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华东六省一市生物化学与分子生物学学会2014年学术交流...

作者： Wei-rui Zhao Jun Huang Chang-jiang Lv Sheng Hu Le-he Mei Department of Chemical and Biological Engineering Zhejiang University School of Biological and Chemical Engineering Zhejiang University of Science and Technology School of Biotechnology and Chemical Engineering Ningbo Institute of TechnologyZhejiang University

Lactic acidis a most important industry product which has been widely used in food,chemical,cosmetic,and ***,lactic acid can be used as a feedstock for the production of poly-lactic acid(PLA),which is a biodegradable,biocompatible and environmentally friendly biopolymer and can be used in surgical sutures,orthopedic implants,drug delivery systems,and disposable consumer *** the increase interest in production of PLA,the production of lactic acid has been further *** acid can be produced by either chemical synthesis or microbial *** with chemical synthesis which provides the racemic lactic acid,fermentation that can produce optically pure L-or D-lactic acid by using different organisms is ***,almost all lactic acid manufactured in the world is produced though fermentation *** to lactic acid-producingRhizopusoryzaecan be tolerant to low pH environment(pH 4.0-6.0)and producelactic acidin a sole isomer with a simple and low-cost media,usingit to produce lactic acid has received a great ***,lactic acid fermentation is a process that inhibited by the reaction *** inhibitive effect of lactic acid is mainly due to the decrease in the pH of the fermentation medium,which results in an incomplete substrate conversion and relatively low concentration of *** way to overcome this disadvantage and to produce high concentration of lactic acid is to neutralization of the lactic acid(H)with addition of a large amount of ***,the low solubility of CaCO would undermine the effectiveness of the neutralizing process and cause problems in the subsequent purification *** decarboxylase(GAD)could catalyze theα-decarboxylation of glutamate to generate neutraly-aminobutyric acid(GABA)by consuming *** is a useful substance and has good prospect of application in food,drug and chemical *** addition,GABA has also been used as a building block to produce a variety of

关键词： lactic acid glutamate decarboxylase γ-aminobutyric acid Rhizopus oryzae

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High-level production of𝛾γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system in Escherichia coli

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Engineering Microbiology 2023年第2期3卷 65-71页

作者： Lili Yao Changjiang Lyu Yuting Wang Sheng Hu Weirui Zhao Hongwei Cao Jun Huang Lehe Mei College of Life Science and Technology Heilongjiang Bayi Agricultural UniversityDaqing 163319China School of Biological and Chemical Engineering Zhejiang University of Science and TechnologyHangzhou 310023China School of Biological and Chemical Engineering Ningbo Tech UniversityNingbo 315100China College of Chemical and Biological Engineering Zhejiang UniversityHangzhou 310027China

Biosynthesis of the functional factor𝛾γ-aminobutyric acid(GABA)in bacteria involves two key proteins an intra-cellular glutamate decarboxylase(GadB)and a membrane-bound antiporter(GadC).Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA *** this study,gadBΔC11 of Lacti-plantibacillus plantarum and gadCΔC41 of Escherichia coli were inserted into the designed double promoter(P T7lac and P BAD)expression ***,*** Lemo21(DE3)was chosen as the host to minimize the toxic effects of GadCΔC41 ***,a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3)cells as biocatalysts was *** total GABA yield reached 829.08 g/L with a 98.7%conversion ratio within 13 h,when engineered *** Lemo21(DE3)cells were concentrated to an OD 600 of 20 and reused for three cycles in a 3 M L-glutamate solution at 37℃,which represented the highest GABA productivity ever ***,expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in ***.

关键词： glutamate decarboxylase Antiporter γ-Aminobutyric acid E.coli Whole-cell biocatalysts

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Activation of dopamine 2 receptors modulates glutamate decarboxylases 65 and 67 during stroke recovery in mice

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Neuroprotection 2023年第2期1卷 160-169页

作者： Daniela Talhada Robert Nilsson Severin Walser Georgios Michalettos Karsten Ruscher Laboratory for Experimental Brain Research Department of Clinical SciencesDivision of NeurosurgeryLund UniversityLundSweden LUBIN Lab-Lunds Laboratorium för Neurokirurgisk Hjärnskadeforskning Department of Clinical SciencesDivision of NeurosurgeryLund UniversityLundSweden

Backgrounds:Treatment with levodopa enhances recovery of lost neurological functions in preclinical stroke models and ***,we studied whether dopamine signaling modulates GABAergic neurotransmission in parvalbumin-positive interneurons after experimental ***:Following block randomization,mice were subjected to experimental stroke induced by photothrombosis(PT).Two days after the insult,mice were treated either with the D1 receptor antagonist by R(+)-SCH-23390(0.1 mg/kg),the selective D1 receptor agonist(R)-(+)-SKF-38393 hydrochloride(1 mg/kg),the D2 receptor agonist R(−)-2,10,11-trihydroxy-Npropyl-noraporphine hydrobromide hydrate(TNPA)(1 mg/kg),the D2 receptor antagonist S-(−)-eticlopride hydrochloride(0.3 mg/kg),or vehicle(saline)by daily intraperitoneal injection for five consecutive days,*** of function was assessed by paw placement and foot fault test before and on Days 2 and 7 after ***:Mice treated with TNPA showed a statistically significant improvement of recovery compared to all other treatment *** of gamma-aminobutyric acid(GABA)was quantified by levels of full-length and cleaved glutamate acid decarboxylase 67 and 65(GAD65 and GAD67)in the peri-infarct area and homotypic regions of the contralateral *** to the other treatments,TNPA significantly reduced the level of the GAD67 isoform both in the ischemic and contralateral *** of GAD65 were found significantly higher in the contralateral hemisphere in TNPA-treated mice after PT accompanied by an increase in the 58 kDa-truncated ***:Our results point toward reduced GABA synthesis in a D2 receptor-mediated mechanism possibly contributing to counteract functional inhibition after stroke.

关键词： calpain dopamine glutamate decarboxylase photothrombosis stroke recovery

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Efect of the Gad system on Actinobacillus succinogenes during acid stress

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Systems Microbiology and Biomanufacturing 2022年第1期2卷 177-185页

作者： Chunmei Chen Qun Zhang Junzhu Qian Dan Wu Pengcheng Chen Pu Zheng The Key Laboratory of Industrial Biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China

The glutamate decarboxylase(Gad)system is an important amino acid-dependent acid resistance system commonly found in *** succinogenes is one of the best natural producers of succinic acid(SA)but lacks glutamate *** study assessed the efects of Gad system introduction into *** recombinant strains gadB-SW and gadBC-SW were constructed by heterologous expression of gadB alone,or gadB together with gadC,*** 1.0 and 1.5 h of acid stress at pH 4.6,cell survival of gadBC-SW was greater than *** growth of gadB-SW and gadBC-SW was both afected by the expression of heterologous proteins and byγ-aminobutyric acid,with gadBC-SW growth reduced at a neutral *** production in acidic conditions was evaluated by a shake fask and by 3-L bioreactor *** results showed gadBC-SW to increase SA production by 8.4%in shake fask compared to the parent strain,*** a 3-L bioreactor batch fermentation under acidic environment,the highest conversion rate of sugar to SA was observed for gadBC-SW,reaching 96%.However,SA concentration by gadBC-SW was only 47 g/L and 31 g/L at pH 6.5 and pH 6.0,*** summary,the introduction of heterologous gadB and gadC into *** not only improved acid tolerance but also infuenced the synthesis of SA and added a metabolic burden.

关键词： Actinobacillus succinogenes Acid stress glutamate decarboxylase Succinic acid glutamate

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Effects of antisense glutamic aciddecarb0xylase oligodeoxynucleotide on epileptic rats induced by pentylenetetrazol

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Chinese Medical Journal 2002年第3期115卷 425-429页

作者：何小华李文鑫王伟阮旭中张梁武汉大学生命科学院生物技术系武汉430070 同济医科大学附属同济医院神经内科武汉430030

OBJECTIVE: To investigate the effects of antisense glutamic acid decarboxylase (GAD(67)) oligodeoxynucleo-tide (ODN) on behavior, seizure threshold and EEG of hippocampus in the epileptic rats induced by pentylenetetrazol (PTZ). METHODS: A model of chronic epilepsy in rats was established by PTZ. The inhibition of GAD(67) mRNA expression in hippocampus was selectively induced by antisense oligodeoxynucleotide of GAD(67). The effect of antisense GAD(67) ODN on behavior, seizure threshold and EEG recording of kindled rats was examined. RESULTS: Antisense GAD(67) ODN could inhibit the expression of GAD(67) mRNA and the concentration of GABA. It also could significantly shorten the latencies of seizure and increase the level of seizure and the frequency of epileptiform discharges. CONCLUSION: The gene of GAD(67) may be an anti-seizure gene, which might inhibit epileptiform discharge. The treatment of epilepsy by GAD(67) gene will have a bright future.

关键词： Animals Electroencephalography Epilepsy glutamate decarboxylase Hippocampus Isoenzymes Kindling (Neurology) Male Oligonucleotides, Antisense Pentylenetetrazole Rats Research Support, Non-U.S. Gov't gamma-Aminobutyric Acid

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On-line near-infrared spectroscopy optimizing and monitoring biotransformation process of γ-aminobutyric acid

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Journal of Pharmaceutical Analysis 2016年第3期6卷 171-178页

作者： Guoyu Ding Yuanyuan Hou Jiamin Peng Yunbing Shen Min Jiang Gang Bai State Key Laboratory of Medicinal Chemical Biology College of Pharmacy Nankai University Tianfin Key Laboratory of Molecular Drug Research Nankai University Tianjin 300071 China

Near-infrared spectroscopy （NIRS） with its fast and nondestructive advantages can be qualified for the real-time quantitative analysis. This paper demonstrates that NIRS combined with partial least squares （PLS） regression can be used as a rapid analytical method to simultaneously quantify L-glutamic acid （L- GIu） and γ-aminobutyric acid （GABA） in a biotransformation process and to guide the optimization of production conditions when the merits of NIRS are combined with response surface methodology. The high performance liquid chromatography （HPLC） reference analysis was performed by the o-phthaldialdehyde pre-column derivatization. NIRS measurements of two batches of 141 samples were firstly analyzed by PLS with several spectral pre-processing methods. Compared with those of the HPLC reference analysis, the resulting determination coefficients （R2）, root mean square error of prediction （RMSEP） and residual predictive deviation （RPD） of the external validation for the L-GIu concentration were 99.5%, 1.62 g/L, and 11.3, respectively. For the GABA concentration, R2, RMSEP, and RPD were 99.8%, 4.00 g/L, and 16.4, respectively. This NIRS model was then used to optimize the biotransformation process through a Box- Behnken experimental design. Under the optimal conditions without pH adjustment, 200 gjL L-GIu could be catalyzed by 7148 U/L glutamate decarboxylase （GAD） to GABA, reaching 99% conversion at the fifth hour. NIRS analysis provided timely information on the conversion from L-GIu to GABA. The results suggest that the NIRS model can not only be used for the routine profiling of enzymatic conversion, providing a simple and effective method of monitoring the biotransformation process of GABA, but also be considered to be an optimal tool to guide the optimization of production conditions.

关键词： Near-intrared spectroscopy L-glutamic acid γ-aminobutyric acid Box-Behnken design glutamate decarboxylase

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Effects of germination and aeration treatment following segmented moisture conditioning on theγ-aminobutyric acid accumulation in germinated brown rice

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International Journal of Agricultural and Biological Engineering 2020年第5期13卷 234-240页

作者： Qiang Zhang Nian Liu Shuangshuang Wang Leiqing Pan College of Engineering Huazhong Agricultural UniversityWuhan 430070China Key Laboratory of Agricultural Equipment in Mid-lower Yangtze River Ministry of AgricultureWuhan 430070China College of Food Science and Technology Nanjing Agricultural UniversityNanjing 210095China

Brown rice was treated by segmented moisture conditioning to reach the suitable water content and aerated with air for *** effects of germination and aeration treatment on theγ-aminobutyric acid(GABA)content in germinated brown rice were *** results showed that the germination rate,glutamic acid,glutamate decarboxylase activity and GABA content in germinated brown rice increased significantly and then decreased with the increase of germination and aeration treatment *** analysis also revealed that there was a significant positive correlation between GABA accumulation and glutamic acid content,glutamate decarboxylase *** results suggested that the aeration treatment during germination following segmented moisture conditioning could contribute to a high GABA content of germinated brown rice.

关键词： germinated brown rice segmented moisture conditioning and aeration treatment GABA glutamic acid glutamate decarboxylase

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Efficient gamma-aminobutyric acid bioconversion by engineered Escherichia coli

Efficient gamma-aminobutyric acid bioconversion by engineere...

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华东地区生物化学与分子生物学学会联合会2017年学术交...

作者： Xinwei Yang Jie Wei Yang Ren Chongrong Ke Jia Chen Songgang Wu Jianzhong Huang National Engineering Research Center of Industrial Microbiology and Fermentation Technology

To construct a simple and efficient process for industrial production of GABA from L-glutamic acid(L-Glu) by whole-cell *** encoding glutamate decarboxylase(GAD) from Lactococcus lactis FJNUGA01 was over-expressed using the most available vector pET-28 a in *** production was enhanced by optimizing culture conditions of the catalyst,such as IPTG concertration,induction temperature and induction *** optimizing,98.4 g GABA I-1 with 96 mol%conversion within 6 h was achieved by whole-cell conversion using the cells induced with 1 mM IPTG for 12 h at30 ℃.Furthermore,when initial concentration of L-Glu was adjusted,204.12 g GABA I with 34 g I h productivity was reached using 2 M L-Glu as the *** the 6 h whole-cell bioconversion process,almost all L-Glu was converted to GABA with a conversion efficiency of-99%.The whole-cell bioconversion for GABA production was easy to construct and *** high conversion efficiency and high productivity made the GABA purification downstream and the whole process more simple and economical for industrial application.

关键词： Escherichia coli Gamma-aminobutyric acid glutamate decarboxylase Whole-cell bioconversion

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