Inhibition of Large Neutral Amino Acid Transporters Suppresses Kynurenic Acid Production Via Inhibition of Kynurenine Uptake in Rodent Brain

The tryptophan metabolite, kynurenic acid (KYNA), is a preferential antagonist of the α7 nicotinic acetylcholine receptor and N-methyl-d-aspartic acid receptor at endogenous brain concentrations. Recent studies have suggested that increases of brain KYNA levels are involved in psychiatric disorders such as schizophrenia and depression, and regulation of KYNA production has become a new target for treatment of these diseases. Kynurenine (KYN), the immediate precursor of KYNA, is transported into astrocytes via large neutral amino acid transporters (LATs). In the present study, the effect of LATs regulation on KYN uptake and KYNA production was investigated in vitro and in vivo using an LATs inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). In the in vitro study, cortical slices of rat brain were incubated with a physiological concentration of KYN and 3 µmol/L–3 mmol/L BCH. BCH inhibited KYNA production and KYN uptake in a dose-dependent manner, and their IC₅₀ values were 90.7 and 97.4 µmol/L, respectively. In the in vivo study, mice were administered KYN (50 mg/kg BW) orally and BCH (200 mg/kg BW) intravenously. Administration of KYN increased brain KYN and KYNA levels compared with the mice treated with vehicle, whereas additional administration of BCH suppressed KYN-induced elevations in KYN and KYNA levels to 50 and 70 % in the brain. These results suggest that inhibition of LATs prevented the increase of KYNA production via blockade of KYN uptake in the brain in vitro and in vivo. LATs can be a target to modulate brain function by regulation of KYNA production in the brain.     

Engineering an ATP-dependent <Emphasis Type="SmallCaps">d</Emphasis>-Ala:<Emphasis Type="SmallCaps">d</Emphasis>-Ala ligase for synthesizing amino acid amides from amino acids

We successfully engineered a new enzyme that catalyzes the formation of d-Ala amide (d-AlaNH₂) from d-Ala by modifying ATP-dependent d-Ala:d-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of d-Ala-d-Ala from two molecules of d-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second d-Ala of d-Ala-d-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for d-AlaNH₂ production. The S293E variant, which was selected as the best enzyme for d-AlaNH₂ production, exhibited an optimal activity at pH 9.0 and 40 °C for d-AlaNH₂ production. The apparent K _m values of this variant for d-Ala and NH₃ were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of d-AlaNH₂ from 10 and 50 mM d-Ala and 3 M NH₄Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.     

An efficient biocatalytic synthesis of imidazole-4-acetic acid

Objective

To develop a new and efficient biocatalytic synthesis method of imidazole-4-acetic acid (IAA) from l-histidine (l-His).

Results

l-His was converted to imidazole-4-pyruvic acid (IPA) by an Escherichia coli whole-cell biocatalyst expressing membrane-bound l-amino acid deaminase (ml-AAD) from Proteus vulgaris firstly. The obtained IPA was subsequently decarboxylated to IAA under the action of H₂O₂. Under optimum conditions, 34.97 mM IAA can be produced from 50 mM l-His, with a yield of 69.9%.

Conclusions

Compared to the traditional chemical synthesis, this biocatalytic method for IAA production is not only environmentally friendly, but also more cost effective, thus being promising for industrial IAA production.

     

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [<Superscript>3</Superscript>H]D-Aspartate Release from Bovine Isolated Retinae

We investigated the pharmacological actions of a slow-releasing H₂S donor, GYY 4137; a substrate for the biosynthesis of H₂S, l-cysteine and its precursor, N-acetylcysteine on potassium (K⁺; 50 mM)-evoked [³H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K⁺-evoked [³H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H₂S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K⁺-evoked [³H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of K_ATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K⁺-induced [³H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K⁺-evoked [³H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K⁺-evoked [³H]D-aspartate release. We conclude that both donors and substrates for H₂S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of K_ATP channels and NO synthase.     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

Characterization of aspartate kinase and homoserine dehydrogenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> IWJ001 and systematic investigation of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine biosynthesis

Previously we have characterized a threonine dehydratase mutant TD^F383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS^{P176S, D426E, L575W} (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK^A279T (encoded by lysC1) and a homoserine dehydrogenase mutant HD^G378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK^A279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD^G378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD^F383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD^G378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.     

Engineering rTCA pathway and C4-dicarboxylate transporter for <Emphasis Type="SmallCaps">l</Emphasis>-malic acid production

l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L^?1, 0.30 g g^?1, and 0.32 g L^?1 h^?1 in the resulting strain W4209 with CaCO₃ as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L^?1, 0.31 g g^?1, and 0.32 g L^?1 h^?1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.     

Kynurenic acid inhibits colon cancer proliferation in vitro: effects on signaling pathways

Kynurenic acid (KYNA), a tryptophan metabolite, inhibits proliferation of several cancer cell lines including colon cancer, renal cancer and glioblastoma cells. Previous studies reported that inhibitory properties of KYNA may be related to interactions of KYNA with cell cycle regulators and signaling proteins. However, the exact molecular interaction of KYNA with signaling pathways in colon cancer cells has not been studied to date. The molecular mechanism of KYNA activity towards colon cancer cells may be of great importance taking into consideration that KYNA is present in several tissues and physiological fluids, including gastrointestinal tract, and it is also present in various products of human diet. In this study, the inhibitory effect of KYNA on activation of phosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways in colon adenocarcinoma HT-29 cells was revealed. KYNA decreased phosphorylation of Akt, ERK 1/2 and p38 kinases in HT-29 cells. Interestingly, the study revealed also unexpected effect of KYNA on Wnt pathway in HT-29 cells. KYNA in millimolar concentrations increased protein expression of β-catenin. However, the nuclear translocation of β-catenin in HT-29 cells exposed to KYNA was not observed. Moreover, KYNA 1 mM increased antiproliferative properties of inhibitors of signaling pathways: wortmannin, PD98059, SB202190 and IWR-1. Taking into consideration these results, KYNA may be seen as a potential chemopreventive agent in colon cancer or supportive agent in standard cancer chemotherapy. However, the interactions between KYNA, Wnt signaling pathway and β-catenin need further studies to exclude potential effect of KYNA on colon carcinogenesis.     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

     

Increased production of plumbagin in <Emphasis Type="Italic">Plumbago indica</Emphasis> root cultures by biotic and abiotic elicitors

Objectives

To evaluate the effects of 12 biotic and abiotic elicitors for increasing the production of plumbagin in Plumbago indica root cultures.

Results

Most elicitors showed minimal effects on the root dry weight, except for 250 mg chitosan l^?1 and 10 mM l-alanine that markedly decreased root biomass by about 40 % compared to the untreated root cultures (5 g l^?1). Treatments with 100 µM AgNO₃ significantly increased intracellular plumbagin production by up to 7.6 mg g^?1 DW that was 4-fold more than the untreated root cultures (1.9 mg g^?1 DW). In contrast, treatments with 150 mg chitosan l^?1, 5 mM l-alanine, and 50 µM 1-naphthol significantly enhanced the extracellular secretion of plumbagin by up to 10.6, 6.9, and 5.7 mg g^?1 DW, respectively, and increased the overall production of plumbagin by up to 12.5, 12.5, and 9.4 mg g^?1 DW, respectively.

Conclusions

Chitosan (150 mg l^?1), l-alanine (5 mM), and 1-naphthol (50 µM) were the best elicitors to enhance plumbagin production in P. indica root cultures.

     

Highly efficient production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from chicory-derived inulin by <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis>

Inulin is a readily available feedstock for cost-effective production of biochemicals. To date, several studies have explored the production of bioethanol, high-fructose syrup and fructooligosaccharide, but there are no studies regarding the production of d-lactic acid using inulin as a carbon source. In the present study, chicory-derived inulin was used for d-lactic acid biosynthesis by Lactobacillus bulgaricus CGMCC 1.6970. Compared with separate hydrolysis and fermentation processes, simultaneous saccharification and fermentation (SSF) has demonstrated the best performance of d-lactic acid production. Because it prevents fructose inhibition and promotes the complete hydrolysis of inulin, the highest d-lactic acid concentration (123.6 ± 0.9 g/L) with a yield of 97.9 % was obtained from 120 g/L inulin by SSF. Moreover, SSF by L. bulgaricus CGMCC 1.6970 offered another distinct advantage with respect to the higher optical purity of d-lactic acid (>99.9 %) and reduced number of residual sugars. The excellent performance of d-lactic acid production from inulin by SSF represents a high-yield method for d-lactic acid production from non-food grains.     

Increased productivity of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid and total turnover number of NAD<Superscript>+</Superscript>/NADH in a one-pot system through enhanced thermostability of <Emphasis Type="SmallCaps">l</Emphasis>-threonine deaminase

Objective

To strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).

Results

l-Threonine deaminase (l-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type l-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable l-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for l-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of l-ABA and total turnover number of NAD⁺/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type l-TD, respectively.

Conclusions

By using the engineered l-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of l-ABA and total turnover number of coenzyme.

     

Expression,purification, and characterization of a membrane-bound <Emphasis Type="SmallCaps">d</Emphasis>-amino acid dehydrogenase from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> JN458

Objectives

To characterize a novel membrane-bound d -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD).

Results

The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with d -alanine the most preferred substrate. The K _m and V _max values for d -alanine were 9 mM and 20 μmol min^?1 mg^?1, respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H₂O₂ and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs.

Conclusions

d-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.

     

Co-expression of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase and catalase in <Emphasis Type="Italic">Escherichia coli</Emphasis> to produce α-ketoglutaric acid by whole-cell biocatalyst

Objectives

To improve the production of α-ketoglutaric acid (α-KG) from l-glutamate by whole-cell biocatalysis.

Results

A novel and highly active l-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 ± 2.7 U mg^?1 at pH 6.0, 35 ^oC. The apparent K_m and V_max values of SmlGOX were 9.3 ± 0.5 mM and 159 ± 3 U mg^?1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H₂O₂, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of α-KG reached 77.4 g l^?1 with a conversion rate from l-glutamate of 98.5% after 12 h.

Conclusions

An efficient method for the production of α-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.

     

Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of <Emphasis Type="SmallCaps">D</Emphasis>-lactate in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale.     

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from <Emphasis Type="Italic">Citreicella</Emphasis> sp. SE45

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS–polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including l-alanine amide and l-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K _m and V _max values for d- and l-ACL were 5.3 and 2.17 mM, and 769 and 558 μmol/min.mg protein, respectively. Moreover, the turn over number (K _cat) and catalytic efficiency (K _cat/K _m ) of purified ACL racemase from Citreicella sp. SE45 using l-ACL as a substrate were 465 S^?1 and 214 S^?1mM^?1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.     

Molecular cloning and enzymological characterization of pyridoxal 5′-phosphate independent aspartate racemase from hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus</Emphasis><Emphasis Type="Italic">litoralis</Emphasis> DSM 5473

We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5′-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on l-cysteic acid and l-cysteine sulfinic acid in addition to d- and l-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. d-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells.     

Semi-industrial scale (30 m<Superscript>3</Superscript>) fed-batch fermentation for the production of <Emphasis Type="SmallCaps">d</Emphasis>-lactate by <Emphasis Type="Italic">Escherichia coli</Emphasis> strain HBUT-D15

d(?)-lactic acid is needed for manufacturing of stereo-complex poly-lactic acid polymer. Large scale d-lactic acid fermentation, however, has yet to be demonstrated. A genetically engineered Escherichia coli strain, HBUT-D, was adaptively evolved in a 15% calcium lactate medium for improved lactate tolerance. The resulting strain, HBUT-D15, was tested at a lab scale (7 L) by fed-batch fermentation with up to 200 g L^?1 of glucose, producing 184–191 g L^?1 of d-lactic acid, with a volumetric productivity of 4.38 g L^?1 h^?1, a yield of 92%, and an optical purity of 99.9%. The HBUT-D15 was then evaluated at a semi-industrial scale (30 m³) via fed-batch fermentation with up to 160 g L^?1 of glucose, producing 146–150 g L^?1 of d-lactic acid, with a volumetric productivity of 3.95–4.29 g L^?1 h^?1, a yield of 91–94%, and an optical purity of 99.8%. These results are comparable to that of current industrial scale l(+)-lactic acid fermentation.     

Cerebralcare Granule<Superscript>®</Superscript>, a Chinese Herb Compound Preparation,Attenuates <Emphasis Type="SmallCaps">d</Emphasis>-Galactose Induced Memory Impairment in Mice

Cerebralcare granule^® (CG) is a preparation of Traditional Chinese Medicine that widely used in China. It was approved by the China State Food and Drug Administration for treatment of headache and dizziness associated with cerebrovascular diseases. In the present study, we aimed to investigate whether CG had protective effect against d-galactose (gal)-induced memory impairment and to explore the mechanism of its action. d-gal was administered (100 mg/kg, subcutaneously) once daily for 8 weeks to induced memory deficit and neurotoxicity in the brain of aging mouse and CG (7.5, 15, and 30 g/kg) were simultaneously administered orally. The present study demonstrates that CG can alleviate aging in the mouse brain induced by d-gal through improving behavioral performance and reducing brain cell damage in the hippocampus. CG prevents aging mainly via suppression of oxidative stress response, such as decreasing NO and MDA levels, renewing activities of SOD, CAT, and GPx, as well as decreasing AChE activity in the brain of d-gal-treated mice. In addition, CG prevents aging through inhibiting NF-κB-mediated inflammatory response and caspase-3-medicated neurodegeneration in the brain of d-gal treated mice. Taken together, these data clearly demonstrates that subcutaneous injection of d-gal produced memory deficit, meanwhile CG can protect neuron from d-gal insults and improve memory ability.     

Enhanced biocatalytic production of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. B-3 with in situ product removal using ion-exchange resin

Bioconversion of dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of l-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated l-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve l-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of dl-ATC for the in situ removal of l-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for l-cysteine and low adsorption for dl-ATC, which is a favorable option. With in situ addition of 60 g L^?1 resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L^?1 of dl-ATC was converted to l-cysteine with 90.4 % of yield and 28.6 mmol L^?1 h^?1 of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of l-cysteine was improved by 2.27-fold using ISPR method.