具有谷氨酸脱羧酶的大肠杆菌固定化细胞

本文研究了用海藻酸钙包埋法制备含谷氨酸脱羧酶固定化细胞的方法以及研究了制备的条件和影响其制备的因素。该法具有包埋细胞活力回收高,方法简便等优点。比较研究了固定化细胞和自然细胞谷氨酸脱羧酶的一些生物化学性质。其中固定化细胞最适pH和pH稳定性增加,最适温度及热稳定性下降;表观米氏常数增大;二价金属离子Zn~(++)、Cu~(++)、Mg~(++)、Fe~(++),Sr~(++)程度不同的抑制酶活性,Ca~(++)激活固定化细胞酶活性,EDTA无抑制作用。对固定化细胞和自然细胞酶活力活化的研究中发现这两种细胞经蒸馏水保温处理后酶活性都上升,且自然细胞酶活的上升较固定化细胞大;而用底物溶液处理后,自然细胞无变化,固定化细胞酶活下降。     

谷氨酸脱羧酶结构及催化机制的研究概述

谷氨酸脱羧酶(glutamate decarboxylase,GAD)是一种磷酸吡哆醛(pyridoxal-5′-phosphate,PLP)依赖性酶,广泛存在于自然界的动植物和微生物中,在酸性环境下发生结构变化,不可逆地催化L-谷氨酸或谷氨酸盐α-脱羧生成γ-氨基丁酸(γ-aminobutyric acid,GABA)。γ-氨基丁酸在人体中作为一种抑制性神经递质,具有重要的生理功能,可以被广泛应用于食品和制药工业中。本文就谷氨酸脱羧酶结构及催化机制的研究进展进行概述。     

谷氨酸脱羧酶的分子改造及酶学性质研究

以大肠杆菌(Escherichia coli)来源的谷氨酸脱羧酶(GadB)为研究对象,通过组合突变,获得了pH适用范围拓宽、催化活力提高和稳定性增强的组合突变体M2。与野生型GadB-WT相比,组合突变体M2的pH适用范围有效拓宽,在pH6.0时催化活力比GadB-WT提高113.43%。之后对含有M2突变体基因重组菌的发酵培养基和诱导条件进行优化,优化后单位培养基酶活力比未优化时提高了104.13%。在此基础上对M2的酶学性质进行测定,测得其最适pH为5.0,最适温度为37℃。通过稳定性测定M2的pH稳定性和热稳定性与野生型GadB-WT相比都有一定程度的增强。M2的动力学参数K_m值为7.316μmol/L,k_cat为13.387 s^-1,k_cat/K_m为1.830 L/(s·μmol)。研究获得的组合突变体M2进一步丰富了催化合成γ-氨基丁酸的GadB突变体酶库,具有良好应用前景。     

豆豉中高产γ-氨基丁酸乳酸菌的筛选及其谷氨酸脱羧酶酶学特性研究

目的从云南传统发酵豆豉中分离和筛选得到高产γ-氨基丁酸（GABA）的乳酸菌,同时研究其分泌产生的GAD酶学特性,以期为GABA的自动化发酵及GAD的固定化生产提供参考依据。方法通过高效液相色谱分析,筛选到一株高产GABA的乳酸菌,并初步研究其GAD酶学特性。结果从云南传统发酵豆豉中筛选得到高产GA-BA的Lactobacillus plantarum YM-4-3（产量高达5.74 mmol/L,即0.592 g/L）,其最适发酵条件为35℃,MSG含量为3%,初始pH 6.0,静置厌氧（5%CO2）发酵96 h;酶学特性研究结果表明,该菌株由来GAD是一种酸性酶,在酸性条件下稳定,最适反应pH为4.0,最适反应温度40℃,辅因子磷酸吡哆醛的最适浓度为200μmol/L。结论云南传统发酵豆豉可作为筛选具有较强GABA转化能力乳酸菌的资源库,该研究将有助于新型乳酸菌发酵豆豉的研发。     

猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

大鼠降结肠上皮细胞γ-氨基丁酸及谷氨酸脱羧酶的表达与意义

目的检测γ-氨基丁酸(gamma-aminobutyric acid,GABA)和谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)在大鼠降结肠上皮的表达及分布特征,并探讨GABA与上皮细胞分化增殖的关系。方法用免疫荧光及激光共聚焦显微扫描技术,检测GABA、GAD65及GAD67在大鼠降结肠上皮中的表达,并以麦芽凝聚素组织化学染色与免疫荧光结合的双重染色显示GABA和GAD65表达细胞的分布特征。同时,用RT-PCR方法检测GAD mRNA的表达。此外,用3H-胸腺嘧啶放射自显影及增殖细胞核抗原(PCNA)免疫组化方法显示降结肠上皮的增殖带。结果RT-PCR显示降结肠粘膜中GAD65及GAD67mRNA均为阳性。GABA及GAD65免疫反应阳性细胞主要分布在降结肠的腔面和隐窝的上1/3上皮细胞的胞浆,而GAD67阳性细胞仅分布腔面,此外,GABA及GAD65阳性染色也见于黏膜固有层。双重染色显示杯状细胞中GABA及GAD65均为阴性3。H-胸腺嘧啶及PCNA标记阳性细胞主要在隐窝的中下段。结论GABA及GAD65分布在大鼠降结肠上皮的成熟带及功能带,GABA系统可能参与上皮细胞的分化与增殖的调节。     

应用拉氏图信息提高短乳杆菌谷氨酸脱羧酶催化性能

谷氨酸脱羧酶(Glutamate decarboxylase,GAD)是用于催化L-谷氨酸脱羧合成γ-氨基丁酸(γ-aminobutyrate,GABA)的唯一酶,提高GAD的催化活力或热稳定性,有利于GABA的高效制备和生产。以热稳定性和活性为筛选目标,通过研究短乳杆菌GAD1407三维模拟结构的拉氏图,确定不稳定氨基酸残基位点K413,采用定点突变的方法构建该位点的突变体,并测定野生型酶和突变酶的热稳定性和活力。结果表明突变酶K413A和突变酶K413I分别在热稳定性和酶活力上获得了提高,突变酶K413A在50℃的半衰期为105 min,是野生酶的2.1倍;突变酶K413I热稳定性没有明显的提高,但其酶活力却得到了有效提高,约为野生型的1.6倍。因此,通过拉氏图提供的结构信息可为利用理性设计提高GAD活性和热稳定性提供指导。     

谷氨酸脱羧酶若干研究进展

谷氨酸脱羧酶是γ－氨基丁酸的合成酶,主要存在脑和胰岛中。因体内存在多种形成的谷氨酸脱羧酶,现无获得各种均一的谷氨酸脱羧酶的 统一方法。谷氨酸脱羧酶的克隆和表达,既弄清了谷氨酸脱羧酶的基因结构与定位,又为谷氨酸脱酶的大规模应用奠定了基础。目前认为谷氨酸脱羧酶是Ⅰ型糖尿病的始动靶抗原,体内注入谷氨酸脱羧酶可预防或延缓ＮＯＤ（ｎｏｎｏｂｅｓｅ ｄｉａｂｅｔｉｃ）小鼠Ⅰ型糖尿病的发生。     

一株产谷氨酸脱羧酶乳酸菌的鉴定及其酶学性质

从28株乳酸菌中筛选到了10株谷氨酸脱羧酶产生菌,其中以菌株Y-2的活性最高,当菌体(湿重)与1%谷氨酸一钠溶液按1∶10混合,于37℃反应12 h,转化液中γ-氨基丁酸浓度为14.52±0.93 mmol/L。通过形态特征、生理生化特征和16S rDNA序列分析鉴定菌株Y-2为唾液链球菌嗜热亚种(Streptococcus salivariussubsp.thermophilus)。同时基于16S rDNA构建了系统进化树,并进行了系统发育分析。S.salivariussubsp.thermophilusY-2谷氨酸脱羧酶的粗酶最适反应温度和pH分别为45℃和5.0,在4~40℃和pH 4.75~5.25范围内较稳定,酶催化反应在0~6 h具有良好的线性。     

Inverse polymerase chain reaction mediated chromosome walking within the human glutamic acid decarboxylase gene

Using inverse polymerase chain reaction (PCR), we have cloned partial intronic sequences from human glutamic acid decarboxylase (GAD) gene. A small 153 bp core region was selected from the GAD cDNA sequence to design outward primers corresponding to its 3′ and 5′ ends. EcoRI digested human DNA which had been circularized by self-ligation and then linearized withSacII was used as a substrate to can.y out PCR. This gave a 900 bp long product which was cloned into pUC19. The sequence analysis of this fragment revealed the presence of introns in the region flanking the selected core DNA. In this work we used this technique to walk into the upsteam region of the GAD gene using sequence information from its cloned cDNA.     

Is there a high molecular weight glutamic acid decarboxylase?

Gel-filtration chromatography from crude extracts of mouse brain indicates the presence of a high molecular weight (HMW) (more than 750 kd) and a low molecular weight (LMW) (87.5 Kd) glutamic acid decarboxylase (GAD) when they are concentrated prior chromatography either by precipitation with salts or ethanol. Kinetically both peaks of GAD-activity exhibit an almost identical Km for L-glutamate. Only LMW-GAD appears if the concentration step is carried out by ultrafiltration or if the extract is chromatographied without the concentrating step. HMW-GAD disappears from the chromatographic profiles if ethanolic extracts of GAD are treated with Triton X-100 before the chromatographic procedure. The sodium sulfate precipitation of a previously separated LMW-GAD gives rise to the reappearance of a HMW-GAD peak. Apparently HMW-GAD does not exist as a different molecular entity; indeed it may be an artefactual aggregation of LMW-GAD.     

Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase

In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.     

Site-directed mutagenesis improves the practical application of L-glutamic acid decarboxylase in Escherichia coli

γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains Escherichia coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production. Practical Application : Enzymatic properties of the GAD from Escherichia coli and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters K_m and V_max of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains E. coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S, and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.     

Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris

The yeast cell factory is a potentially useful source of proteins in general. They include glutamic acid decarboxylase (GAD), which is one of the major autoantigens for Type 1 diabetes. We have created a hybrid form of GAD consisting of amino acids 1–101 of the human GAD67 protein fused to amino acids 96–585 of the human GAD65 protein, and have modified this to include a C-terminal hexa-Histidine (H6) tag sequence. This hybrid GAD67/65-H6 was expressed in two yeast hosts: constitutively under the control of the plasmid phosphoglycerate kinase promoter (PGK1) in Saccharomyces cerevisiae, and inducibly under the control of the chromosomal alcohol oxidase promoter (AOX1) in Pichia pastoris. Enzymatically active hybrid GAD was prepared from yeast lysates by purification either on an affinity column based on the GAD-1 monoclonal antibody, or by metal-affinity chromatography. The purified GAD67/65-H6 was radiolabelled with iodine-125 and tested with Type 1 diabetes sera in a radioimmunoprecipitation assay, and results were compared with those using untagged GAD67/65 and those using porcine brain GAD. The results of enzymatic and immunological assays show hybrid GAD67/65 is isolated at high specific activity and moderate yield, and the addition of the H6 tag sequences or the choice of yeast strain did not appreciably affect enzyme activity, percentage recovery of GAD, protein purification, or the utility in diagnosis of diabetes in terms of specificity and sensitivity to the various sera.     

Activity-dependent cleavage of brain glutamic acid decarboxylase 65 by calpain

Previously, we reported that l-glutamic acid decarboxylase isoform 65 (GAD65) could be cleaved in vitro to release a stable truncated form which lacks amino acid 1-69 from the N-terminus, GAD65(Delta1-69). However, whether such a truncated form is also present under certain physiological conditions remains elusive. In the present study, we showed that, upon sustained neuronal stimulation, GAD65 could be cleaved into a truncated form in a rat synaptosomal preparation. This truncated form had similar electrophoretic mobility to purified recombinant human GAD65(Delta1-69). Furthermore, we demonstrated that this conversion was calcium dependent. Calcium-chelating reagents such as EDTA and 1,2-bis-(o-aminphenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetra-acetoxy-methyl ester prevented the cleavage of GAD65. In addition, our data suggested that calpain, a calcium-dependent cysteine protease, is activated upon neuronal stimulation and could be responsible for the conversion of full-length GAD65 to truncated GAD65 in the brain. Moreover, calpain inhibitors such as calpain inhibitor I or calpastatin could block the cleavage. Results of our in vitro cleavage assay using purified calpain and immunopurified rat GAD65 also supported the idea that GAD65 could be directly cleaved by calpain.     

Identification and functional analysis of truncated human glutamic acid decarboxylase 65

Human brain glutamate decarboxylase 65 (hGAD65) was found to exist as full-length and truncated forms when the glutathione S-transferase-tagged hGAD65 fusion protein was subjected to factor Xa cleavage. The truncated form is produced by cleavage at arginine 69 based on N-terminal amino acid sequence analysis, and has a molecular weight of 58 kD. It is resistant to further factor Xa cleavage or mild trypsin treatment and is more active and more stable than the full-length form. Both the full-length and truncated forms of GAD are also observed in brain preparations in the presence of protease inhibitors. Furthermore, full-length GAD could be converted to the truncated form by endogenous proteases, suggesting that the conversion of full-length to truncated GAD mediated by endogenous protease may represent an important mechanism in the regulation of GABA biosynthesis in the brain.     

Post-translational modification of glutamic acid decarboxylase 67 by intermittent hypoxia: evidence for the involvement of dopamine D1 receptor signaling

Intermittent hypoxia (IH) associated with sleep apnea leads to cardio-respiratory morbidities. Previous studies have shown that IH alters the synthesis of neurotransmitters including catecholamines and neuropeptides in brainstem regions associated with regulation of cardio-respiratory functions. GABA, a major inhibitory neurotransmitter in the CNS, has been implicated in cardio-respiratory control. GABA synthesis is primarily catalyzed by glutamic acid decarboxylase (GAD). In this study, we tested the hypothesis that IH like its effect on other transmitters also alters GABA synthesis. The impact of IH on GABA synthesis was investigated in pheochromocytoma 12 cells, a neuronal cell line which is known to express active form of GAD67 in the cytosolic fraction and also assessed the underlying mechanisms contributing to IH-evoked response. Exposure of cell cultures to IH decreased GAD67 activity and GABA level. IH-evoked decrease in GAD67 activity was caused by increased cAMP - protein kinase A (PKA) - dependent phosphorylation of GAD67, but not as a result of changes in either GAD67 mRNA or protein expression. PKA inhibitor restored GAD67 activity and GABA levels in IH treated cells. Pheochromocytoma 12 cells express dopamine 1 receptor (D1R), a G-protein coupled receptor whose activation increased adenylyl cyclase activity. Treatment with either D1R antagonist or adenylyl cyclase inhibitor reversed IH-evoked GAD67 inhibition. Silencing D1R expression with siRNA reversed cAMP elevation and GAD67 inhibition by IH. These results provide evidence for the role of D1R-cAMP-PKA signaling in IH-mediated inhibition of GAD67 via protein phosphorylation resulting in down-regulation of GABA synthesis.