Purification and biochemical characterisation of a novel glutamate decarboxylase from rice bran

BACKGROUND: Glutamate decarboxylase (GAD) is a useful enzyme whose main function is to catalyse the irreversible α‐decarboxylation of L ‐glutamate to produce γ‐aminobutyric acid. The cheap and abundant rice‐processing by‐product rice bran contains a high amount of GAD, the purification and characterisation of which have not yet been reported. In this study, research on rice bran GAD was initiated. RESULTS: Rice bran GAD was purified to homogeneity via a combined purification protocol of ammonium sulfate fractionation, ion exchange chromatography and two gel filtrations, with a purification fold of 128.6 and an activity recovery of 21.3%. The enzyme was active at pH 5.5 and 40 °C and retained 80% of its original activity in the pH range 5–9 and the temperature range 30–50 °C. GAD activity was significantly enhanced in the presence of Ca²⁺ but strongly inhibited by Ag⁺, Hg²⁺, sodium dodecyl sulfate and CH₃COOH. Kinetic determination of the apparent K_m for L ‐glutamate and pyridoxal 5′‐phosphate gave values of 27.4 mmol L^?1 and 1.16 µmol L^?1 respectively. CONCLUSION: Considering that rice bran is cheap and commercially available and that rice bran GAD is relatively stable, the development of cost‐effective rice bran GAD‐related functional foods would seem to be feasible. Copyright © 2010 Society of Chemical Industry     

一株高产3-羟基丁酮枯草芽孢杆菌的构建

以产3-羟基丁酮枯草芽孢杆菌(Bacillus subtilis)TH-49(Val-)和产α-乙酰乳酸脱羧酶地衣芽孢杆菌(Bacillus licheniformis)AD-30为亲本菌株,以聚乙二醇为融合促进剂,进行原生质体融合获得融合子,融合子经再生、筛选等过程,最终获得高产3-羟基丁酮菌株HB-32,该菌株3-羟基丁酮产量高达49.64 g/L,比菌株TH-49(Val-)提高了61.8%,且遗传稳定性良好。进一步采用随机扩增多态性DNA(RAPD)技术,从分子水平上分析了高产3-羟基丁酮菌株HB-32与亲本菌株基因组的变化。结果表明,枯草芽孢杆菌(B. subtilis)TH-49(Val-)和地衣芽孢杆菌(B. licheniformis)AD-30原生质体融合成功,提高了融合子HB-32 3-羟基丁酮产量。     

阿魏酸对粪肠球菌和屎肠球菌产酪胺机制的影响

摘 要：研究阿魏酸对高产酪胺的粪肠球菌XL-M66和屎肠球菌XL-M76生长、基因表达以及产酪胺的影响。利用反转录实时荧光定量聚合酶链式反应技术分析2 株菌在阿魏酸作用下的酪氨酸脱羧途径相关基因表达情况,并使用高效液相色谱法检测2 株肠球菌培养48 h期间酪胺积累量。结果表明：未添加酪氨酸底物时,阿魏酸对酪氨酸脱羧酶（tyrosine decarboxylase,tyrDC）和酪氨酸/酪胺透性酶（tyrosine/tyramine permease,tyrP）基因的转录影响不大（P＞0.05）,但能促进酪氨酰-tRNA合成酶（tyrosyl-tRNA synthetase,tyrS）基因的转录（P＜0.05）。反之存在酪氨酸时,阿魏酸对tyrS基因表达的影响不大（P＞0.05）,却能显著抑制tyrDC和tyrP基因的表达（P＜0.05）。同时,阿魏酸能显著抑制粪肠球菌XL-M66和屎肠球菌XL-M76的生长（P＜0.05）,最终使得酪胺产量分别降低27.0%和19.9%。     

Development of a histidine decarboxylase medium and its application to detect other amino acid decarboxylases

A liquid medium is described which can be used to detect histidine decarboxylase activity in bacterial pure cultures within 24 h. It can also be used to detect the presence of histamine-producing Enterobacteriaceae in mixed cultures, e.g. in environmental swab specimens by colour change within 48 h. A limited number of tests with tuna fish meat show that this medium may also be used for a semi-quantitative detection of histamine producers, for example by utilizing the MPN technique. Comparative examinations with Møller's decarboxylase medium using 122 Enterobacteriaceae strains demonstrated that the medium can also be used to prove the decarboxylation of lysine, ornithine and arginine. The advantage of the new medium is the faster indication of positive reactions.     

有机溶剂透性化处理谷氨酸脱羧酶工程菌制备γ-氨基丁酸的研究

为了降低细胞壁和细胞膜的传质阻力对全细胞催化过程的不利影响,采用有机溶剂对表达重组谷氨酸脱羧酶(GAD)的工程菌BL21(DE3)-p ET28a-gad B进行了透性化处理。结果表明,菌体GAD表观催化活力的改善程度与有机溶剂的介电系数和疏水性具有较强相关性,低介电常数(6),高疏水性(log P0.68)的有机溶剂可以有效提高工程菌的GAD催化活性,其中最有效的透性化试剂为二甲苯(介电常数为2.4,log P为3.1)。当二甲苯的用量为5μL?mg?1(干重细胞)、处理时间5 min时,菌体表观催化活力达到最优值,为7.94 U?mg?1(干重细胞),是处理前表观催化活力的12.4倍。为了充分利用菌体的催化活力,采用海藻酸钙包埋的方法对二甲苯处理过的菌体进行固定化,结果发现固定化的透性化菌体可以较好地保持催化活力,反复催化10次后发现,第10次的GABA产率为第1次GABA产率的72.04%。研究为系统的选择有机溶剂来有效提高GAD工程菌表观催化活性提供了参考,同时制备的固定化的透性化菌体显示了良好工业应用前景。     

羧基化磁性微球固定化谷氨酸脱羧酶

通过化学共沉淀法结合高锰酸钾氧化制备羧基化Fe₃O₄磁性微球,以该磁性微球作为载体,固定化谷氨酸脱羧酶。利用热重分析（TGA（、透射电镜（TEM（及振动样品磁强计（VSM（对羧基化磁性微球进行表征,结果表明该磁性微球磁含量约为95.1%,粒径均一,呈近似球形且具有超顺磁性。通过对固定化酶进行傅里叶红外光谱（FT-IR（、VSM和X射线衍射（XRD（分析,确定磁性微球载体与谷氨酸脱羧酶分子间形成酰胺键,实现共价结合且固定化酶前后粒子晶形完整,均具有良好的磁响应能力和超顺磁性。与游离谷氨酸脱羧酶相比,固定化酶的热稳定性和酸碱耐受性均有不同程度的提高,且制备的固定化酶重复使用10批后相对酶活力仍大于90%。     

Some molecular properties of glutamate decarboxylase from rice germ

Glutamate decarboxylase (EC 4.1.1.15, GAD) is a pyridoxal 5′-phosphate (PLP) dependent enzyme, which catalyses the irreversible α-decarboxylation of l-glutamic acid to γ-aminobutyric acid (GABA). GAD was purified 186-fold from rice germ. Ultraviolet–visible spectra showed that the rice germ holoGAD presented a weak peak at 420 nm, but the inactivated apoGAD did not. The holoGAD also exhibited a strong peak at 308 nm and a weak peak at 336 nm in its fluorescence emission spectrum. The apoGAD led to a 20% increase in the fluorescence emission at 308 nm. The contents of the secondary structure elements of the holoGAD and apoGAD were estimated from the values of the mean residue ellipticity based on the CD spectra. The holoGAD had a greater β-sheet content than the apoGAD (39% versus 27%), whereas both had a similar α-helix content (13% versus 14%). These findings confirmed that a slight conformational change had occurred when PLP bound to the apoGAD to form the holoGAD. Chemical modifications of the GAD by some selected reagents indicated that histidine residue(s) might be involved in the enzymatic functions, but were not essential for the enzyme activity. The study also suggested there was one arginine residue in the GAD active site, and most likely at the substrate-binding site.     

Genetic screening of biogenic amines production capacity from some lactic acid bacteria strains

There is an increasing interest for using lactic acid bacteria (LAB) as a starter and adjunct cultures for producing novel foods with particular functional traits. The ability of the starter to produce biogenic amines (BA) should be taken into account wherein protective starters should be selected to avoid hygienic and toxicological risks. This work aimed to study the possibilities of forming BA (histamine, putrescine, agmatine and tyramine) from thirty two LAB strains belonging to species of the genera Lactobacillus and Streptococcus that used in food products as well as strains isolated from healthy breast-fed infants. The analytical protocol involved using polymerase chain reaction (PCR) and thin layer chromatography (TLC) techniques to determine the ability of LAB strains to form BA. The presence of key genes involved in the biosynthetic pathways of the BA was also assessed by PCR. Six LAB strains gave positive results for putrescine production wherein the maximum level was 14.6 mg/kg. Six strains gave positive results for histamine production (maximum level was 31.7 mg/kg) and were positive for the presence of histidine decarboxylase (HDC) gene. Seven strains exhibited positive results for tyramines production (maximum level was 2.85 mg/kg) and were positive for the presence of tyrosine decarboxylase (TDC) gene. Eight strains gave positive results for agmatine production (maximum level was 174.5 mg/kg) and were positive for the presence of dihydrolase (deiminase) gene that responsible for agmatine formation. It could be concluded that the microorganisms used in food and dairy production should be screened carefully by PCR for their ability to produce BA.     

南瓜谷氨酸脱羧酶酶学特性及γ-氨基丁酸富集条件

对生鲜南瓜(Cucurbita moschata)所含谷氨酸脱羧酶(Glutamate decarboxylase,GAD)的最适反应温度、最适pH、热稳定性和冷冻稳定性等酶学特性进行研究,并利用其富集γ-氨基丁酸(γ-Aminobutyric acid,GABA),探索了反应时间、南瓜品种、缓冲体系、南瓜及味精添加量、料水比对富集效果的影响。结果表明,南瓜GAD最适反应温度为30~35 ℃,最适pH为5.8。南瓜GAD对热比较敏感,50 ℃保温30 min,酶活力损失20%,70 ℃以上保温30 min可导致酶活力完全丧失。冷冻对GAD影响较小,但长期冷冻仍会导致酶活力损失,冷冻8周酶活力损失36%。GABA最适富集条件为:以pH5.8,0.2 mol/L磷酸氢二钠-柠檬酸缓冲液作为反应溶液,南瓜与缓冲液比例为1:3,南瓜添加量25%,味精添加量1.5%,30 ℃反应18 h,反应液中GABA浓度可达7633.2 mg/L,转化率为93.3%,单位质量南瓜GABA富集量为30.5 mg/g,与未富集时相比提高了132倍。