RESUMO
Astaxanthin has 550 times more antioxidant activity than vitamin E, so it can scavenge free radicals in vivo and improve body immunity. However, the poor stability of astaxanthin becomes a bottleneck problem that limits its application. Herein, Haematococcus pluvialis (H. pluvialis) as a raw material was used to extract astaxanthin, and the optimal extraction conditions included the extraction solvent (EA:EtOH = 1:6, v/v), extraction temperature (60 °C), and extraction time (70 min). The extracted astaxanthin was then loaded using lecithin to form corresponding liposomes via the ethanol injection method. The results showed that the particle size and zeta potential of the prepared liposomes were 105.8 ± 1.2 nm and -38.0 ± 1.7 mV, respectively, and the encapsulation efficiency of astaxanthin in liposomes was 88.83%. More importantly, the stability of astaxanthin was significantly improved after being embedded in the prepared liposomes.
Assuntos
Lipossomos , Xantofilas , Xantofilas/isolamento & purificação , Xantofilas/química , Lipossomos/química , Tamanho da Partícula , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Clorófitas/química , Clorofíceas/químicaRESUMO
BACKGROUND: Ophiorrhiza pumila (Rubiaceae) is capable of producing camptothecin (CPT), one monoterpene indole alkaloid extensively employed in the treatment of multiple cancers. Transcription factors (TFs) GATA are a group of transcription regulators involved in plant development and metabolism, and show the feature of binding to the GATA motif within the promoters of target genes. However, GATA TFs have not been characterized in O. pumila. RESULT: In this study, a total of 18 GATA genes classified into four subfamilies were identified, which randomly distributed on 11 chromosomes of O. pumila. Synteny analysis of GATA genes between O. pumila and other plant species such as Arabidopsis thaliana, Oryza sativa, Glycine max, Solanum lycopersicum, Vitis vinifera, and Catharanthus roseus genomes were analyzed. Tissue expression pattern revealed that OpGATA1 and OpGATA18 were found to be correlated with ASA, MK, CPR and GPPS, which were highly expressed in leaves. OpGATA7, showed high expression in roots as most of the CPT biosynthetic pathway genes did, suggesting that these OpGATAs may be potential candidates regulating CPT biosynthesis in O. pumila. CONCLUSIONS: In this study, we systematically analyzed the OpGATA TFs, and provided insights into the involvement of OpGATA TFs from O. pumila in CPT biosynthesis.
Assuntos
Camptotecina , Rubiaceae , Vias Biossintéticas , Raízes de Plantas/genética , Rubiaceae/genética , Rubiaceae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Phenolic acids and tanshinones are main bioactive compounds produced in Salvia miltiorrhiza widely used in treatment of cardiovascular diseases, which could be promoted by abscisic acid elicitation. However, the regulation mechanism remained to be elucidated. An ABA-inducible IIa WRKY transcription factor (TF) named SmWRKY34 exhibiting high homology with AtWRKY40 was isolated. SmWRKY34 exhibited a negative role on phenolic acids and tanshinones by directly regulating SmRAS and SmGGPPS. Moreover, ABA-responsive bZIP TF member named SmbZIP3 expressing significantly in SmWRKY34 transcriptome was screened. SmWRKY34 showed a negative regulatory role on SmbZIP3. SmbZIP3 acted as a positive regulator in the biosynthesis of phenolic acids and tanshinones by targeting SmTAT and two tanshinone-promoting TFs SmERF128 and SmMYB9b. Taken together, we identify a new module WRKY34-bZIP3 involved in ABA signaling that manipulates phenolic acid and tanshinone accumulation, shedding new insights in metabolic engineering application in S. miltiorrhiza.
Assuntos
Salvia miltiorrhiza , Abietanos , Regulação da Expressão Gênica de Plantas , Hidroxibenzoatos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Salvia miltiorrhiza/genética , Salvia miltiorrhiza/metabolismoRESUMO
A new type of vapreotide-templated Ag/Au bimetallic nanoshells (Vap@Ag/AuNSs) were successfully designed and fabricated based on polypeptide-directed mineralization and hierarchical self-assembly mechanisms under mild synthetic conditions. The nanoparticles with polypeptides serving as a core and coated Ag/Au bimetallic nanoshells exhibit diverse advantages, such as excellent biocompatibility, tumor targeting and low-cost. The Vap@Ag/AuNSs share excellent dispersibility, uniform size (120 nm) and a positive zeta potential (36.74 ± 4.49 mV), hence they easily accumulate in negatively charged tumor tissue. The results of thermal imaging, temperature variation assays and photothermal conversion efficiency (41.6%) indicated that Vap@Ag/AuNSs have excellent photothermal conversion capability. Based on their photothermal response, as well as biocompatibility determined by MTT assay, the prominent anti-tumor effects of Vap@Ag/AuNSs have been verified by fluorescein diacetate staining. Therefore, Vap@Ag/AuNSs are novel and promising candidates for photothermal tumor therapy.
Assuntos
Antineoplásicos/química , Ouro/química , Minerais/química , Nanoconchas/química , Prata/química , Somatostatina/análogos & derivados , Materiais Biocompatíveis/química , Somatostatina/química , TemperaturaRESUMO
In the past decades, the microbial fuel cell (MFC) technology has caught the attention of the scientific community for its potential in transforming petroleum hydrocarbon (PHC) pollutants directly into electricity through microbial catalyzed anodic. The microbe was one of the most important factors that both influence MFCs and PHC degradation. Here we aimed to identify new microbes to expand the list of microbial species which are both electrogenic and diesel hydrocarbon degrading. In this text, we depicted a strain of microbe named E2, isolated from on the anode surface of MFC, and using diesel as sole carbon source. E2 exhibited electrochemical activity in cyclic voltammetry curve, implicating that it had electrogenic ability. E2 degraded about 50% diesel (3.26â¯g/L) in maximum during 8 days. Pyrosequencing of 16S rRNA gene of E2 revealed E2 was a sub-strain of Vibrio. Corresponding to salt and alkali tolerant properties of vibrio, the optimal condition for E2 in degrading diesel was 3%-4% in salinity, and pH 8-9 in mineral medium. Collectively, as a member of Gammaproteobacteria class, E2 was novel marine microbe both electricity generation and diesel degradation, which may attract its future application toward artificial microbial community construction in MFC in promoting the PHC pollution removal.
Assuntos
Fontes de Energia Bioelétrica , Poluentes Ambientais , Petróleo , Biodegradação Ambiental , Eletrodos , Hidrocarbonetos , RNA Ribossômico 16SRESUMO
We conducted industrial scale γ-polyglutamic acid (γ-PGA) production by Bacillus subtilis (B. subtilis) LX and modeled its microbial growth kinetics based on a logistic regression. We found that the use of a three-layer impeller including a lower semicircular disc impeller and two-layers of six-wide-leaf impellers were able to both increase γ-PGA yields and decrease fermentation time as compared with two-layer Rushton impellers. Indeed, our results revealed that the optimal γ-PGA yield (20.67 ± 2.19 g/L) was obtained after 40 hr in the impeller retrofitted fermenter, and this yield was 29.7% higher than that in Rushton impellers fixed fermenter. The microbial growth kinetics of B. subtilis LX in this system were established, and the model was consistent with the experimental data (R2 = 0.924) suggesting that it was suitable for describing the microbial growth kinetics underlying γ-PGA production on an industrial scale. In addition, biomass yield (Yx/s-glucose), γ-PGA yield (Yp/s-glucose), γ-PGA yield (Yp/s-glutamate), and the correlation between γ-PGA production and B. subtilis LX (Yp/x) were found to be 0.043, 0.133, 0.743, and 3.090 g/g, respectively, in the impeller retrofitted fermenter, as compared with 0.036, 0.103, 0.629, and 2.819 g/g, respectively, in the two-layer Rushton impeller fermenter.
Assuntos
Bacillus subtilis/metabolismo , Reatores Biológicos/microbiologia , Ácido Poliglutâmico/análogos & derivados , Biomassa , Fermentação , Cinética , Ácido Poliglutâmico/biossínteseRESUMO
The SnRK gene family is the chief component of plant stress resistance and metabolism through activating the phosphorylation of downstream proteins. S. miltiorrhiza is widely used for the treatment of cardiovascular diseases in Asian countries. However, information about the SnRK gene family of S. miltiorrhiza is not clear. The aim of this study is to comprehensively analyze the SnRK gene family of S. miltiorrhiza and its response to phytohormone. Here, 33 SmSnRK genes were identified and divided into three subfamilies (SmSnRK1, SmSnRK2 and SmSnRK3) according to phylogenetic analysis and domain. SmSnRK genes within same subgroup shared similar protein motif composition and were unevenly distributed on eight chromosomes of S. miltiorrhiza. Cis-acting element analysis showed that the promoter of SmSnRK genes was enriched with ABRE motifs. Expression pattern analysis revealed that SmSnRK genes were preferentially expressed in leaves and roots. Most SmSnRK genes were induced by ABA and MeJA treatment. Correlation analysis showed that SmSnRK3.15 and SmSnRK3.18 might positively regulate tanshinone biosynthesis; SmSnRK3.10 and SmSnRK3.12 might positively regulate salvianolic acid biosynthesis. RNAi-based silencing of SmSnRK2.6 down-regulated the biosynthesis of tanshinones and biosynthetic genes expression. An in vitro phosphorylation assay verified that SmSnRK2.2 interacted with and phosphorylated SmAREB1. These findings will provide a valuable basis for the functional characterization of SmSnRK genes and quality improvement of S. miltiorrhiza.
RESUMO
Phenolic acids are one of the major secondary metabolites accumulated in Salvia miltiorrhiza with various pharmacological activities. Moderate drought stress can promote the accumulation of phenolic acids in S. miltiorrhiza, while the mechanism remains unclear. Therefore, we performed transcriptome sequencing of S. miltiorrhiza under drought treatment. A total of 47,169 unigenes were successfully annotated in at least one of the six major databases. Key enzyme genes involved in the phenolic acid biosynthetic pathway, including SmPAL, SmC4H, Sm4CL, SmTAT, SmHPPR, SmRAS and SmCYP98A14, were induced. Unigenes annotated as laccase correlated with SmRAS and SmCYP98A14 were analyzed, and seven candidates that may be involved in the key step of SalB biosynthesis by RA were obtained. A total of 15 transcription factors significantly up-regulated at 2 h and 4 h potentially regulating phenolic acid biosynthesis were screened out. TRINITY_DN14213_c0_g1 (AP2/ERF) significantly transactivated the expression of SmC4H and SmRAS, suggesting its role in the regulation of phenolic acid biosynthesis. GO and KEGG enrichment analysis of differential expression genes showed that phenylpropanoid biosynthesis and plant hormone signal transduction were significantly higher. The ABA-dependent pathway is essential for resistance to drought and phenolic acid accumulation. Expression patterns in drought and ABA databases showed that four PYLs respond to both drought and ABA, and three potential SnRK2 family members were annotated and analyzed. The present study presented a comprehensive transcriptome analysis of S. miltiorrhiza affected by drought, which provides a rich source for understanding the molecular mechanism facing abiotic stress in S. miltiorrhiza.
RESUMO
Inaccessibility of deep-seated malignant cells in the central region of tumors and uncontrollable tumor recurrence represent a significant challenge for conventional synergistic cancer therapy. Herein, we designed a novel nanoplatform based on hierarchical drug release for deep cascade cancer therapy including localized photothermal therapy, systematic chemotherapy, and elicited immune responses. Methods: The first-step chemotherapy could be carried out by polydopamine (PDA) releasing doxorubicin (DOX) in the specific microenvironment of lysosomes (pH 5.5). The branched gold nanoshells and PDA converted the light to heat efficiently to accomplish the second-step photothermal therapy and collapsed biomimetic vesicles (BVs) to release paclitaxel (PTX), which promoted the third-step of chemotherapy and triggered immune responses. Results: After 10 days of treatment, there were no obvious residual tumors in tumor-bearing mice. Significantly, 10 days after stopping treatment, mice in the drug immune-therapeutic group showed little tumor recurrence (1.5 times) compared to substantial recurrence (20 times) in the conventional treatment group. Conclusion: The hierarchical drug release and cascade therapeutic modality enhance the penetration of drugs deep into the tumor tissue and effectively inhibit recurrence. This cascade therapeutic modality provides a novel approach for more effective cancer therapy.
Assuntos
Carcinoma/terapia , Sistemas de Liberação de Medicamentos , Tratamento Farmacológico/métodos , Hipertermia Induzida/métodos , Imunoterapia/métodos , Nanocompostos/administração & dosagem , Fototerapia/métodos , Animais , Antineoplásicos/administração & dosagem , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Liberação Controlada de Fármacos , Ouro/administração & dosagem , Células HeLa , Humanos , Fatores Imunológicos/administração & dosagem , Camundongos , Modelos Biológicos , Transplante de Neoplasias , Paclitaxel/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Transplante Heterólogo , Resultado do TratamentoRESUMO
In this study, an antitumor drug delivery system, gold nanoshell coated wedelolactone liposomes (AuNS-Wed-Lip), were designed and synthesized. In the drug delivery system, wedelolactone liposome and gold-nanoshell were linked by l-cysteine, which had been shown an effective nanocarrier for antitumor drug delivery, on-demand drug release, and phototherapy under near-infrared (NIR) light irradiation. It was capable of absorbing 780-850â¯nm NIR light and converting light energy to heat rapidly. The hyperthermia promoted wedelolactone release rapidly from the systems. The release amount of AuNS-Wed-Lip under NIR irradiation reached up to 97.34% over 8â¯h, achieving the on-demand drug release. Moreover, a high inhibition rate up to 95.73% for 143B tumor cells by AuNS-Wed-Lip upon laser irradiation at 808â¯nm was observed. The excellent inhibition efficacy was also displayed in vivo antitumor study with S180 tumor-bearing mice. The results demonstrated that AuNS-Wed-Lip, as an antitumor drug delivery system, achieved chemo-photothermal synergetic effect, which has great potential in cancer therapy.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Cumarínicos/química , Cumarínicos/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Ouro/química , Lipossomos/química , Nanoconchas/química , Animais , Camundongos , Fototerapia , Sarcoma/tratamento farmacológicoRESUMO
With the rapid development of photothermal therapy (PTT) in cancer treatment, it is necessary to obtain effective plasma-responsive tunable photothermal transducing agents. Inspired by the peptide-directed hierarchical mineralized Ag nanocages (Ag NCs), scientists designed a new duel-template cascade preparation method, and novel unique multi-branched gold nanoshells (BGSs) were successfully prepared under mild conditions using green strategy. The length, density and diameter of the branches were tuned, which led to the adjustment of the surface plasma response of the nanostructure. Because of the hierarchical structure and anisotropic surface, an obvious red shift of the local surface plasmon resonance spectrum was observed for the branched Au nanoshells. The excellent photothermal conversion efficiency (70.9%) and photo-induced heating responsive curves proved the superior photothermal conversion performance and photothermal stability of BGSs. The in vitro and in vivo results indicated that the heat generated by the intense NIR absorption of BGSs can selectively destroy cancer cells under laser irradiation. The nanostructures with ultrastrong absorption have promising prospects in tumor therapy.
Assuntos
Nanoconchas , Neoplasias/terapia , Fototerapia/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Ouro/química , Células HEK293 , Células HeLa , Humanos , Camundongos , Nanoconchas/química , Nanoconchas/uso terapêutico , Fenômenos Físicos , Prata/químicaRESUMO
A new glutamate-dependent γ-polyglutamic acid (γ-PGA) producer Z3 isolated from soil samples in Daxinganling forest region of China was identified, and its optimal medium components were investigated using response surface methodology. Strain Z3 was identified as Bacillus velezensis by physiology and biochemistry and 16S rDNA sequence analysis. This is the first report of glutamate-dependent B. velezensis with the ability to synthesize γ-PGA. Then, the optimum γ-PGA yield (5.58 g/L) was achieved with glutamate 86 g/L, glucose 36 g/L, yeast extract powder 5.5 g/L, and NaH2PO4 7.5 g/L. Furthermore, activities of enzymes participating in glutamate synthesis were assessed, and the results showed that lower ketoglutaric dehydrogenase activity (KGDH) and higher glutamate dehydrogenase activity (GDH) resulted in higher γ-PGA yield. Identification of glutamate-dependent γ-PGA producer named B. velezensis Z3 enriches microbiological resources with γ-PGA-producing capacity. B. velezensis optimization of nutrients and analysis of enzymes activities will not only help to increase γ-PGA productivity but also to understand the γ-PGA synthesis mechanism in B. velezensis Z3.
Assuntos
Bacillus/crescimento & desenvolvimento , Bacillus/metabolismo , Meios de Cultura/química , Ácido Poliglutâmico/metabolismo , Bacillus/genética , Bacillus/isolamento & purificação , Técnicas de Tipagem Bacteriana , China , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Florestas , Glutamato Desidrogenase/análise , Complexo Cetoglutarato Desidrogenase/análise , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Microbiologia do SoloRESUMO
Based on the self-assembly properties of vapreotide acetate (Vap), one kind of novel vapreotide acetategold nanoflowers (Vap-AuNFs) was fabricated for the first time by biomimetic mineralization method using Vap as a template. The Vap-AuNFs possessed anisotropic structure with a large absorption cross-section, which were face-centered cubic crystalline, exhibiting a remarkable monodisperse, narrow size (154â¯nm) distribution and good stability in aqueous solution. The apparent anisotropy of the gold nanostructure with high molar extinction coefficient can cause significantly higher plasmon absorption of Vap-AuNFs in the near infrared (NIR) region compared with Au nanoparticles (AuNPs), so the nanocomplex can induce remarkably enhanced photothermal conversion efficiency under NIR light irradiation. Breathtakingly, Vap-AuNFs exhibited superior biocompatibilities compared to AuNPs, as well as enhanced Hela cells lethality under NIR irradiation. This novel method was simple, low cost and green for the design and preparation of anisotropic gold nanoflowers with outstanding NIR laser-induced local hyperthermia, highlighting their potential applications in biomedical fields.
Assuntos
Ouro , Hipertermia Induzida , Nanopartículas Metálicas , Neoplasias/terapia , Fototerapia , Somatostatina/análogos & derivados , Ouro/química , Ouro/farmacologia , Células HeLa , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Neoplasias/metabolismo , Neoplasias/patologia , Somatostatina/química , Somatostatina/farmacologiaRESUMO
AIM: We developed octreotide-modified magnetic liposomes (OMlips) as dual-targeting drug carriers to enhance the drug accumulation in tumor site. MATERIALS & METHODS: Octreotide acts as a modified ligand for receptor-mediated targeting and the coated Fe3O4 nanoparticles offer the magnetic targeting property. SSTR2 overexpressed A549 cells and S180 cells were chosen to explore the targeting ability and antitumor effect of the oleanolic acid (OA)-loaded OMlips in vitro and in vivo. RESULTS: The OMlips platform significantly improves the targeting, penetrating and accumulation of OA at the SSTR2 overexpressed cells and SSTR2-positive tumor-bearing mice. CONCLUSION: The OA-loaded OMlips have better antitumor effect and lower systemic toxicity. Such a receptor-mediated and magnetically-orienting dual-targeting drug nanocarriers may have great potentials in clinical practice.
Assuntos
Antineoplásicos/química , Lipossomos/química , Nanopartículas de Magnetita/química , Receptores de Somatostatina/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Feminino , Óxido Ferroso-Férrico/química , Humanos , Camundongos , Transplante de Neoplasias , Octreotida/química , Ácido Oleanólico/química , Tamanho da Partícula , Distribuição TecidualRESUMO
BACKGROUND: Astaxanthin, a naturally occurring carotenoid pigment molecule, displays strong antioxidant, anti-cancer, and immunity-enhancing properties, and is often utilized in food, biomedical, cosmetic, and other industries. Free astaxanthin has better solubility than astaxanthin esters (Ast-E), and is a useful auxiliary ingredient in health foods and medicines. Our goal was to establish an improved enzymatic method for preparation of free astaxanthin from natural sources (e.g., the microalga Haematococcus pluvialis), to expand the potential applications of free astaxanthin. RESULTS: The alkaline lipase gene proalip and its propeptide were cloned and successfully fusion-expressed in Pichia pastoris X-33. The recombinant lipase was termed Lipase-YH. Through optimization of culture conditions (medium formulation, pH, added methanol concentration), cell growth (OD600) and secreted enzyme activity respectively reached to 280 and 2050 U/mL in a 50-L autofermentor. Activity of Lipase-YH enzyme powder was about 40,000 U/g. Hydrolysis of Ast-E (extracted from H. pluvialis) by Lipase-YH occurred in aqueous phase, and reaction conditions were optimized based on emulsification method and enzyme/substrate ratio. The highest enzymatic reaction rate was observed for substrate concentration 200 µg/mL, with maximal free astaxanthin yield (80%) at 1 h, and maximal Ast-E hydrolysis rate 96%, as confirmed by TLC, HPLC, and mass spectroscopy. CONCLUSION: A novel, efficient enzymatic process was developed for production of free astaxanthin through hydrolysis of Ast-E. Lipase activity was enhanced, and production cost was greatly reduced. The unique structure of free astaxanthin allows linkage to various functional compounds, which will facilitate development of novel pharmaceutical and food products in future studies.
RESUMO
AIM: To prepare and validate the efficacy of a novel targeted drug delivery system - octreotide (Oct)-modified oleanolic acid (OA) liposomes. MATERIALS & METHODS: OA liposomes (OA-L) were prepared by an ethanol injection method. The Oct-modified OA liposomes (O-OA-L) were synthesized and their properties were evaluated. Cellular uptake and competition inhibition studies were performed. RESULTS: The OA vesicles presented satisfactory particles size (100-200 nm), stability and drug release characteristics; their ζ potential was -1.42 ± 0.08 mV. The O-OA-L had a higher cell proliferation inhibitory activity and cellular uptake than that of OA-L for somatostatin receptor-positive A549 cells. CONCLUSION: The O-OA-L, as a promising agent, can target actively the lesion site with safe and high-efficiency pattern.
Assuntos
Sistemas de Liberação de Medicamentos , Lipossomos/química , Neoplasias Pulmonares/tratamento farmacológico , Ácido Oleanólico/administração & dosagem , Células A549 , Animais , Liberação Controlada de Fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipossomos/administração & dosagem , Camundongos , Octreotida/administração & dosagem , Octreotida/química , Ácido Oleanólico/química , Receptores de Somatostatina/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stimuli-responsive drug delivery and release have a great significance in cancer therapy. Herein, a multifunctional responsive drug carrier was designed and developed by loading resveratrol (Res) in chitosan (CTS) modified liposomes, and coated by gold nanoshells (GNS@CTS@Res-lips). The resultant GNS@CTS@Res-lips possess broad near-infrared (NIR) absorbance, high capability, stability, and also high photothermal conversion ability for efficient photothermal therapy (PTT) applications. In addition, the GNS@CTS@Res-lips exhibit the on-demand pH/photothermal-sensitive drug release, and a high loading capacity of Res. Under NIR laser irradiation, the drug delivery system could significantly enhance the cellular uptake of drugs. More importantly, compared to the single chemotherapy or PTT, the carriers with NIR irradiation displayed a higher therapeutic effect for HeLa cells. Therefore, the GNS@CTS@Res-lips with a combination of chemotherapy and PTT will show great potential for application in cancer therapy.
RESUMO
CaCl(2) was used as a novel additive to enhance poly-(γ-glutamic acid) (γ-PGA) production by Bacillus subtilis strain CGMCC 2108. Addition of CaCl(2) to medium effectively reduced viscosity of culture broth, and increased consumption of extracellular glutamate by 11.4%, leading to γ-PGA yield of 9.07 g/l, compared to 7.88 g/l in control. CaCl(2) also increased activities of three key enzymes around the crucial 2-oxoglutarate branch of the γ-PGA biosynthesis pathway: isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), and 2-oxoglutarate dehydrogenase complex (ODHC). In particular, GDH activity was increased more than 8-fold, indicating that more 2-oxoglutarate was directed to synthesis of glutamate, the substrate of γ-PGA. Interestingly, the molecular weight of γ-PGA remained constant regardless of CaCl(2) addition.