Gamma-Aminobutyric Acid Production Using Immobilized Glutamate Decarboxylase Followed by Downstream Processing with Cation Exchange Chromatography

We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step.     

Expression, immobilization and enzymatic properties of glutamate decarboxylase fused to a cellulose-binding domain

Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA), was fused to the cellulose-binding domain (CBD) and a linker of Trichoderma harzianum endoglucanase II. To prevent proteolysis of the fusion protein, the native linker was replaced with a S(3)N(10) peptide known to be completely resistant to E. coli endopeptidase. The CBD-GAD expressed in E. coli was successfully immobilized on Avicel, a crystalline cellulose, with binding capacity of 33 ± 2 nmol(CBD-GAD)/g(Avicel) and the immobilized enzymes retained 60% of their initial activities after 10 uses. The results of this report provide a feasible alternative to produce GABA using immobilized GAD through fusion to CBD.     

Overexpression of Glutamate Decarboxylase in Transgenic Tobacco Plants Deters Feeding by Phytophagous Insect Larvae

Gamma-aminobutyrate (GABA) is a ubiquitous four-carbon, non-protein amino acid synthesized by glutamate decarboxylase. Previous research suggests that the endogenous synthesis of GABA, a naturally occurring inhibitory neurotransmitter at neuromuscular junctions, serves as a plant resistance mechanism against invertebrate pests. In this study, two homozygous transgenic tobacco lines constitutively overexpressing a single copy of a full-length chimeric glutamate decarboxylase cDNA and possessing enhanced capacity for GABA accumulation (GAD plants), a homozygous transgenic line lacking the gene insert, and wild-type tobacco were employed. Tobacco budworm larvae were presented with plantattached wild type and transgenic leaves for 4 hr in a feeding preference study. Larvae consumed six to twelve times more leaf tissue from wild-type plants than from GAD plants. These results suggest that leaf GABA accumulation, which is known to occur in response to insect larval walking and feeding, represents a rapidly deployed localresistance mechanism.     

Antiepileptic Potential of Matrine via Regulation the Levels of Gamma-Aminobutyric Acid and Glutamic Acid in the Brain

Our present study aimed to determine the antiepileptic activity of matrine, and explore the possible molecular mechanism. To evaluate the antiepileptic activity of matrine, seizures in mice induced by PTZ and MES were established, then the pentobarbital sodium-induced anaesthetizing time and locomotor activity tests in mice were also carried out. For the molecular mechanism investigations, contents of aspartic acid (Asp), gamma-aminobutyric acid (GABA), glutamic acid (Glu), glycine (Gly) in seizures mice were determined; then, the chronic seizures rats induced by PTZ were prepared, and western blotting was used to determine the expressions of GAD 65, GABA_A and GABA_B in the brains. In the results, matrine showed significant antiepileptic effects on seizures mice induced by MES and PTZ. Moreover, the pentobarbital sodium-induced anaesthetizing time and locomotor activity tests were also demonstrated that matrine had obvious antiepileptic effects. Additionally, our results revealed that after treatment with matrine, contents of GABA can be elevated, and the contents of Glu were obviously decreased. Furthermore, western blotting revealed that the mechanism regarding the antiepileptic effect of may be related to the up-regulations of GAD 65 and GABA_A in the brain. Collectively, we suggested that matrine can be developed as an effective antiseptic drug.     

Profile of miR-23 Expression and Possible Role in Regulation of Glutamic Acid Decarboxylase during Postnatal Retinal Development

As neurotransmitter, GABA is fundamental for physiological processes in the developing retina. Its synthesis enzymes are present during retinal development, although the molecular regulatory mechanisms behind the changes in expression are not entirely understood. In this study, we revealed the expression patterns of glutamic acid decarboxylase 67(GAD67) and its coding gene (GAD1) and its potential miRNA-dependent regulation during the first three postnatal weeks in rat retina. To gain insight into the molecular mechanisms, miRNA-sequencing supported by RT-qPCR and in situ hybridization were carried out. GAD1 expression shows an increasing tendency, peaking at P15. From the in silico-predicted GAD1 targeting miRNAs, only miR-23 showed similar expression patterns, which is a known regulator of GAD1 expression. For further investigation, we made an in situ hybridization investigation where both GAD67 and miR-23 also showed lower expression before P7, with the intensity of expression gradually increasing until P21. Horizontal cells at P7, amacrine cells at P15 and P21, and some cells in the ganglion cell layer at several time points were double labelled with miR-23 and GAD67. Our results highlight the complexity of these regulatory networks and the possible role of miR-23 in the regulation of GABA synthesizing enzyme expression during postnatal retina development.     

Amidination of lipase with hydrophobic imidoesters

Lipase fromCandida rugosa was chemically modified by amidination with imidoester hydrochlorides of different hydrophobicity. The modified enzyme showed a higher ester synthesis activity but a lower ester hydrolysis activity compared with the native enzyme. The maximum specific activity of the modified enzyme depended on its degree of derivatization. Benzene was found to be the best solvent for the synthesis reaction. The optimal temperature for the reaction was not affected by modification of the lipase. The modified lipase was more thermostable and solvent-stable than the native enzyme. When fatty acids of different carbon chainlength were tested as substrates in the synthesis of esters with the modified lipase, the highest activity was observed with myristic acid and propanol.     

Ultrasonic extraction of ferulic acid from Angelica sinensis

In this paper, the extraction of ferulic acid, a pharmacologically active ingredient from the root of Angelica sinensis with ultrasonic extraction was investigated. Percolation and supercritical fluid extraction (SFE) were also employed to make comparisons with ultrasonic extraction. Three variables, which including the concentration of solvent, the ratio of solvent volume to sample (mL/g), and extraction time, were found to have great influence on ultrasonic extraction. The optimum extraction conditions were using pure ethanol with a ratio of solvent volume to sample 8:1 (mL/g) and extraction time of 30 min. Under the optimum extraction conditions, the extraction yield could reach 6.5% mass fraction, which was higher than that of SFE process with ethanol as co‐solvent and nearly a content of ferulic acid 1.0%; both the yield and the content of ferulic acid were higher than those obtained by percolation. Moreover, the time of ultrasonic extraction was significantly shortened. Overall, Ultrasonic extraction was shown to be highly efficient in the extraction of ferulic acid from Angelica sinensis.     

Strategies of pH control and glucose‐fed batch fermentation for production of succinic acid by Actinobacillus succinogenes CGMCC1593

BACKGROUND: Succinic acid is an important precursor of numerous products, including pharmaceuticals, feed additives, green solvents, and biodegradable polymers. In this work, strategies of pH control and glucose‐fed batch fermentation for producing succinic acid using Actinobacillus succinogenes CGMCC1593 were carefully optimized. RESULTS: The production of succinic acid was stable within the pH range 6.0–7.2. Both cell growth and succinic acid production were inhibited by high concentrations of sodium and calcium ions, while there was no significant inhibition by magnesium ions. With an initial glucose concentration of 25 g L^?1, and glucose concentration was maintained between 10 and 15 g L^?1 during the course of fed batch fermentation, succinic acid concentration, productivity and yield were 60.2 g L^?1, 1.3 g L^?1 h^?1 and 75.1%, respectively. CONCLUSION: Of all the neutralization reagents used for pH control of A. succinogenes CGMCC1593, solid MgCO₃ was the most satisfactory. With increase of initial glucose concentration, the time course showed a longer growth lag period and the maximum biomass declined, while more carbon was diverted to succinate synthesis. The results obtained in this study should be helpful for the design of a highly efficient succinic acid production process. Copyright © 2008 Society of Chemical Industry     

Characterization of a novel glutamate decarboxylase from Streptococcus salivarius ssp. thermophilus Y2

BACKGROUND: γ‐Aminobutyric acid with several well‐known physiological functions is biosynthesized via the irreversible α‐decarboxylation of L ‐glutamate catalysed by glutamate decarboxylase (GAD). Although Streptococcus salivarius ssp. thermophilus has been widely applied to the dairy, the characterization of its GAD has not been reported. In this paper, the purification and the characterization of S. salivarius ssp. thermophilus GAD were investigated. RESULTS: GAD was purified 22‐fold from crude protein extracts with a yield of 7.8% in five steps. The final preparation gave a single band on SDS‐PAGE. The molecular weight of GAD determined by SDS‐PAGE and gel filtration was 46.9 kDa and 103.6 kDa, respectively, indicating that the enzyme exists as a dimmer of homological subunits. The optimum temperature and pH of GAD was 55 °C and pH 4.0, respectively. The enzyme reacted only with L ‐glutamate among 19 α‐amino acids with apparent K_m at 2.3 mmol L^?1 and did not react with D ‐glutamic acid. Activity of the enzyme could significantly be activated by 5 mmol L^?1 of BaCl₂ and inhibited by FeSO₄, ZnSO₄, CuSO₄, MnSO₄, Na₂SO₄, AgNO₃, CoCl₂, LiCl and KCl, respectively. The N‐terminal amino acid sequence of GAD was NH₂‐MNEKLFREI. CONCLUSION: Both the characterization and the deduced amino sequence (ABI31651) showed the purified enzyme was a novel GAD. Copyright © 2008 Society of Chemical Industry     

Refolding of recombinant N-acetyl-d-glucosamine 2-epimerase by a fed-batch process

N-Acetyl-d-glucosamine 2-epimerase is one of the key enzymes for the enzymatic synthesis of N-acetylneuraminic acid, a sialic acid and a critical precursor for the synthesis of some antiviral agents. Overexpression of the recombinant epimerase in Escherichia coli led to the formation of protein inclusion bodies. Refolding of guanidine HCl-solubilized protein by direct dilution resulted in the formation of soluble oligomers, mediated probably by hydrophobic interactions. The extent of aggregation of protein subunits into inactive oligomers could be efficiently reduced by employing fed-batch refolding process, in which the solubilized proteins were added continuously at a pre-determined rate. The yields of soluble proteins decreased with the feeding rates. The addition of glutathione into refolding buffer at certain stage of the refolding process could enhance the yield of soluble proteins more than two-fold, possibly by resolving the inadvertently formed disulfide bridges among the protein subunits that contain 10 cysteine residues each. Folding aids such as l-arginine and glycerol were found effective in increasing the yield of soluble proteins and the specific activity of the refolded proteins. Under the optimal condition, a specific activity of 0.47 IU/mg was obtained with an activity recovery yield of ca. 30%. The specific activity of the refolded proteins was significantly lower than that of the native protein, 1.23 IU/mg, indicating that more information concerning the 3D structure of native N-acetyl-d-glucosamine 2-epimerase and the role of its cofactor, ATP, for catalytic activity is needed for the development of a more efficient refolding process.     

Efficient Synthesis of Phenylacetate and 2-Phenylethanol by Modular Cascade Biocatalysis

The green and sustainable synthesis of chemicals from renewable feedstocks by a biotransformation approach has gained increasing attention in recent years. In this work, we developed enzymatic cascades to efficiently convert l -phenylalanine into 2-phenylethanol (2-PE) and phenylacetic acid (PAA), l -tyrosine into tyrosol (p-hydroxyphenylethanol, p-HPE) and p-hydroxyphenylacetic acid (p-HPAA). The enzymatic cascade was cast into an aromatic aldehyde formation module, followed by an aldehyde reduction module, or aldehyde oxidation module, to achieve one-pot biotransformation by using recombinant Escherichia coli. Biotransformation of 50 mM l -Phe produced 6.76 g/L PAA with more than 99 % conversion and 5.95 g/L of 2-PE with 97 % conversion. The bioconversion efficiencies of p-HPAA and p-HPE from l -Tyr reached to 88 and 94 %, respectively. In addition, m-fluoro-phenylalanine was further employed as an unnatural aromatic amino acid substrate to obtain m-fluoro-phenylacetic acid; >96 % conversion was achieved. Our results thus demonstrated high-yielding and potential industrial synthesis of above aromatic compounds by one-pot cascade biocatalysis.     

Impact of Dysfunctional Feed-Forward Inhibition on Glutamate Decarboxylase Isoforms and γ-Aminobutyric Acid Transporters

Absence seizures are associated with generalised synchronous 2.5–4 Hz spike-wave discharges causing brief and sudden alteration of awareness during childhood, which is known as childhood absence epilepsy (CAE). CAE is also associated with impaired learning, psychosocial challenges, and physical danger. Absence seizures arise from disturbances within the cortico-thalamocortical (CTC) network, including dysfunctional feed-forward inhibition (FFI); however, the precise mechanisms remain unclear. In epileptic stargazers, a genetic mouse model of CAE with chronic seizures, levels of γ-aminobutyric acid (GABA), and expression of GABA receptors are altered within the CTC network, implicating altered GABAergic transmission in absence seizures. However, the expression of GABA synthesising enzymes (GAD65 and GAD67) and GABA transporters (GAT-1 and 3) have not yet been characterised within absence seizure models. We found a specific upregulation of GAD65 in the somatosensory cortex but not the thalamus of epileptic stargazer mice. No differences were detected in GAD67 and GAT-3 levels in the thalamus or somatosensory cortex. Then, we assessed if GAD65 upregulation also occurred in Gi-DREADD mice exhibiting acute absence seizures, but we found no change in the expression profiles of GAD65/67 or GAT-3. Thus, the upregulation of GAD65 in stargazers may be a compensatory mechanism in response to long-term dysfunctional FFI and chronic absence seizures.     

离子束注入改造壬二酸生产菌株的初步研究

采用离子束注入的方法对产壬二酸菌种进行诱变筛选,获得一株壬二酸产率达到35.81g.L-1的菌株,壬二酸产率较出发菌株提高了74.17%。     

Fatty Acid Profile and Bioactive Compound Extraction in Purple Viper's Bugloss Seed Oil Extracted with Green Solvents

Oil extraction from seeds of purple viper's bugloss (Echium plantagineum) was carried out using different solvents (chloroform:methanol, n-hexane, ethanol, 2-propanol and ethyl acetate) at room temperature and also using Randall extraction. Extraction yields were calculated and oils were analyzed in terms of fatty acid profiles and distribution among lipid classes, total polyphenol content, oxygen radical absorbance capacity (ORAC) and phytosterol content. No considerable differences were found on fatty acid profiles and distribution in oils regardless of the solvent and temperature used for the extraction. However, ethanol combined with Randall extraction (85 °C for 1 hour) offered the best results in terms of total polyphenol content (20.9 mg GAE/100 g oil), ORAC (468.0 μmol TE/100 g oil), and phytosterol amount (437.2 mg identified phytosterols/100 g oil) among all assayed extraction methods. A higher extraction temperature led to significantly higher concentrations of bioactive compounds and ORAC values in the oil when ethanol or 2-propanol were used as extracting solvent, but that was not the case using n-hexane except for the concentrations of β-sitosterol and stigmasterol, which were significantly higher using Randall extraction than room temperature extraction with n-hexane. Ethanol is classified as a “green solvent,” and it could be considered a suitable option to produce oil from E. plantagineum seeds with a higher antioxidant capacity and bioactive compound concentration than the current commercial oil, which is usually extracted with n-hexane.     

Permeabilization of Escherichia coli with ampicillin for a whole cell biocatalyst with enhanced glutamate decarboxylase activity

The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability barrier against the diffusion of substrates and products. Although common chemical or physical permeabilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, as well as impede large scale processing. To increase membrane permeability and cellbound glutamate decarboxylase(GAD) activity of recombinant Escherichia coli(BL21(DE3)-p ET28a-gad B) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or surfactants to the culture media. Ampicillin was the most effective at improving cell-bound GAD activity of the BL21(DE3)-p ET28a-gad B, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 μg·ml-1. Using this concentration,the cell biomass did decrease by 40.58%, but the cell-bound GAD activity of BL21(DE3)-p ET28a-gad B and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment of BL21(DE3)-p ET28a-gad B cells with 5 μg·ml-1ampicillin resulted in structural changes to the cell envelope,but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol(PVA), alginate, and boric acid, the transformation rate of γ-aminobutyric acid(GABA) at the 10 th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after30 days of storage at 4 °C.     

Ultrasonic extraction of ferulic acid from Ligusticum chuanxiong

The extraction of ferulic acid, a pharmacologically active ingredient from the root of Ligusticum chuanxiong, with ultrasonic extraction was investigated. Percolation and supercritical fluid extraction (SFE) were employed to make comparisons with ultrasonic extraction. Three variables, which included the concentration of solvent, the ratio of solvent volume/sample (mL/g), and extraction time, were found to have a great influence on ultrasonic extraction. The optimum extraction was with pure ethanol with a solvent volume/sample ratio 8:1 (mL/g) and extraction time of 30 min. Under the optimum extraction conditions, the extraction yield could reach 8.8% which was higher than that using SFE with ethanol as co-solvent and a content of ferulic acid of 0.7%; both the yield and the content were higher than those obtained by percolation. Ultrasonic extraction significantly shortened the time required for the extraction process. Overall, ultrasonic extraction was shown to be highly efficient in the extraction of ferulic acid from Ligusticum chuanxiong.     

Well‐defined,environment‐friendly synthesis of polypeptides based on phosgene‐free transformation of amino acids into urethane derivatives and their applications

In this study, both naturally occurring and artificial amino acids were successfully transformed into the corresponding urethane derivatives using diphenyl carbonate. The urethanes thus prepared could be efficiently cyclized into amino acid N‐carboxyanhydrides (NCAs) without the requirement of phosgene. In addition, the presence of primary amines converted the urethane derivatives into NCAs and initiated the ring‐opening polymerization of the in situ formed NCAs, allowing for the well‐defined synthesis of polypeptides. These polypeptides contained initiating ends functionalized by an amine‐derived residue and propagating ends bearing the reactive amino group. By precise control of the structures of the polypeptides, various polypeptide conjugates such as block copolymers and graft copolymers were successfully synthesized as designed, and their applications in antifouling coatings against proteins, drug delivery systems and biosensors were demonstrated. © 2019 Society of Chemical Industry     

Higher Increase in Plasma DHA in Females Compared to Males Following EPA Supplementation May Be Influenced by a Polymorphism in ELOVL2: An Exploratory Study

Young adult females have higher blood docosahexaenoic acid (DHA), 22:6n-3 levels than males, and this is believed to be due to higher DHA synthesis rates, although DHA may also accumulate due to a longer half-life or a combination of both. However, sex differences in blood fatty acid responses to eicosapentaenoic acid (EPA), 20:5n-3 or DHA supplementation have not been fully investigated. In this exploratory analysis, females and males (n = 14–15 per group) were supplemented with 3 g/day EPA, 3 g/day DHA, or olive oil control for 12 weeks. Plasma was analyzed for sex effects at baseline and changes following 12 weeks' supplementation for fatty acid levels and carbon-13 signature (δ¹³C). Following EPA supplementation, the increase in plasma DHA in females (+23.8 ± 11.8, nmol/mL ± SEM) was higher than males (−13.8 ± 9.2, p < 0.01). The increase in plasma δ¹³C-DHA of females (+2.79 ± 0.31, milliUrey (mUr ± SEM) compared with males (+1.88 ± 0.44) did not reach statistical significance (p = 0.10). The sex effect appears driven largely by increased plasma DHA in the AA genotype of females (+58.8 ± 11.5, nmol/mL ± SEM, n = 5) compared to GA + GG in females (+4.34 ± 13.5, n = 9) and AA in males (−29.1 ± 17.2, n = 6) for rs953413 in the ELOVL2 gene (p < 0.001). In conclusion, EPA supplementation increases plasma DHA levels in females compared to males, which may be dependent on the AA genotype for rs953413 in ELOVL2.     

粪肠球菌HX-3-6产γ-氨基丁酸发酵条件的优化

目的优化粪肠球菌HX-3-6产γ-氨基丁酸(γ-Aminobutyric acid,GABA)的发酵条件,提高GABA的产量。方法通过单因素试验和正交试验对产GABA的粪肠球菌HX-3-6发酵条件进行优化,采用Plackett-Burman(PB)试验设计法筛选产GABA的培养基主要影响因素,应用Box-Behnken设计及响应面分析对影响发酵产GABA的主要培养基因素进行优化;用最终优化的配方进行3次验证试验。结果最佳发酵条件为底物浓度30 g/L,起始pH 5.5,发酵时间72 h,温度35℃;PB法筛选出了培养基中有显著效应的4个因素为MnSO4﹒H2O、NaCl、柠檬酸三胺和葡萄糖,经Box-Behnken设计及响应面分析,确定该4个主要影响因素的最佳浓度分别为0.100 785、0.224 459、0.215 355及12.335 95 g/L。3次验证GABA的产量平均可达16.027 g/L,比优化前(11.006 g/L)提高了45.62%。结论优化的粪肠球菌HX-3-6发酵条件和培养基可显著提高GABA产量,为其工业化生产奠定了基础。     

QUANTIFICATION OF GALLIC ACID AND ELLAGIC ACID FROM THE SEED OF CORNUS OFFICINALIS BY UHPLC METHOD AND THEIR ANTIOXIDANT ACTIVITY

Gallic acid and ellagic acid have been identified in the seed of Cornus officinalis by the use of an ultrahigh performance liquid chromatography (UHPLC) method coupled with a diode array detector (DAD). The water extract of C. officinalis seed contained the highest gallic acid content (3.03 ± 0.10 mg/g seed), which was followed by aqueous methanol extract (2.43 ± 0.10 mg/g seed) and aqueous ethanol extract (1.53 ± 0.25 mg/g seed). But a higher concentration (12.32 ± 0.45 mg/g seed) of ellagic acid was obtained from extraction with aqueous methanol than with aqueous ethanol (11.03 ± 0.42 mg/g seed) and water (7.28 ± 0.16 mg/g seed). After heat treatment and acid hydrolysis, C. officinalis seed had higher concentrations of gallic acid and ellagic acid, contributing to more potent antioxidant activity. The results demonstrated a rich source of gallic acid and ellagic acid in C. officinalis seed, which might provide a novel source of these natural antioxidants.