Gamma-aminobutyrate (GABA) is an important chemical in the pharmaceutical field. GABA-producing lactic acid bacteria (LAB) offer the opportunity of developing this health-oriented product. In this study, the gadA, gadB, gadC, gadCB, and gadCA gene segments of Lactobacillus brevis were cloned into pMG36e, and strain Lb. brevis/pMG36e-gadA was selected for thorough characterization in terms of GABA production after analysis of GAD activities. Subsequently, a physiology-oriented engineering strategy was adopted to construct an FoF1-ATPase deficient strain NRA6 with higher GAD activity. As expected, strain NRA6 could produce GABA at a concentration of 43.65 g/L with a 98.42% GABA conversion rate in GYP fermentation medium, which is 1.22-fold higher than that obtained by the wild-type strain in the same condition. This work demonstrates how the acid stress response mechanisms of LAB can be employed to develop cell factories with improved production efficiency and contributes to research into the development of the physiology-oriented engineering.Keywords: FoF1-ATPase; GABA; GAD system; Lb. brevis; physiology-oriented engineering;

•N-CECS was synthesized from squid pen β-chitin as starting materials.•The adsorption capacity of Cu(II) correlated well with the DS of N-CECS.•The highest adsorption capacity of Cu(II) among three N-CECS reached 207.5 mg g−1.•Chemisorption was the rate-controlling step for Cu(II) adsorbed onto N-CECS.Chitosan was prepared by N-deacetylation of squid pens β-chitin, and N-carboxyethylated chitosan (N-CECS) with different degrees of substitution (DS) were synthesized. DS values of N-CECS derivatives calculated by 1H nuclear magnetic resonance (NMR) spectroscopy were 0.60, 1.02 and 1.46, respectively. The adsorption capacity of Cu(II) by N-CECS correlated well with the DS and pH ranging from 3.2 to 5.8. The maximum Cu(II) adsorption capacity (qm) of all three N-CECS at pH 5.4 was 207.5 mg g−1, which was 1.4-fold higher than that of chitosan. The adsorption equilibrium process was better described by the Langmuir than Freundlich isotherm model. Adsorption of Cu(II) ion onto N-CECS followed a pseudo-second order mechanism with chemisorption as the rate-limiting step. In a ternary adsorption system, the adsorption capacity of Cu(II) by N-CECS also presented high values, and qm for Cu(II), Cd(II), and Pb(II) were 150.2, 28.8, and 187.9 mg g−1, respectively.

•SP850 resin was selected as the most suitable resin for sulforaphane purification.•The Redlich-Peterson equation could describe the adsorption behavior.•The adsorption of sulforaphane was a physical, exothermic and spontaneous process.•Sulforaphane diffused quickly in the macropore at the beginning of the adsorption process.The adsorption equilibrium, kinetic and thermodynamic of sulforaphane (SF) adsorption onto macroporous resin in aqueous phase were studied. The SP850 resin was screened as the appropriate resin for SF purification. From the equilibrium studies, the Redlich-Peterson model was found to be the best for description of the adsorption behavior of SF onto SP850 resin, followed by the Freundlich model and the Langmuir model. Batch equilibrium experiments demonstrated that, in the examined temperature range, the equilibrium adsorption capacity of SP850 resin decreased with increasing adsorption temperature. Thermodynamics studies indicated that the adsorption of SF was a physical, exothermic, and spontaneous process. The adsorption kinetics revealed that the pseudo-second-order kinetic model was suitable to characterize the kinetics of adsorption of SF onto SP850. Finally, the intra-particle diffusion model demonstrated that SF diffused quickly into macropores, and that diffusion slowed down in the meso- and micropores.

Chitosan was prepared by alkaline N-deacetylation of β-chitin from squid pens, and N-carboxyethylated derivatives (N-CECS) with different degrees of substitution (DS) were synthesized. The carboxyethylation of the polysaccharide was identified by Fourier transform infrared, 1H and 13C nuclear magnetic resonance (NMR), and X-ray diffraction analyses. The DS of the derivatives was calculated by 1H NMR and elemental analysis. All three N-CECS samples showed good water solubility at pH > 6.5. The antioxidant properties and bile acid binding capacity of the derivatives were studied in vitro. The highest bile acid binding capacity of all N-CECS reached 36.9 mg/g, which was 2.6-fold higher than that of chitosan. N-CECS showed a stronger scavenging effect on 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical ability, and EC50 values were below 2 mg/mL. The scavenging ability of N-CECS against superoxide radicals correlated well with the DS and concentration of N-CECS. These results indicated that N-carboxyethylation is a possible approach to prepare chitosan derivatives with desirable in vitro biochemical properties.

Cytochrome P450 BM-3 (A74G/F87V/L188Q) could catalyze indole to produce indigo. To further improve this capability, random mutagenesis was performed on the heme domain of P450 BM-3 (A74G/F87V/L188Q) with error-prone PCR. A single mutant V445A was selected out from the error-prone library and exhibited the highest specific activity toward indole among the mutants obtained. The kinetic parameters of V445A were also highly improved. Compared with the parent enzyme, the turnover rate (kcat) of V445A was increased by 7.5 times, while its Km value decreased by 9.2 %. Consequently, the catalytic efficiency (kcat/Km) of V445A was raised to 8.2 times than that of the parent enzyme. Moreover, alanine was confirmed as the best amino acid substitution by saturated mutagenesis in Val445 position. Three-dimensional structure analysis was also used to rationalize the effect on the enzyme properties of the mutation. This study showed that random mutagenesis was efficient to identify mutants with potential values in industry and increased our insight into P450 BM-3.

Chitosans were prepared by H2O2 oxidative depolymerization from squid pens with low molecular weights (LMW) of 13,025, 7011, 4169, 2242 and 963 Da. The bile acid binding capacities and antioxidant properties of LMW chitosans were studied in vitro. LMW chitosans exhibited stronger bile acid binding capacities than that of chitosan. The scavenging ability of LMW chitosans against DPPH radicals improved with increasing concentration, and EC50 values were below 1.3 mg/mL. The EC50 values of LMW chitosans against hydroxyl radicals ranged from 0.93 to 3.66 mg/mL. All LMW chitosans exhibited a strong ferrous ion chelating effect and reducing power. At 1 mg/mL, the scavenging ability of chitosan-963 towards superoxide radicals was 67.76%. These results indicated that LMW chitosans which have stronger bile acid binding capacity and antioxidant activities may act as potential antioxidants in vitro.Highlights► LMW chitosans exhibited good bile acid binding capacity and antioxidant ability. ► The highest bile acid binding capacity of five LMW chitosans reached 63.53 mg/g. ► Increasing LMW chitosans concentration improved chelating effects and reducing power. ► EC50 value of LMW chitosans against hydroxyl radicals ranged from 0.93 to 3.66 mg/mL.

Chitosan was prepared by alkaline N-deacetylation of squid cartilage β-chitin and carboxymethylated derivatives with different degrees of substitution (DS) were synthesized. The DS of the derivatives calculated by pH titration were 0.64, 0.81, 1.0, 1.33 and 1.59. The antioxidant properties and bile acid binding capacity of the derivatives were studied in vitro. The carboxymethylation of chitosan caused enhancement of bile acid binding capacity. At 1 mg/mL, carboxymethyl chitosan (CMCS) showed a stronger scavenging effect than chitosan towards DPPH radicals. The CMCS scavenging effect on superoxide radicals was stronger than that of chitosan, and EC50 values were below 5.6 mg/mL. The effectiveness of reducing power correlated with the DS of CMCS. At 1.2 mg/mL, the ability of CMCS to chelate ferrous iron was 100%, whereas that of chitosan was only 22.34%. This suggested that carboxymethylation is a possible approach to obtain chitosan derivatives with desirable biological properties.

On an industrial scale, the production of γ-aminobutyric acid (GABA) from the cheaper sodium L-glutamate (L-MSG) is a valuable process. By entrapping Lactobacillus brevis cells with higher glutamate decarboxylase (GAD) activity into Ca-alginate gel beads, the biotransformation conditions of L-MSG to GABA were optimized with the immobilized cells. The cells obtained from a 60-h culture broth showed the highest biotransformation efficiency from L-MSG to GABA. The optimal cell density in gel beads, reaction pH and temperature were 11.2 g dry cell weight (DCW) l−1, 4.4 and 40°C respectively. The thermal stability of immobilized cells was significantly higher than free cells. Under the optimized reaction conditions, the yield of GABA reached above 90% during the initial five batches and the yield still remained 56% in the tenth batch. Continuous production of GABA was realized with a higher yield by incorporating cell re-cultivation using the packed bed reactor.

P450 BM3 mutant can catalyze indole to indoxyl, and indoxyl can dimerize to form indigo. But the reaction catalyzed by P450 BM3 requires NADPH, as coenzyme regeneration is very important in this system. As we know, when glucose dehydrogenase oxidizes glucose to glucolactone, NADH or NADPH can be formed, which can contribute to NADPH regeneration in the reaction catalyzed by P450 BM3. In this paper, a recombinant Escherichia coli BL21 (pET28a (+)-P450 BM3-gdh0310) was constructed to co-express both P450 BM3 gene and glucose dehydrogenase (GDH) gene. To improve the expression level of P450 BM3 and GDH in E. coli and to avoid the complex and low-efficiency refolding operation in the purification procedure, the expression conditions were optimized. Under the optimized conditions, the maximum P450 BM3 and GDH activities amounted to 8173.13 and 0.045 U/mg protein, respectively. Then bioconversion of indole to indigo was carried out by adding indole and glucose to the culture after improved expression level was obtained under optimized conditions, and 2.9 mM (760.6 mg/L) indigo was formed with an initial indole concentration of 5 mM.

Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E. coli. Operational conditions for maximum production were determined with twelve parameters under consideration: the concentration of FeCl3, induction at OD578 (optical density measured at 578 nm), induction time and inoculum concentration. Initially, Plackett-Burman (PB) design was used to evaluate the process variables relevant in relation to P450 BM-3 production. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM-3 production was improved to 57.90×10−3 U/ml by the best combinations of the physicochemical parameters at optimum levels of 0.12 mg/L FeCl3, inoculum concentration of 2.10%, induction at OD578 equal to 1.07, and with 6.05 h of induction.

•GADc expression in E. coli increased GAD activity 2.6-fold but inhibited growth.•GAD-expressing cells were permeabilized with chemicals or heat.•These treatments increased GAD activity by at least 10-fold.•A solid-state system for efficient bacterial expression of GAD was developed.γ-Aminobutyric acid (GABA) is widely used as a pharmaceutical, nutraceutical, and as a precursor for synthesizing materials for industrial use. Bacterial cells can be exploited for use as biocatalysts with the potential to synthesize compounds such as GABA with greater efficiency, safety, and economy. However, efforts to use biocatalysts must overcome the permeability barrier of the cell envelope. Therefore, to produce a whole-cell biocatalyst with enhanced cell-associated glutamate decarboxylase (GAD) activity, we overexpressed the Glutamate (Glu)-GABA antiporter (GadC) in Escherichia coli (BL21(DE3)-pET28a-gadB). The cell-associated GAD activity of the transformants was higher by a factor of 2.6 in comparison; however, expression of GadC inhibited growth. Therefore, we permeabilized the cells using either organic solvents, surfactants, or heat. Permeabilization with organic solvents increased cell-associated GAD activity as a function of their hydrophobicity, and hexane was the most effective, increasing cell-associated GAD activity by a factor of 9.65 (6.72 U/mg). The surfactants Triton X-100, CHAPS, NP-40, OGP, and Brij-35 enhanced cell-associated GAD activity, and Triton X-100 was the most effective, increasing cell-associated GAD activity by a factor of 10.8 (7.53 U/mg). Heating BL21(DE3)-pET28a-gadB at 70 °C for 30 min increased cell-associated GAD activity by a factor of 13.1. GAD did not leak from the permeabilized cells under optimum conditions. When the heat-permeabilized cells were immobilized on Ca-alginate gel beads, the biotransformation ability of beads maintained over 60% of their initial ability after 20 consecutive batches, and the beads retained 90% of their initial activity after 30 days of storage. These approaches for improving cell permeability to enhance cell-associated GAD activity show great promise for decreasing the cost of industrial production of GABA.Download full-size image

Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of l-glutamate to form γ-aminobutyrate (GABA), which is in great demand in some foods and pharmaceuticals. In our previous study, gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51H with high activity [55.4 mmol·L− 1·min− 1·(mg protein)− 1, 120% higher than that of the wild type at pH 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out, and three specific mutants, N-terminal truncated GAD, Q51P, and Q51L, were identified. The kinetic parameters of the three mutants and Q51H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD.A novel key amino acid residue site (Q51) affecting glutamate decarboxylase (GAD) activity and expression level remarkably was identified in this study. This site is distant from active sites according to the homology model of GAD but situated on a helix at the end of the N-terminal region (residues 1–59). One mutant Q51H with an activity of 1.2 times the wild type activity was obtained by directed evolution. The possible reason might be that the substitution will give rise to hydrogen bonding interactions with L47 and E55 and therefore alter the flexibility and position of the adjacent loop and improve the activity.Download full-size image

Engineering the regioselectivity of enzymes to fulfill application needs is an important goal of protein engineering. To create biocatalysts suitable for the biosynthesis of indirubin (a drug for chronic myelogenous leukemia and a novel promising anticancer agent), cytochrome P450 BM-3 was engineered by site-directed saturation mutagenesis at position D168 to alter its hydroxylation regioselectivity towards indole. One mutant, D168W, was created. It primarily produces indirubin (∼90%) whereas the parent enzyme primarily forms indigo (∼85%). Docking calculations showed that the mutation altered the orientation of indole, and that the C-2 of the indole pyrrole ring was closer to the heme iron of P450 BM-3 than the C-3. The mutation possibly shifted the hydroxylation preference of P450 BM-3 for indole from the C-3 to C-2, which may be responsible for the reversal of distribution of the product yield. This mutant yielded high-purity indirubin and may be a good starting point for the biosynthesis of indirubin.Research highlights▶ The regioselectivity of P450 BM-3 to indole is altered by saturation mutagenesis. ▶ The saturation mutagenesis is carried out at a non-active site. ▶ A mutant primarily produces high-purity indirubin (∼90%) has been created. ▶ The mutant is different to other P450 BM-3 variants mainly produce indigo.

In order to facilitate the preparation of paeoniflorin (PF) and albiflorin (AF), two chief bioactive constituents in Paeonia lactiflora Pall (PL), induction and culture of callus from PL were studied. With a modified woody plant medium supplemented with 0.5 mg·L− 1 6-benzylaminopurine, 1.0 mg·L− 1 naphthylacetic acid, 0.1 mg·L− 1 thidiazuron and 30 g·L− 1 sucrose, callus was induced from four kinds of explants: leaf, stems, petiole, and root. The potency to form callus varies between different explants and leaf explants exhibits the highest capacity (100%). On the other hand, root-derived callus (R-callus) produces the highest level of total amount of PF and AF, 31.8 mg·g− 1 dry mass, which is higher than the corresponding level in the root of field cultivated PL. Furthermore, the time needed is only 40 days, remarkably shorter than the cultivation time of PL, about 4–5 years. Higher accumulation levels of PF and AF with shorter production time indicate that callus culture of PL is a promising powerful tool for production of PF and AF in the future.In this work, induction and culture of callus from Paeonia lactiflora Pall (PL) were studied to facilitate the preparation of paeoniflorin (PF) and albiflorin (AF), two chief bioactive constituents in PL. Callus was induced from four kinds of explants of PL (leaf, stems, petiole, and root) with a modified woody plant medium (WPM) and the presence of PF and AF in callus was demonstrated by HPLC and HPLC/ESI–MS. Proliferation, productivity, and genetic stability of different explants-derived callus were investigated. The root-derived callus culture produced PF and AF in a much higher level than the root of cultivated PL but the cultivation time was shorten from 4–5 years to about 40 days. The study validates the feasibility of preparation of PF and AF through callus culture of PL and provides a time-and cost-efficient alternative for the production of PF and AF.Download full-size image

Glutamate decarboxylase (GAD) from Lactobacillus brevis is a very promising candidate for biosynthesis of GABA and various other bulk chemicals that can be derived from GABA. However, no structure of GAD of this origin has been reported to date, which limits enzyme engineering strategy to improve its properties for better use in production of GABA. Bacterial GAD exhibits an acidic pH optimum and there is often a sharp pH dependence. In the present work, site-directed mutagenesis was performed to delete the C-terminal residues of GAD to generate a mutant, designated as GADΔC, which exhibited extended activity toward near-neutral pH compared to the wild type. Comparison of the UV–visible, fluorescence and Circular Dichroism spectra of the mutant with those of the wild type revealed that the microenvironment of the active site had been changed. Based on the homology model, we speculated that the substrate entrance was probably enlarged in GADΔC. These results provide evidence for the important role of C-terminal region in the pH-dependent regulation of enzyme activity, and the resulting mutant would be useful in a bioreactor for continuous production of GABA.Highlights► We construct the homology model of GAD form Lactobacillus brevis CGMCC 1306. ► We delete fourteen C-terminal amino acid residues of the enzyme. ► Fluorescence and CD spectra of the mutant show different properties from those of WT. ► The mutant exhibits extended activity toward near-neutral pH compared to WT. ► The C-terminal region is essential to the pH-dependent control of enzyme activity.

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of l-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pKa to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.Highlights► A high-throughput colorimetric assay to measure the activity of GAD. ► We monitor the absorbance change of bromocresol green at 620 nm. ► A pH difference of 2.5 units does not prevent this method from application. ► This method allows the determination of reaction kinetic parameters. ► Improvements in enzyme activity can be detected with a low coefficient of variance.