C, which was 5 ◦C higher than that of the parent enzyme (Figure1). The increase in the optimum reaction temperature indicated that the thermal stability of the mutant enzyme G395S was better than that of the parent enzyme. Microorganisms 2020, 8, x FOR PEER REVIEW 5 of 12 Microorganisms 2020 8 Microorganisms, ,2020 519, 8, x FOR PEER REVIEW 5 of 12 5 of 11

110 WT a a 100 G395S 110 b 90 WT c a a 100 G395S 80 b d 90 c 70 e 80 d e 60 f 70 e 50 e 60 f 40 50 30 Relative activity (%) activity Relative 40 20 30 Relative activity (%) activity Relative 10 20 0 10 30 35 40 45 o 0 Temperature ( C) 30 35 40 45 Temperature (oC) Figure 1. Effects of temperature on the activities of the tryptophan synthase mutant and wild type. Relative activity of the tryptophan synthase mutant (G395S) and wild type (WT) catalyzing reactions Figure 1. Effects of temperature on the activities of the tryptophan synthase mutant and wild type. at differentRelative temperatures activity of the (30, tryptophan 35, 40, and synthase 45 ◦C), mutant L-serine (G395S) concentration and wild type of(WT) 50 catalyzing mmol/L, reactions pH of 8, 12 h. Figure 1. Effects of temperature on the activities of the tryptophan synthase mutant and wild type. The Errorat bars differe werent temperatures mean values (30, of 35 three, 40, and independent 45 °C), L-serine replicate. concentration Themeaning of 50 mmol/L, of letters pH of a,8, 12 b, c,h. d, e, f, Relative activity of the tryptophan synthase mutant (G395S) and wild type (WT) catalyzing reactions The Error bars were mean values of three independent replicate. The meaning of letters a, b, c, d, e, f, in the figureat differe legendnt temperatures indicates (30, significant 35, 40, and di ff45erences. °C), L-serine concentration of 50 mmol/L, pH of 8, 12 h. in the figure legend indicates significant differences. The Error bars were mean values of three independent replicate. The meaning of letters a, b, c, d, e, f, 3.4. Directed Evolution in the figure legend indicates significant differences. 3.4. Directed Evolution Error-prone PCR was used to construct a random mutant library. After screening 15,000 mutant 3.4. DirectedError- Epronevolution PCR was used to construct a random mutant library. After screening 15,000 mutant strains, the positive mutant strain G395S/A191T was obtained. The enzyme activity of the highly active strains, the positive mutant strain G395S/A191T was obtained. The enzyme activity of the highly positive mutantError-prone G395S PCR/A191T was used was to 4.8 construct times a higher random than mutant that library. of the After parent screening enzyme, 15,000 indicating mutant that strains,active thepositive positive mutant mutant G395S/A191T strain G395S/A191T was 4.8 times was higher obtained than. thatThe ofenzyme the parent activity enzyme, of the indicating highly sites 191 and 395 were the key sites affecting the enzyme activity of TrpB (Figure2). The OD600 values activethat sitespositive 191 mutantand 395 G395S/A191T were the key siteswas 4.8affecting times higherthe enzyme than thatactivity of the of parentTrpB (Figure enzyme, 2). indicatingThe OD600 for the growththatvalues sites for of191 the and growth wild 395 were type of the the and wild key G395S sitestype affectingand and G395S G395S the and enzyme/A191T G395S/A19 activity mutants1T of mutants TrpB were (Figure were measured. measured. 2). The OD600 There There was no differencevalueswas in OD600nofor difference the growth value in duringof OD600 the wild thevalue type growth during and G395S ofthe the growth and three G395S/A19 of strains, the three1T and strains,mutants no di and ffwereerences no measured. differences in the There morphologyin the of the threewasmorphology no strains difference were of the in observed threeOD600 strains value under were during a observed microscope. the growth under of Compared a the microscope three strains, with. Compared theand parent, no withdifferences the parent, mutant in the the enzyme −1 −1 morphologymutant enzyme of the (G395S three strains/A191T) were had observeda Km of 0.21 under mM anda microscope a catalytic. Comparedefficiency k withcat/Km theof 5.38 parent,1 mM1 the∙s , (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM− s− , which was which was 4.8 times higher than that of the parent (Table 3). · −1 −1 4.8 timesmutant higher enzyme than (G395S that of /A191T) the parent had a (TableKm of 0.213). mM and a catalytic efficiency kcat/Km of 5.38 mM ∙s , which was 4.8 times higher than that of the parent (Table 3). a 100 a 100 80

60 bc 40

c c bc Relative activity (%) activity Relative 40 d d c c 20 e Relative activity (%) activity Relative d d f 20 f 0 e f f WT 0 G395S

G395S/P56L WT G395S/T190AG395S/A191T G395S/K167FG395S/V325TG395S/Q90GG395S/H313S G395S

G395S/P56L Figure 2. Relative activities of glycineG395S/T190A-G395S/A191TsubstitutedG395S/K167F mutantsG395S/V325T G395S/Q90G of tryptophanG395S/H313S synthase . The reaction

conditions for the mutated enzyme were an L-serine concentration of 50 mmol/L, a reaction FigureFigure 2. Relative 2. Relative activities activities of glycine-substitutedof glycine-substituted mutants mutants of oftryptophan tryptophan synthase synthase.. The reaction The reaction temperature of 40 °C , pH of 8, 12 h. The Error bars were mean values of three independent replicate. conditionsconditions for the for mutated the mutated enzyme enzyme were an were L-serine an L-serine concentration concentration of 50 ofmmol 50 mmol/L,/L, a reaction a reaction temperature The meaning of letters a, b, c, d, e, f, in the figure legend indicates significant differences. temperature of 40 °C , pH of 8, 12 h. The Error bars were mean values of three independent replicate. of 40 ◦C, pH of 8, 12 h. The Error bars were mean values of three independent replicate. The meaning The meaning of letters a, b, c, d, e, f, in the figure legend indicates significant differences. of letters a, b, c, d, e, f, in the figure legend indicates significant differences.

Table 3. Kinetic parameters of tryptophan synthase and its mutants.

1 1 1 Mutations Km (mM) kcat (s ) kcat/Km (mM s ) Fold Change over WT − − · − WT 0.31 0.11 0.35 0.09 1.12 0.07 1 ± ± ± G395S 0.32 0.14 0.42 0.11 1.31 0.10 1.16 ± ± ± G395S/A191T 0.21 0.10 1.13 0.12 5.38 0.11 4.80 ± ± ± Mean values of three independent replicate standard deviation. ± Microorganisms 2020, 8, x FOR PEER REVIEW 6 of 12

Table 3. Kinetic parameters of tryptophan synthase and its mutants.

Mutations Km (mM) kcat (s−1) kcat/Km (mM−1∙s −1) Fold Change over WT WT 0.31 ± 0.11 0.35 ± 0.09 1.12 ± 0.07 1 MicroorganismsG395S2020 , 8, 519 0.32 ± 0.14 0.42 ± 0.11 1.31 ± 0.10 1.16 6 of 11 G395S/A191T 0.21 ± 0.10 1.13 ± 0.12 5.38 ± 0.11 4.80 Mean values of three independent replicate ± standard deviation. 3.5. Synthesis of L-tryptophan 3.5. Synthesis of L-tryptophan The mutated enzymes reacted at diﬀerent pH values, and the results showed that the mutant enzymeThe G395S mutated/A191T enzymes had the reacted highest at activitydifferent at pH pH values, 8. The and optimal the results pH value showed of the that mutant the mutant enzyme G395Senzyme and G395S/A191T the parental enzyme had the washighest also activity 8, but the at pH activities 8. The ofoptimal the mutant pH value enzyme of the G395S mutant and enzyme parental enzymeG395S were and thelower parental than that enzyme of the was mutant also enzyme8, but the G395S activities/A191T of the(Figure mutant3). These enzyme results G395S showed and thatparental the mutant enzyme enzyme were lower G395S than/A191T that maintainedof the mutant high enzyme activity G395S/A191T in the L-serine (Figure concentration 3). These results range ofshowed 25, 50, andthat 75the mmolmutant/L, enzyme while G395S/A191T the optimalL-serine maintained concentration high activityof in thethe L mutant-serine concentration enzyme G395S andrange parent of 25, enzyme 50, and was 75 mmol/L, approximately while the 50 optimal mmol L/L-serine (Figure concentration4). Under theof the reaction mutant conditions enzyme G395S of an L-serineand parent concentration enzyme was of 50approximately mmol/L, the 50 production mmol/L (Figure of L-tryptophan 4). Under the was reaction determined conditions by anof an Amino L- serine concentration of 50 mmol/L, the production of L-tryptophan was determined by an Amino Acid Analyzer (Hitachi L-8900, Japan). Synthesis of L-tryptophan by the mutant enzyme G395S/A191T Acid Analyzer (Hitachi L-8900, Japan). Synthesis of L-tryptophan by the mutant enzyme was higher than that by the mutant enzyme G395S and parental enzyme (Figure5). G395S/A191T was higher than that by the mutant enzyme G395S and parental enzyme (Figure 5).

G395S/A191T a 100 WT b G395S b

80 c

c 60 d

e e

e 25 e e e f f f 20 f g g

Relative activity (%) activity Relative 15

0 5 6 7 8 9 10 pH FigureFigure 3. E3ff. ectsEffects of pH of onpH tryptophan on tryptophan synthase synthase activity. activity. pH stability pH stability of tryptophan of tryptophan synthase synthase at diff erentat pHdifferent values (5, pH 6, values 7, 8, 9, (5, 10), 6, L-serine7, 8, 9, 10), concentration L-serine concentration of 50 mmol of/L, 50 40 mmol/L,◦C, 12 h.40 The°C , Error12 h. T barshe Error were bars mean valueswere ofmean three values independent of three independent replicate. The replicate meaning. The ofmeaning letters of a, letters b, c, d, a, e,b, f,c, g,d, ine, f, the g, in figure the figure legend Microorganisms 2020, 8, x FOR PEER REVIEW 7 of 12 indicateslegend significantindicates significant differences. differences.

a a 100 G395S/A191T ab WT G395S 80 b

c 60 d e e g e e g e 20 g f

Relative activity (%) activity Relative f f f

0 25 50 75 100 125 150 L-Serine (mmol/L) Figure 4. Effects of the L-serine concentration on tryptophan synthase activity. Relative activity of Figure 4. Effects of the L-serine concentration on tryptophan synthase activity. Relative activity of tryptophantryptophan synthase synthase at at di differentfferent L-serine L-serine concentrations concentrations (25, (25, 50, 50, 75, 75, 100, 100, 125, 125, 150150 mmolmmol/L),/L), 4040 ◦°CC,, pH of 8,of 12 8, h. 12 The h. T Errorhe Error bars bars were were mean mean values values of of three three independent independent replicate.replicate. T Thehe meaning meaning of ofletters letters a,b,c,d,e,f,g,a,b,c,d,e,f, ing, in the the figure figure legend legend indicates indicates significant significantdi differences.fferences.

Figure 5. Synthesis of L-tryptophan by the three strains. The reaction conditions for three tryptophan synthase recombinant strains (A: G395S /A191T; B: G395S; C: Wild strain) were an L-serine concentration of 50 mmol/L, reaction temperature of 40 °C, pH of 8, 12 h. Red line was the integral line.

3.6. Identification of L-tryptophan The infrared spectrum of L-tryptophan is shown in Figure 6, with the introduction of amino and carboxyl groups. The 1H-NMR spectrum of L-tryptophan is shown in Figure 7. 1H NMR (500 MHz, D2O): δ 3.23(s, 3H), 3.41 (d, 2H), 3.97 (t, 1H), 7.12 (d, 1H), 7.21 (d,1H), 7.45 (m, H), 7.64 (m, H). The theoretical molecular weight of L-tryptophan is 204 Da. As shown in Figure 8, a signal at m/z 205 appeared in the mass spectrum of the reaction mixture, which is consistent with ionized L-tryptophan (Figure 8).

Microorganisms 2020, 8, x FOR PEER REVIEW 7 of 12

a a 100 G395S/A191T ab WT G395S 80 b

c 60 d e e g e e g e 20 g f

Relative activity (%) activity Relative f f f

0 25 50 75 100 125 150 L-Serine (mmol/L)

Figure 4. Effects of the L-serine concentration on tryptophan synthase activity. Relative activity of tryptophan synthase at different L-serine concentrations (25, 50, 75, 100, 125, 150 mmol/L), 40 °C , pH of 8, 12 h. The Error bars were mean values of three independent replicate. The meaning of letters Microorganisms 2020, 8, 519 7 of 11 a,b,c,d,e,f, g, in the figure legend indicates significant differences.

Figure 5. Synthesis of L-tryptophan by the three strains. The reaction conditions for three tryptophan synthaseFigure recombinant 5. Synthesis of strains L-tryptophan ((A): G395S by the /threeA191T; strains. (B): Th G395S;e reaction (C ):conditions Wild strain) for three were tryptophan an L-serine synthase recombinant strains (A: G395S /A191T; B: G395S; C: Wild strain) were an L-serine concentration of 50 mmol/L, reaction temperature of 40 ◦C, pH of 8, 12 h. Red line was the integral line. concentration of 50 mmol/L, reaction temperature of 40 °C, pH of 8, 12 h. Red line was the integral 3.6. Identiﬁcationline. of L-tryptophan

The3.6. infraredIdentification spectrum of L-tryptophan of L-tryptophan is shown in Figure6, with the introduction of amino and 1 1 carboxyl groups.The infrared The spectrumH-NMR of spectrum L-tryptophan of L-tryptophan is shown in Figure is shown 6, with in the Figure introduction7. H NMRof amino (500 and MHz, D2O):carboxylδ 3.23(s, groups 3H),. 3.41The 1H (d,-NMR 2H), spectrum 3.97 (t, 1H),of L-tryptophan 7.12 (d, 1H), is shown 7.21 in (d,1H), Figure7.45 7. 1H (m, NMR H), (500 7.64 MHz, (m, H). The theoreticalD2O): δ 3.2 molecular3(s, 3H), 3.41 weight (d, 2H), of L-tryptophan3.97 (t, 1H), 7.12 is (d, 204 1H), Da. 7.21 As (d,1H), shown 7. in45 Figure (m, H),8, 7. a64 signal (m, H). at mThe/z 205 appearedtheoretical in the molecular mass spectrum weight of of the L-tryptophan reaction mixture, is 204 Da. which As shown is consistent in Figure with 8, a ionized signal at L-tryptophan m/z 205 Microorganisms 2020, 8, x FOR PEER REVIEW 8 of 12 (Figureappeared8). in the mass spectrum of the reaction mixture, which is consistent with ionized L-tryptophan Microorganisms 2020, 8, x FOR PEER REVIEW 8 of 12 (Figure 8).70 70

70 70 60 60

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40 % 40

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30 30

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30 30

20 20 Transmittance 20 Transmittance 20

10 10

Transmittance Transmittance 10 10 0 0

04000 3500 3000 2500 2000 1500 1000 500 04000 3500 3000 2500 2000 1500 1000 500

-1 -1 4000 3500 3000 Wavenumbers(cm2500 2000 ) 1500 1000 500 4000 3500 3000 Wavenumbers(cm2500 2000 ) 1500 1000 500 -1 -1 Wavenumbers(cm(A) ) Wavenumbers(cm(B) ) (A) (B) Figure 6. FTIR spectrum of L-tryptophan. (A: Reaction product; B: Standard substance). FigureFigure 6. FTIR 6. FTIR spectrum spectrum of L-tryptophan.of L-tryptophan ((. (AA):: R Reactioneaction product product;; B: (SBtandard): Standard substance substance).).

Figure 7. 1H-NMR spectrum of L-tryptophan. Figure 7. 1H-NMR spectrum of L-tryptophan. Figure 7. 1H-NMR spectrum of L-tryptophan.

Figure 8. Mass spectrum of L-tryptophan. Figure 8. Mass spectrum of L-tryptophan.

Microorganisms 2020, 8, x FOR PEER REVIEW 8 of 12

70 70

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50 50

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40 % 40

（

30 30

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Transmittance Transmittance 10 10

0 0

4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500

-1 -1 Wavenumbers(cm ) Wavenumbers(cm ) (A) (B)

Figure 6. FTIR spectrum of L-tryptophan. (A: Reaction product; B: Standard substance).

Microorganisms 2020, 8, 519 8 of 11 Figure 7. 1H-NMR spectrum of L-tryptophan.

Microorganisms 2020, 8, x FOR PEER REVIEW 9 of 12 FigureFigure 8. Mass 8. Mass spectrum spectrum of L of-tryptophan. L-tryptophan. 3.7. Mutation Analysis 3.7. Mutation Analysis Appropriate modifications of the amino acid residues in the substrate channel can promote the recognitionAppropriate and modifications binding abilities of the aminoof the acid enzyme. residues Through in the asubstrate combination channel of can temperature promote the factor recognitionanalysis and and binding substrate abilities channel of the localization, enzyme. Through key amino a combination acid residues of temperature that affect factor both the analysis thermal andstability substrate and channel catalytic localization, activity of key the amino enzyme acid can residues be identified. that aff ectIn combination both the thermal with stabilitysaturation and site- catalyticspecific activity mutation of the andenzyme screening, can be identified. target mutant In combination enzymes with with saturation improved site-specific thermal stability mutation and andcatalytic screening, activity target can mutant be obtained enzymes from with all improved the possible thermal mutations. stability Chimera1.13 and catalytic was activity used to can analyze be obtainedthe substrate from all channel the possible space mutations.of the enzyme, Chimera1.13 and it was was found used that to the analyze amino the acid substrate at position channel 395 was spacelocated of the in enzyme,a loop involved and it wasin subs foundtrate that channel the aminoformation acid (F atigure position 9). After 395 the was mutation located of in this a loop amino involvedacid from in substrate glycine to channel serine, formation the spatial (Figure orientation9). After freedom the mutation of the main of this chain amino was acid reduced, from glycinethe rigidity to serine,of the theenzyme spatial was orientation increased, freedom and the of thermal the main stability chain was of the reduced, enzyme the was rigidity improved. of the enzymeAlanine at wasposition increased, 191 and was the mutated thermal to stabilitythreonine, of thewhich enzyme may have was improved.changed the Alanine folding at direction position of 191 the was loop, mutatedthus changing to threonine, the shape which and may size have ofchanged the substrate the folding channel direction entrance of and the loop,increasing thus changingenzyme activity the shape(Figure and size9). of the substrate channel entrance and increasing enzyme activity (Figure9).

Figure 9. Relationship between the substrate channel and the 191 and 395 binding sites. Figure 9. Relationship between the substrate channel and the 191 and 395 binding sites. 4. Discussion 4. Discussion According to the structural information of proteins and sequence comparisons of homologous proteins,According using a rational to the structural selection information of amino acid of proteins residues and as targets sequence combined comparisons with site-speciﬁcof homologous proteins, using a rational selection of amino acid residues as targets combined with site-specific mutation of key amino acids, we can screen and obtain target mutants [26–28]. Rational design can improve the substrate specificity of an enzyme by remodeling its active pocket, which is the structural domain of the enzyme responsible for its catalytic function and usually consists of a hydrophobic core. The amino acids in the active pocket can be divided into catalytic amino acids and substrate channel amino acids. Substrate molecules enter the active pocket and interact with the substrate channel amino acids, placing the substrate molecules in a favorable conformation for catalysis and allowing completion of the catalytic reaction under the action of the catalytic amino acids. Changing the substrate specificity of the enzyme can expand the range of enzyme substrates by remodeling the active pocket of the enzyme to form a different conformation with the substrate molecule or by accepting other substrates. The size of the active pocket directly affects the entry and stability of substrate molecules but can also hinder the entry of substrate molecules and prevent the enzyme from accepting more substrates for catalytic reactions. Therefore, the substrate specificity of the enzyme can be modified by regulating the size of its active pocket. For a smaller active pocket, a site- specific mutation can increase the size of the active pocket to accommodate larger substrate molecules, increasing the substrate specificity of the enzyme molecule. The microenvironment of the active pocket, such as its polarity, is critical to the activity of the enzyme. Different environments lead to preferences for different substrates. According to the spatial structure of an enzyme and the properties of its active pocket, it can be rationally designed to allow the target substrate while maintaining its relative activity and stability to better complete the catalytic reaction. By reducing the

Microorganisms 2020, 8, 519 9 of 11 mutation of key amino acids, we can screen and obtain target mutants [26–28]. Rational design can improve the substrate specificity of an enzyme by remodeling its active pocket, which is the structural domain of the enzyme responsible for its catalytic function and usually consists of a hydrophobic core. The amino acids in the active pocket can be divided into catalytic amino acids and substrate channel amino acids. Substrate molecules enter the active pocket and interact with the substrate channel amino acids, placing the substrate molecules in a favorable conformation for catalysis and allowing completion of the catalytic reaction under the action of the catalytic amino acids. Changing the substrate specificity of the enzyme can expand the range of enzyme substrates by remodeling the active pocket of the enzyme to form a different conformation with the substrate molecule or by accepting other substrates. The size of the active pocket directly affects the entry and stability of substrate molecules but can also hinder the entry of substrate molecules and prevent the enzyme from accepting more substrates for catalytic reactions. Therefore, the substrate specificity of the enzyme can be modified by regulating the size of its active pocket. For a smaller active pocket, a site-specific mutation can increase the size of the active pocket to accommodate larger substrate molecules, increasing the substrate specificity of the enzyme molecule. The microenvironment of the active pocket, such as its polarity, is critical to the activity of the enzyme. Different environments lead to preferences for different substrates. According to the spatial structure of an enzyme and the properties of its active pocket, it can be rationally designed to allow the target substrate while maintaining its relative activity and stability to better complete the catalytic reaction. By reducing the polarity of the active pocket, the activity of an enzyme against competitive substrates can be reduced, which is beneficial to accumulating target products in metabolic engineering and improving the specificity of enzyme catalysis. By changing the charge state of the active pocket amino acids, the mutated enzyme has a specificity different from that of the wild type. To analyze ethanol dehydrogenase CpRCR, the active pocket was analyzed, and it was found that F285, W286, and W116 constituted the substrate binding pocket of the enzyme. The activity of mutant enzymes was increased by a rational design to increase the size of the substrate-binding pocket [29]. During the study of LfAmDHs, it was found that the two amino residues A113 and T134, located at the distal end of the substrate binding pocket, blocked the opening of the substrate binding pocket, so LfAmDH could only bind smaller substrates. The mutant was obtained by a rational design using site-specific mutations, and the size of the substrate-binding pocket increased the substrate spectrum of the enzyme [30]. Three key regions of lipase CALB were studied to determine its key amino acids: D223 and A281. D223V/A281S was obtained by site-specific saturation mutation, which increased the selectivity of the final product, methyl succinate from 8% to more than 99% [31]. The ketoisopentane decarboxylase of Lactococcus lactis was mutated by rational design. When V461 was mutated into serine, the specificity of the downstream ketoic acid was reduced, which was conducive to the production of the precursor material amyl alcohol [32]. Kaempferol was produced by the metabolic engineering of Saccharomyces cerevisiae [33]. B-FITTER software was used to analyze the crystal structure of tryptophan synthetase to determine the temperature factor (B-factor) of each amino acid locus and to identify the adverse effects on the thermal stability of the enzyme caused by mutations of the key amino acid G395. Then, at this site, fixed-point saturated mutations were performed to build a mutant library, and from the saturated mutant library, screening mutation was found to result in better thermal stability of the enzyme (G395S). The reaction temperature of the mutant enzyme G395S was 5 ◦C higher than that of the parent enzyme. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme G395S.

5. Conclusions

Compared with the parent, the mutant enzyme G395S /A191T had a Km of 0.21 mM and a catalytic 1 1 eﬃciency Kcat /Km of 5.38 mM s , which was 4.8 times higher than that of the parent. The conditions − · − for L-tryptophan synthesis by the mutated enzyme were an L-serine concentration of 50 mmol/L, a reaction temperature of 40 ◦C, a pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. Microorganisms 2020, 8, 519 10 of 11

Author Contributions: L.X. and F.H. performed the experiments and analyzed the data; Z.D. coordinated and supervised this study; L.X. and Z.W. drafted and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Anhui Wanbei Industrial Innovation Team Project (Anhui Talents Office (2017)No. 2), the project of Suzhou University (2018XJXS02; 2019XJSN05; 2019XJZY10), the Anhui Research Project (17030801022; 2018sjjd044; 2018mooc388; KJ2018A0447; 190808QC146), and the National Innovation and Entrepreneurship Training Program for College Students (201910379046). Conflicts of Interest: The authors declare no conflict of interest.

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