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.     

谷氨酸脱羧酶工程菌的发酵工艺及酶学性质

对谷氨酸脱羧酶(GAD)工程菌DM201的发酵及转化条件进行优化,分析了发酵过程中异丙基-β-D-巯基半乳糖苷(IPTG)诱导条件、转化体系pH、底物浓度和金属离子等因素对谷氨酸脱羧酶活力的影响。发酵过程中最佳IPTG诱导条件为:浓度0.4mmol/L、时间5h、温度30℃;转化温度45℃,pH=4.0,ρ(L-谷氨酸)≈110g/L,镁离子在50mmol/L和5mmol/L浓度时对谷氨酸脱羧酶酶活有稳定作用,铜锌离子对其酶活的抑制作用显著。     

Peptide Antibody Reactivity to Homologous Regions in Glutamate Decarboxylase Isoforms and Coxsackievirus B4 P2C

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.     

饱和定点突变拓宽谷氨酸脱羧酶催化pH范围的研究

谷氨酸脱羧酶(GAD)是生物法制备γ-氨基丁酸GABA以及一些含氮化合物的关键酶,其催化活力受p H值调控。研究GAD的pH值调控性质具有重要的理论和应用价值。研究通过氨基酸序列比对,发现了一个影响GAD 1407催化p H特性的关键位点:S307。通过对这个位点进行饱和突变,构建突变文库,从中筛选出催化pH范围拓宽的突变酶S307N,并对该突变酶的酶学性质进行了研究。为了解GAD 1407结构与功能之间的相互关系提供了一定的依据。     

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.     

Adsorption Behavior of 3-phenoxybenzoic Acid by Lactobacillus Plantarum and Its Potential Application in Simulated Digestive Juices

3-PBA is a major degradation intermediate of pyrethroids. Its widespread existence in the environment poses a severe threat to the ecosystem and human health. This study evaluated the adsorption capacity of L. plantarum RS20 toward 3-PBA. Batch adsorption experiments indicated that the optimal adsorption conditions were a temperature of 37 °C and initial pH of 6.0–8.0, under which the removal rate was positively correlated with the cell concentration. In addition, there was no link between the incubation time and adsorption rate. The kinetic study showed that the adsorption process fitted well with the pseudo-second-order model, and the adsorption isotherms could be described by both Langmuir and Freundlich equations. Heat and acid treatments showed that the ability of strain RS20 in removing 3-PBA was independent of microbial vitality. Indeed, it was involved with chemisorption and physisorption via the cell walls. The cell walls made the highest contribution to 3-PBA removal, according to the adsorption experiments using different cellular components. This finding was further reconfirmed by SEM. FTIR spectroscopy analysis indicated that carboxyl, hydroxyl, amino groups, and –C–N were the functional sites for the binding of 3-PBA. The co-culture experiments showed that the adsorption of strain RS20 enhanced the degradation of 3-PBA by strain SC-1. Strain RS20 could also survive and effectively remove 3-PBA in simulated digestive juices. Collectively, strain RS20 could be employed as a biological detoxification agent for humans and animals by eliminating 3-PBA from foods, feeds, and the digestive tract in the future.     

没食子酸脱羧酶及酶法制备焦性没食子酸研究进展

对没食子酸脱羧酶及酶法制备焦性没食子酸研究进展进行了介绍和综述,重点介绍了产没食子酸脱羧酶的微生物种类、发酵时间、底物浓度、pH值、温度、金属离子,氮源及接种量等对微生物发酵产高活性没食子酸脱羧酶的影响因素,并简述了酶法制备焦性没食子酸的方法,指出了今后的研究方向。     

双酶级联生物合成D-赖氨酸

建立了氨基酸消旋酶和赖氨酸脱羧酶双酶级联高效生产D-赖氨酸的方法。利用氨基酸消旋酶全细胞催化消旋L-赖氨酸得到DL-赖氨酸,去除氨基酸消旋酶后再加入赖氨酸脱羧酶,将消旋产物中的L-构型脱羧生成1,5-戊二胺和CO2,剩下D-赖氨酸;最终,通过阳离子交换树脂吸附洗脱得到D-赖氨酸。经考察双酶级联催化的最佳条件为:反应温度40℃,0.2 mol/L磷酸钾缓冲溶液(p H=5.8),底物L-赖氨酸质量浓度为50 g/L,氨基酸消旋酶全细胞和赖氨酸脱羧酶全细胞质量浓度均为10 g/L,4 mmol/L磷酸吡哆醛,0.1 g/L Triton X-100,反应时间12 h,其中消旋反应2 h和脱羧反应10 h,最终D-赖氨酸收率达42%,对映体过量值(e.e.值)=98%。     

谷氨酸脱氢酶发酵工艺的正交试验设计

谷氨酸脱氢酶是谷氨酸生物合成途径中的关键酶。为提高谷氨酸棒杆菌S0615发酵产谷氨酸脱氢酶的酶活,运用正交试验设计对其发酵培养基与培养条件进行了优化研究。结果表明,优化的培养基组成为:尿素0.8%,葡萄糖5%,玉米浆0.8%;优化的培养条件为pH值7.2,温度35℃,搅拌转速350 r/min,通气量1.5 L/min,采用恒定pH值控制尿素流加。在此发酵工艺条件下,谷氨酸脱氢酶的酶活可达到35.87 U/g。     

Metal Ion Promiscuity and Structure of 2,3-Dihydroxybenzoic Acid Decarboxylase of Aspergillus oryzae

Broad substrate tolerance and excellent regioselectivity, as well as independence from sensitive cofactors have established benzoic acid decarboxylases from microbial sources as efficient biocatalysts. Robustness under process conditions makes them particularly attractive for preparative-scale applications. The divalent metal-dependent enzymes are capable of catalyzing the reversible non-oxidative (de)carboxylation of a variety of electron-rich (hetero)aromatic substrates analogously to the chemical Kolbe-Schmitt reaction. Elemental mass spectrometry supported by crystal structure elucidation and quantum chemical calculations verified the presence of a catalytically relevant Mg²⁺ complexed in the active site of 2,3-dihydroxybenoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao). This unique example with respect to the nature of the metal is in contrast to mechanistically related decarboxylases, which generally have Zn²⁺ or Mn²⁺ as the catalytically active metal.     

生物化工品乳酸及其下游产品的研究和开发

乳酸是一种重要的有机酸及生物化工品,以乳酸为原料可以得到多种衍生物,如聚乳酸、乳酸盐以及乳酸酯等。较详细地评述了乳酸的主要生产工艺和市场,介绍了几种主要的乳酸衍生物的研究和开发,比较了不同的生产路线,并展望了乳酸及其衍生物的研发方向。     

Engineering of Leuconostoc citreum for Efficient Bioconversion of Soy Isoflavone Glycosides to Their Aglycone Forms

Soy isoflavones are phytochemicals that possess various beneficial physiological properties such as anti-aging, anti-tumor, and antioxidant properties. Since soy isoflavones exist in glycoside forms, their bioavailability requires initial hydrolysis of the sugar moieties bound to them to be efficiently absorbed through the gut epithelium. Instead of conventional chemical hydrolysis using acids or organic solvents, alternative strategies for enhancing the bioavailability of soy isoflavones using biological methods are gaining attention. Here, we engineered Leuconostoc citreum isolated from Korean kimchi for efficient bioconversion of soy isoflavone glycosides into their aglycone forms to enhance their bioavailability. We first constructed an expression module based on the isoflavone hydrolase (IH)-encoding gene of Bifidobacterium lactis, which mediates conversion of isoflavone glycosides to aglycone forms. Using a high copy number plasmid and bicistronic expression design, the IH was successfully synthesized in L. citreum. Additionally, we determined enzymatic activity of the IH using an in vivo β-glucosidase assay and confirmed its highly efficient bioconversion efficiency for various types of isoflavone glycosides. Finally, we successfully demonstrated that the engineered L. citreum could convert isoflavone glycosides present in fermented soymilk into aglycones.     

Phenotypic and Safety Assessment of the Cheese Strain Lactiplantibacillus plantarum LL441, and Sequence Analysis of its Complete Genome and Plasmidome

This work describes the phenotypic typing and complete genome analysis of LL441, a dairy Lactiplantibacillus plantarum strain. LL441 utilized a large range of carbohydrates and showed strong activity of some carbohydrate-degrading enzymes. The strain grew slowly in milk and produced acids and ketones along with other volatile compounds. The genome of LL441 included eight circular molecules, the bacterial chromosome, and seven plasmids (pLL441-1 through pLL441-7), ranging in size from 8.7 to 53.3 kbp. Genome analysis revealed vast arrays of genes involved in carbohydrate utilization and flavor formation in milk, as well as genes providing acid and bile resistance. No genes coding for virulence traits or pathogenicity factors were detected. Chromosome and plasmids were packed with insertion sequence (IS) elements. Plasmids were also abundant in genes encoding heavy metal resistance traits and plasmid maintenance functions. Technologically relevant phenotypes linked to plasmids, such as the production of plantaricin C (pLL441-1), lactose utilization (pLL441-2), and bacteriophage resistance (pLL441-4), were also identified. The absence of acquired antibiotic resistance and of phenotypes and genes of concern suggests L. plantarum LL441 be safe. The strain might therefore have a use as a starter or starter component in dairy and other food fermentations or as a probiotic.     

利用糖蜜废水产乳酸细菌的分离与鉴定

为获得可利用糖蜜废水产生乳酸的细菌,采用厌氧Hungate培养技术,从糖蜜废水处理生物反应器中获得活性污泥,分离到一株乳酸菌MD-9。对该株细菌进行了形态学特征、生理生化指标、16S rRNA基因序列分析等研究。结果表明与最相近的种属Lactobillus fermnetus的16S rRNA基因序列同源性为99.6%。该株细菌为兼性厌氧杆菌,葡萄糖代谢特征为专性异型发酵,可以利用糖蜜废水产生乳酸。     

均匀设计在美拉德反应研究中的应用

均匀设计是一种应用于多因素、多水平实验研究的好方法。本文利用均匀实验设计对谷氨酸与葡萄糖的美拉德反应进行研究,建立了几种重要的影响因素及美拉德反应程度之间的关系。     

Dorsolateral Prefrontal Cortex Glutamate/Gamma-Aminobutyric Acid (GABA) Alterations in Clinical High Risk and First-Episode Schizophrenia: A Preliminary 7-T Magnetic Resonance Spectroscopy Imaging Study

Converging lines of evidence suggest that an imbalance between excitation and inhibition is present in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia (SCZ). Gamma-aminobutyric-acid (GABA) and, to a lesser extent, glutamate (Glu) abnormalities were reported in the DLPFC of SCZ patients, especially on the right hemisphere, by post-mortem studies. However, in vivo evidence of GABA, Glu, and Glu/GABA DLPFC abnormalities, particularly on the right side and the early stages of illness, is limited. In this preliminary study, we utilized 7-Tesla magnetic resonance spectroscopic imaging (MRSI) to investigate bilateral Glu/Creatine (Cre), GABA/Cre, and Glu/GABA in the DLPFC of sixteen first episode schizophrenia (FES), seventeen clinical high risk (CHR), and twenty-six healthy comparison (HC) subjects. FES and CHR had abnormal GABA/Cre and Glu/GABA in the right DLPFC (rDLPFC) compared with HC participants, while no differences were observed in the left DLPFC (lDLPFC) among the three groups. Furthermore, HC had higher Glu/GABA in rDLPFC compared to lDLPFC (R > L), whereas the opposite relationship (R < L) was observed in the DLPFC Glu/GABA of FES patients. Altogether, these findings indicate that GABA/Cre and Glu/GABA DLPFC alterations are present before illness manifestation and worsen in FES patients, thus representing a putative early pathophysiological biomarker for SCZ and related psychotic disorders.     

乳酸菌微生物燃料电池产电性能及产电机理研究

采用双室微生物燃料电池（MFC）,以乳酸菌为产电微生物,并以葡萄糖为唯一的电子供体,研究MFC的产电性能以及乳酸菌MFC产电机理。在30 ℃下,底物浓度为1.5 g/L时,该MFC的开路电压稳定在500 mV。实验条件下测得该MFC的最大功率密度为393.23 mW/m²,内阻约为500 Ω。利用气相色谱分析乳酸菌MFC产电过程中代谢产物的含量变化,实验数据表明无论是不参与产电的正常代谢途径还是产电过程中,都涉及到乳酸菌的同型乳酸发酵途径、异型乳酸发酵的经典途径和双歧杆菌发酵途径。在乳酸菌MFC运行过程中人为添加乙醇,该实验结果显示乙醇不利于乳酸菌产电,表明乳酸菌的异型乳酸发酵途径是乳酸菌进行产电的关键代谢途径。     

Pre-Ischemic Treadmill Training for Prevention of Ischemic Brain Injury via Regulation of Glutamate and Its Transporter GLT-1

Pre-ischemic treadmill training exerts cerebral protection in the prevention of cerebral ischemia by alleviating neurotoxicity induced by excessive glutamate release following ischemic stroke. However, the underlying mechanism of this process remains unclear. Cerebral ischemia-reperfusion injury was observed in a rat model after 2 weeks of pre-ischemic treadmill training. Cerebrospinal fluid was collected using the microdialysis sampling method, and the concentration of glutamate was determined every 40 min from the beginning of ischemia to 4 h after reperfusion with high-performance liquid chromatography (HPLC)-fluorescence detection. At 3, 12, 24, and 48 h after ischemia, the expression of the glutamate transporter-1 (GLT-1) protein in brain tissues was determined by Western blot respectively. The effect of pre-ischemic treadmill training on glutamate concentration and GLT-1 expression after cerebral ischemia in rats along with changes in neurobehavioral score and cerebral infarct volume after 24 h ischemia yields critical information necessary to understand the protection mechanism exhibited by pre-ischemic treadmill training. The results demonstrated that pre-ischemic treadmill training up-regulates GLT-1 expression, decreases extracellular glutamate concentration, reduces cerebral infarct volume, and improves neurobehavioral score. Pre-ischemic treadmill training is likely to induce neuroprotection after cerebral ischemia by regulating GLT-1 expression, which results in re-uptake of excessive glutamate.     

Construction of a Chimeric Biosynthetic Pathway for the De Novo Biosynthesis of Rosmarinic Acid in Escherichia coli

Hydroxycinnamic acid esters (HCEs) are widely‐distributed phenylpropanoid‐derived plant natural products. Rosmarinic acid (RA), the most well‐known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, microbial production of HCEs could be a sustainable, controlled means of production. Through the overexpression of a six‐enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA, and the related HCE isorinic acid (IA), in Escherichia coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We demonstrated HCE biosynthesis using three plant rosmarinic acid synthase (RAS) orthologues, which exhibited different levels of HCE biosynthesis but produced the same ratio of IA to RA. This work serves as a proof‐of‐concept for a microbial production platform for HCEs by using a modular biosynthetic approach to access diverse natural and non‐natural HCEs.