Rapid one-step separation and purification of recombinant phenylalanine dehydrogenase in aqueous two-phase systems

Background: Phenylalanine dehydrogenase (PheDH; EC 1.4.1.20) is a NAD+-dependent enzyme that performs the reversible oxidative deamination of L-phenylalanine to phenylpyruvate. It plays an important role in detection and screening of phenylketonuria (PKU) diseases and production of chiral intermediates as well. The main goal of this study was to find a simple and rapid alternative method for purifying PheDH. Methods: The purification of recombinant Bacillus sphaericus PheDH was investigated in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The influences of system parameters including PEG molecular weight and concentration, pH and (NH4)2SO4 concentration on enzyme partitioning were also studied. The purity of enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results: A single extraction process was developed for separation and purification of recombinant PheDH from E. coli BL21 (DE3). The optimized conditions for partitioning and purification of PheDH were 9% (w/w) PEG-6,000 and 16% (w/w) (NH4)2SO4 at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were achieved 58.7, 135%, 94.42%, 491.93 and 9828.88 U/mg, respectively. Also, the Km values for L-phenylalanine and NAD+ in oxidative deamination were 0.21 and 0.13 mM, respectively. Conclusion: The data presented in this paper demonstrated the potential of ATPS as a versatile and scaleable process for downstream processing of recombinant PheDH.     

木薯苹果酸脱氢酶基因克隆和表达分析

为从木薯块根中获得苹果酸脱氢酶(MDH)基因,并研究其在转录水平的表达变化规律,利用RACE技术从木薯“华南8号”块根中克隆得到了苹果酸脱氢酶基因,其cDNA全长1 175 bp,包含999 bp的开放阅读框,共编码332个氨基酸.从木薯“华南8号”中获得的MDH氨基酸序列,与其它物种的该序列相似度达84％～94％,包含细胞质苹果酸脱氢酶中高度保守的NAD结合基元“TGAAGQI”和催化基元“IWGNH”.木薯MDH基因与块根淀粉合成相关,在块根发育前期表达量较低,膨大期表达量较高,膨大后期表达量逐渐降低.     

CO2倍增和UV-B辐射增强对大豆根际氨氧化细菌数量及土壤酶活的影响

以根瘤超着生大豆突变体0498和完全不结瘤的大豆突变体0701为材料,在人工气候室内模拟大气CO2浓度倍增以及UV-B辐射增强的复合逆境胁迫条件,研究其对大豆根际氨氧化细菌数量和根际土壤酶活的影响。结果表明:与对照相比,CO2浓度倍增能显著提高大豆根际氨氧化细菌的数量,增加土壤中脲酶、转化酶、酸性磷酸酶和碱性磷酸酶的活性,对完全不结瘤的0701影响更加显著;UV-B辐射增强对氨氧化细菌数量无显著影响,对土壤酶活存在一定的抑制作用。超量根瘤的存在,使CO2倍增和UV-B辐射增强对上述指标的影响得到一定的缓解。     

Coccolithophores: functional biodiversity, enzymes and bioprospecting

Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an 'in house' enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.     

iTRAQ-based quantitative proteomics analysis reveals the molecular mechanism of controlled atmosphere storage for Tibetan hull-less barley (Hordeum vulgare L) preservation

Controlled atmosphere storage (CAS) is an effective method for cereal grain preservation, but its mechanism is yet to be completely elucidated. In this study, the molecular mechanism of CAS was evaluated for Tibetan hull-less barley preservation using iTRAQ quantitative proteomics, Western blot, and physicochemical and individual volatiles analyses. In barley stored at 25 °C, compared with non-controlled atmosphere storage (NCAS), CAS altered the expression of 116 proteins by > 2.0 fold. Among these proteins, 82 downregulated proteins were mainly involved in carbon metabolism, protein and amino acid metabolism, and lipid and fatty acid degradation. Physicochemical analyses suggest that CAS could reduce the severity of abiotic stress. Bioinformatic and molecular biology analyses reveal that CAS could decrease the rates of carbon metabolism, protein and amino acid metabolism, and lipid and fatty acid degradation by downregulating some key proteins (Beta-amylase, 6-phosphofructokinase 1, isocitrate dehydrogenase, glutamate decarboxylase, 1-pyrroline-5-carboxylate dehydrogenase, seryl-tRNA synthetase, leucyl-tRNA synthetase, aspartyl-tRNA synthetase, aldehyde dehydrogenase (NAD+), and alcohol dehydrogenase), accompanied by significant reductions in oxidative dehydrogenation reactions (p < 0.05). Together, a favorable environment provided by CAS ultimately mitigated physiological abnormalities, nutrient and energy consumption, and flavor deterioration in the barley. The proteomics analysis results were confirmed by Western blot, and physicochemical and individual volatiles analyses.     

The first characterised wheat (Triticum aestivum L.) member of the nudix hydrolase family shows specificity for NAD(P)(H) and FAD

We cloned the first nudix hydrolase of wheat (Triticum aestivum L.) (TaNUDT1) and expressed it using Escherichia coli as expression host. Properties of the purified His₆-tagged protein were studied. The sequence codes for a nudix hydrolase with a molecular mass of around 43x10³ and a predicted pI of 5.68. The characteristic residues of the nudix box were highly conserved in the wheat enzyme sequence, and TaNUDT1 is most probably targeted to the peroxisomes. In the presence of dithiothreitol and MnCl₂, the enzyme hydrolyses the nicotinamide coenzymes NAD(P)(H) as could be predicted by the presence of a conserved amino acid array C-terminal to the nudix box, and the enzyme has higher affinity towards the reduced forms of the coenzymes. In light of previous reports of nudix enzymes and the physiological concentration of their substrates, we found its K_m values towards some of the coenzymes to be relatively high. As NAD(P)(H) and FAD play crucial roles in cell metabolism, extensive hydrolysis could be detrimental to cell functioning. We speculate that a decreased enzyme affinity may point to a built-in restriction on coenzyme hydrolysis in vivo. Furthermore, by hydrolysing the cofactors of several redox enzymes, this enzyme may well affect several wheat based processes.     

Endogenous redox agents and enzymes that affect protein network formation during breadmaking – A review

During breadmaking, wheat gluten proteins form a continuous network which is stabilized by disulfide bonds and modified by thiol/disulfide interchange reactions. This gluten network results in visco-elastic dough that holds together the other dough components and assists in retaining carbon dioxide. Wheat flour contains several components, enzyme co-factors and enzymes which can affect the formation and properties of the gluten network and, hence, the dough and bread characteristics. We present a brief overview of our current knowledge of the fate of gluten proteins during breadmaking, and how they are affected by endogenous wheat components (e.g. glutathione, cysteine and NAD(P)(H)) and enzyme systems (e.g. tyrosinase, peroxidase, the NADP-dependent thioredoxin and glutathione enzyme systems, protein disulfide isomerase, lipoxygenase, catalase and dehydrogenases).     

山东茶园土壤高活性解钾细菌的筛选鉴定及肥效研究

为了筛选到适合山东茶园土壤环境的解钾菌株,以提高土壤钾素的高效转化效率,本研究经过菌株富集、分离、纯化等步骤,从山东茶园土壤中分离出9株解钾细菌。通过有效钾含量比较和菌株分类,筛选出1株活性最强菌株K2,经鉴定K2菌株为枯草芽孢杆菌（Bacillus subtilis）。发酵试验表明,菌株K2最适宜生长的条件为pH值6.0,温度35℃,以麦芽糖、淀粉、玉米粉为碳源,酵母膏为氮源。施用该菌剂后土壤速效钾和速效磷含量比对照最大分别提高28.40%和28.49%。茶叶产量最大提高36.30%,同时茶叶中氨基酸含量显著增加,酚氨比值降低,有利于茶叶品质的提升。菌株K2可能是一株茶园土壤高效解钾细菌,可作为后续进一步研究茶园专用微生物菌剂的参考。     

大豆脂肪酸脱氢酶GmFAD3C-1基因的克隆及功能分析

研究通过对大豆脂肪酸脱氢酶GmFAD3家族中4个关键酶基因序列进行比对,与对照JN18相比,大豆低亚麻酸突变体MT72中GmFAD3C-1基因在起始密码子后+966bp处存在一个碱基位点的缺失（G→?）,产生移码突变,导致氨基酸序列上产生较大变化（该突变基因命名为gmfad3c-1）。构建GmFAD3C-1基因超表达及CRISPR/Cas9编辑载体,利用花粉管通道法获得T₂转化植株。脂肪酸脱氢酶酶活测定结果显示,超表达植株T₂籽粒中,酶活与对照相比上升47.62%~78.85%,编辑载体T₂籽粒中,酶活与对照相比下降25.67%~47.11%。脂肪酸相对含量测定的结果进一步表明,GmFAD3C-1基因的表达与植株亚麻酸含量密切相关。     

花生线粒体型苹果酸脱氢酶基因AhMMDH1 的结构与表达分析

苹果酸脱氢酶（malate dehydrogenase，MDH）是生物体内十分重要的一类酶，参与植物生长、花粉和果实发 育等重要生物过程的调控。当前花生中苹果酸脱氢酶在种子后熟和萌发过程中的作用研究较少。本研究克隆了 花生AhMMDH1 基因，其ORF大小为1038bp，编码345个氨基酸，是定位于线粒体的疏水蛋白。三维结构分析发现， AhMMDH1以苹果酸脱氢酶（M链）和乙醛酸前体（G链）的异源二聚体形式存在，其M链在41-47位点出现NAD+结 合位点。qRT-PCR分析发现，该基因在花中表达量较高，干种子次之，茎叶中表达痕量；不同发育时期10 ~70 d（新 鲜收获种子）种子表达水平均明显低于干种子；种子吸水16h表达量明显升高，胚根突破种皮时（吸水36h）达到最 高，随后表达量明显下降。推测该基因在种子后熟和萌发过程中起重要作用。本研究为进一步研究AhMMDH1 的 生物功能提供了依据。     

烟酰胺腺嘌呤二核苷酸(NAD)合成途径和水稻叶片早衰

叶片是植物进行光合作用的主要场所,其衰老由内源遗传发育信号和外界环境胁迫所启动,是一个非常复杂有序的调控过程。烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD)是脱氢酶的辅酶,在糖酵解、糖异生、三羧酸循环以及呼吸链等代谢中发挥着不可替代的作用。最新研究表明,水稻NAD生物合成参与调控沉默信息调控因子Sirtuins的生物活性、组蛋白H3K9去乙酰化、植物激素茉莉酸(JA)和叶片衰老。本文综述了有关水稻叶片衰老的细胞生理特征、Sirtuins酶活、NAD生物合成以及水稻早衰的OsSRT1-NAD调控途径和OsSRT1-Me OH-JA调控途径,以期阐明水稻叶片衰老的分子机理及其调控途径,为高产育种提供相应的理论参考。     

蚯蚓生物有机培肥技术(FBO)对茶园土壤微生物特征及酶活性的影响

连续5年对蚯蚓生物有机培肥的茶园土壤微生物特征以及酶活性变化情况进行了研究,分析了蚯蚓生物有机培肥技术(FBO)对茶园土壤微生物特征及酶活性的影响。结果表明,蚯蚓生物有机培肥处理(100% FBO)可以显著增加0~20 cm土层微生物量碳、呼吸速率、微生物熵、细菌、真菌和放线菌等表征土壤微生物数量和活性的指标,并可以显著提升过氧化氢酶、脲酶、转化酶和碱性磷酸酶的活性(P0.05)。在20~40 cm土层,100%FBO处理的微生物和酶活性相关指标也高于CK,但多数未达到显著水平。蚯蚓生物有机培肥技术可以显著增加表层土壤微生物的数量和活性,并提升与土壤养分转化密切相关的几类酶的活性,有利于受损茶园土壤生态系统的恢复和重建,全面提升土壤质量。     

Dietary Broccoli Sprouts Protect Against Myocardial Oxidative Damage and Cell Death During Ischemia-Reperfusion

Cruciferous vegetables are known for antioxidant and anti-carcinogenic effects. In the current study we asked whether dietary broccoli sprouts can protect the heart from ischemia-reperfusion. Rats were fed either control diet (sham and control groups) or a diet mixed with 2% dried broccoli sprouts for 10 days. After 10 days the isolated hearts were subjected to ischemia for 20 min and reperfusion for 2 h, and evaluated for cell death, oxidative damage, and Nrf2-regulated phase 2 enzyme activities. Broccoli sprouts feeding inhibited markers of necrosis (lactate dehydrogenase release) and apoptosis (caspase-3 activity) by 78–86%, and decreased indices of oxidative stress (thiobarbituric acid reactive substances and aconitase inactivation) by 82–116%. While broccoli sprouts increased total glutathione and activities of the phase 2 enzymes glutamate cysteine ligase and quinone reductase in liver, they did not affect these in ischemic-reperfused heart. While the mechanism is not clear, the results show that a relatively short dietary treatment with broccoli sprouts can strongly protect the heart against oxidative stress and cell death caused by ischemia-reperfusion.     

华南地区酸雨对花生土壤微生物的影响

本文通过盆栽试验研究了不同酸度酸雨处理对华南地区花生土壤细菌、放线茵、真菌数量的影响。结果表明，pH3．5及以下酸雨处理对花生土壤真菌数量具有较大的抑制作用；在绝大多数生长时期，pH4．0及以下酸雨处理对花生土壤放线茵数量具有较大的抑制作用；pH4．5及以下酸雨处理在花生生长前期对土壤细菌数量具有促进作用，在花生生长中后期却具有较大的抑制作用。酸雨对土壤三大微生物类群的影响表现出不同的规律，具有一定的复杂性。     

稻蟹共作模式生态效应研究进展

主要从土壤、水环境和杂草防控三个方面,对目前我国稻蟹共作模式下田间生态的研究现状进行了总结与分析.提出了几种目前比较常见的稻蟹共作模式,即有机稻蟹和常规稻蟹两种普遍模式.分析了稻蟹共作对土壤理化性质、土壤酶活性和根际微生物区系的影响.养蟹稻田可以显著降低土壤容重,而总孔隙度及全氮、全磷、碱解氮、有效磷、速效钾和土壤有机质含量都显著增加;显著提高0~10 cm土壤脲酶、蛋白酶、脱氢酶以及过氧化氢酶的活性;土壤根际微生物以细菌为主要菌群,氨化细菌、反硝化细菌、嫌气性纤维素分解菌、好气性自生固氮菌、亚硝酸细菌数量显著高于单作水稻;与常规稻蟹相比,有机稻蟹的土壤性状表现更好.稻蟹模式下浮游动植物的多样性和生物量显著低于单作水稻,而水体中溶解氧(DO)明显偏低,硝酸盐、磷酸盐含量显著高于不养蟹稻田.稻田养蟹能够显著抑制稻田4种优势杂草的密度,对于稻田杂草的防控效果明显.     

草铵膦对麦田土壤微生物数量及酶活性的影响

为给草铵膦的科学使用提供理论依据,设置5个不同草铵膦施用量(0、2、4、6、8L·hm^-2),研究了草铵膦对麦田土壤微生物(细菌、放线菌、真菌)数量及其土壤酶(过氧化氢酶、磷酸酶、脲酶、脱氢酶)活性的影响。结果表明,随着草铵膦施用量的增加,土壤微生物数量随着小麦生育期的推进呈现先增加后减少趋势;在草铵膦施用量为6L·hm^-2时,微生物数量均高于其他处理;细菌和放线菌数量较对照增加,其增加幅度随着小麦生育期的推进逐渐增大,成熟期达到显著水平;喷施草铵膦对真菌数量影响不显著。在低浓度(2、4和6L·hm^-2)草铵膦处理下,被测土壤酶活性均随着草铵膦施用量的增加而增大;在高浓度(8L·hm^-2)草铵膦处理下,酶活性均受到抑制,其中过氧化氢酶与其他处理差异显著。在本试验的条件下,推荐麦田草铵膦的施用量为4～6L·hm^-2。     

Protein supplementation of formic acid- and enzyme-treated silages. 2. Nitrogen and amino acid digestion

Two silages were made from primary growth perennial ryegrass and ensiled after the application of either formic acid or an enzyme mixture of cellulase and hemicellulase. Silage analysis showed both silages to be well preserved with low pH of 3·70 and 3·62 for the formic and enzyme treatments respectively. Formic acid-treated silage had a higher total amino acid concentration than enzyme-treated silage. The silages were offered to growing steers either as the sole diet or supplemented with rapeseed meal at 60 g or 120 g fresh weight kg⁻¹ silage DM offered, in a 6 × 6 Latin square arrangement.
Non-ammonia nitrogen and microbial nitrogen flows at the duodenum (g d⁻¹) were significantly ( P < 0·05) increased by supplementation of enzyme-treated silage compared with formic acid-treated silage (enzyme, 83·6, 58·7; enzyme + 60 g, 101·7, 75·3; enzyme + 120 g, 112·5, 80·7; formic, 91·9, 63·7; formic + 60g, 88·3, 67·9; formic + 120 g, 95·5, 67·1) respectively. Efficiencies of microbial protein synthesis were increased for supplemented enzyme-treated silage diets and values were reduced for supplemented formic acid-treated silage diets compared with the silage only diets (enzyme, 27·9; enzyme + 60 37·7; enzyme + 120 g, 38·6; formic, 33·7; formic + 60g, 31·2; formic + 120 g, 28·8). Total amino acid flow at the duodenum increased with supplementation of both silages; however, microbial amino acid flow increased significantly ( P < 0·05) with supplementation of enzyme-treated silage compared with formic acid-treated silage diets. Significantly greater amounts of cystine, methionine, alanine, valine and aspartic acid entered the small intestines of animals receiving supplemented enzyme silages compared with supplemented formic acid silages.     

水稻不同生育期的土壤微生物量和酶活性的变化

 研究了全生育期淹水栽培条件下，水稻不同生长阶段的土壤微生物生物量C、土壤微生物生物量N、呼吸作用强度、代谢商、土壤酶活性及水稻叶绿素、脯氨酸、过氧化物酶（POD）活性的变化特征。随着水稻生长，土壤微生物生物量C、土壤微生物生物量N、呼吸作用强度表现为先升后降，到成熟期有所回升的变化规律，且在水稻不同生育阶段差异显著，而土壤微生物代谢商一直下降。土壤脲酶活性表现为先升后降，而酸性磷酸酶与脱氢酶活性则表现为先降后升再降的变化规律。脲酶及酸性磷酸酶活性在水稻移栽后的30 d左右形成峰值，而脱氢酶活性则在50 d左右形成峰值，且在水稻不同生育阶段差异显著。随着水稻生长，水稻叶片中的叶绿素含量表现为先降后升的变化规律，在移栽后70 d左右形成峰值，然后急剧下降，POD的活性则表现为逐渐增强，而脯氨酸含量表现为逐渐下降，不过上述水稻生理指标与土壤各生物化学指标间不存在显著的相关性。试验结果表明在水稻 土壤 微生物相互作用的体系中，土壤微生物量和酶活性明显受到水稻生长发育的影响。     

An In-Silico Comparative Study of Lipases from the Antarctic Psychrophilic Ciliate Euplotes focardii and the Mesophilic Congeneric Species Euplotes crassus: Insight into Molecular Cold-Adaptation

Cold-adapted enzymes produced by psychrophilic organisms have elevated catalytic activities at low temperatures compared to their mesophilic counterparts. This is largely due to amino acids changes in the protein sequence that often confer increased molecular flexibility in the cold. Comparison of structural changes between psychrophilic and mesophilic enzymes often reveal molecular cold adaptation. In the present study, we performed an in-silico comparative analysis of 104 hydrolytic enzymes belonging to the family of lipases from two evolutionary close marine ciliate species: The Antarctic psychrophilic Euplotes focardii and the mesophilic Euplotes crassus. By applying bioinformatics approaches, we compared amino acid composition and predicted secondary and tertiary structures of these lipases to extract relevant information relative to cold adaptation. Our results not only confirm the importance of several previous recognized amino acid substitutions for cold adaptation, as the preference for small amino acid, but also identify some new factors correlated with the secondary structure possibly responsible for enhanced enzyme activity at low temperatures. This study emphasizes the subtle sequence and structural modifications that may help to transform mesophilic into psychrophilic enzymes for industrial applications by protein engineering.     

杉木纯林及杉桐混交林土壤酶活性研究

以3~5 a生杉木(Cunninghamia lanceolata)—千年桐(Aleurites montana)混交林与杉木纯林2种营造模式下不同土层(0~10、10~20、20~30 cm)土壤作为研究对象,探讨杉木纯林和杉桐混交林(Cunninghamia lanceolataAleurites montana mixed forest)的土壤脲酶(URE)、过氧化氢酶(CAT)、酸性磷酸酶(ACP)和蔗糖酶(INV)活性变化规律。结果表明:(1)在同一造林模式同一林龄不同土层间,4种酶在土壤表层含量最高,随着土层加深,各林下土壤ACP、URE和INV均呈明显下降趋势,CAT活性无差异性。(2)同一造林模式同一土层不同林龄间,杉木纯林和杉桐混交林土壤酶活性均随林龄的增大而变大,但不同酶的变化趋势不同。(3)同一林龄同一土层不同造林模式间,杉桐混交林的土壤ACP、URE及INV活性总体高于杉木纯林。(4)不同林地和土层的土壤酶活性之间存在着极显著的相关性(p<0.01)。混交模式下的林地土壤酶活性均高于杉木纯林模式,土壤生物学性质更加完善。