Trehalose synthesis by sequential reactions of recombinant maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase from Brevibacterium helvolum

A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium Brevibacterium helvolum. The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides. Recombinant BvMTS, BvMTH, and fusion gene BvMTSH, constructed by insertion of an adenylate in the overlapping region, were expressed in Escherichia coli. Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose. The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose. Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units. The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes. The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production. Addition of alpha-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.     

Crystal structure of maltooligosyltrehalose trehalohydrolase from Deinococcus radiodurans in complex with disaccharides

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranose) is a non-reducing diglucoside found in various organisms that serves as a carbohydrate reserve and as an agent that protects against a variety of physical and chemical stresses. Deinococcus radiodurans possesses an alternative biosynthesis pathway for the synthesis of trehalose from maltooligosaccharides. This reaction is mediated by two enzymes: maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase). Here, we present the 1.1A resolution crystal structure of MTHase. It consists of three major domains: two beta-sheet domains and a conserved glycosidase (beta/alpha)8 barrel catalytic domain. Three subdomains consisting of short insertions were identified within the catalytic domain. Subsequently, structures of MTHase in complex with maltose and trehalose were obtained at 1.2 A and 1.5 A resolution, respectively. These structures reveal the importance of the three inserted subdomains in providing the key residues required for substrate recognition. Trehalose is recognised specifically in the +1 and +2 binding subsites by an extensive hydrogen-bonding network and a strong hydrophobic stacking interaction in between two aromatic residues. Moreover, upon binding to maltose, which mimics the substrate sugar chain, a major concerted conformational change traps the sugar chain in the active site. The presence of magnesium in the active site of the MTHase-maltose complex suggests that MTHase activity may be regulated by divalent cations.     

Over-expression of BvMTSH,a fusion gene for maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase,enhances drought tolerance in transgenic rice

Plant abiotic stress tolerance has been modulated by engineering the trehalose synthesis pathway. However, many stress-tolerant plants that have been genetically engineered for the trehalose synthesis pathway also show abnormal development. The metabolic intermediate trehalose 6-phosphate has the potential to cause aberrations in growth. To avoid growth inhibition by trehalose 6-phosphate, we used a gene that encodes a bifunctional in-frame fusion (BvMTSH) of maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH) from the nonpathogenic bacterium Brevibacterium helvolum. BvMTS converts maltooligosaccharides into maltooligosyltrehalose and BvMTH releases trehalose. Transgenic rice plants that over-express BvMTSH under the control of the constitutive rice cytochrome c promoter (101MTSH) or the ABA-inducible Ai promoter (105MTSH) show enhanced drought tolerance without growth inhibition. Moreover, 101MTSH and 105MTSH showed an ABA-hyposensitive phenotype in the roots. Our results suggest that over-expression of BvMTSH enhances drought-stress tolerance without any abnormal growth and showes ABA hyposensitive phenotype in the roots. [BMB Reports 2014; 47(1): 27-32]     

Bifunctional recombinant fusion enzyme between maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase of thermophilic microorganism Metallosphaera hakonensis

MhMTS and MhMTH are trehalose (alpha-D-glucopyranosyl- [1,1]-alpha-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused inframe in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around 70 degrees and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at 70 degrees for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above 80 degrees . The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.     

Identification of maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase enzymes catalysing trehalose biosynthesis in Anabaena 7120 exposed to NaCl stress

Anabaena 7120 cells were exposed to NaCl (25-175 mM) stress. Maximum growth was recorded in media containing 150mM NaCl. Short-term exposure (48h) of the cyanobacterial biomass to 150mM NaCl, induced highest trehalose level (37mM). Control cells lacking NaCl did not show any trace of trehalose as ascertained by NMR and HPLC analysis. Trehalose biosynthesis observed with NaCl plus high temperature (40 degrees C) indicated that its production was specifically triggered by NaCl, not temperature. The increase in trehalose level during NaCl stress was the result of overexpression of the trehalose-forming enzymes maltooligosyltrehalose synthase (MTSase), EC 5.4.99.15 (114kDa) and maltooligosyltrehalose trehalohydrolase (MTHase), EC 3.2.1.141 (68 kDa) as evidenced by SDS-PAGE analysis. To our knowledge this is the first report of induced trehalose biosynthesis in Anabaena 7120 during salt-stress, accompanied by identification of MTSase and MTHase enzymes on gel. It is suggested that Anabaena 7120 cells synthesize the osmolyte trehalose to withstand osmotic fluctuations.     

发菜藻蓝蛋白分离纯化的研究

以发菜为材料,比较了提取液类型和饱和硫酸铵浓度对藻蓝蛋白提取的影响,并对藻蓝蛋白的提取程序和部分特性进行了研究。结果表明:50 mmol/L KP缓冲液(pH值7.2)是合适的提取液,体积分数为40%~50%饱和硫酸铵盐析效果优于其它浓度。经过DEAE-Toyopeal 650 S离子交换层析和SuperdexTM200凝胶过滤层析后,藻蓝蛋白纯度达6.2,最大吸收峰位于615 nm,荧光发射峰位于649 nm,由α和β2个亚基组成,其分子质量分别为18 051.17和19 142.27 Da。因此,发菜藻蓝蛋白分离纯化较为理想的程序为:藻粉→50 mmol/L KP缓冲液(pH值7.2)浸泡→French pressure(1 500 kg/cm2)破碎细胞→40%~50%饱和硫酸铵盐析→DEAE-Toyopeal 650 S离子交换层析→SuperdexTM200凝胶过滤层析→较纯的藻蓝蛋白。     

Molecular cloning of a PR-5 like protein gene from cherry tomato and analysis of the response of this gene to abiotic stresses

LePR-5, a putative PR5 like protein gene was amplified from a cherry tomato (Lycopersicon esculentum), which encodes a precursor protein of 250 amino acid residues, and shares high degrees of homology with a number of other PR5 genes. Expression of LePR-5 in different tomato organs was analyzed with Semi-quantitative RT–PCR, showing that LePR-5 expressed at different levels in leaves, stems, roots, flowers and fruits. In addition, expression of LePR-5 under different abiotic stresses was carried out at different time points. Three of the four tested abiotic stimuli, ethophen, salicylic acid and methyl jasmonate, triggered a significant induction of LePR-5 after treatment. However, LePR-5 was weaker induced by abscisic acid than by others. The positive responses of LePR-5 to the three abiotic stimuli suggested that LePR-5 may play an important role in response to abiotic stresses, and it may also be involved in plant defense system against pathogens. In addition, different expression patterns between tomato fruit and seedling suggested that LePR-5 may play a distinctive role in the defensive system protecting tomato fruit and seedling.     

发状念珠藻胞外多糖的纯化与性质分析

采用DEAE阴离子交换层析和Sephadex G100凝胶层析对液体悬浮培养发状念珠藻胞外多糖进行纯化, 得到两个组分NFPS1和NFPS2。对组分NFPS2进行理化性质分析, 并与野生发状念珠藻多糖NFPS0的性质进行对比。结果表明二者具有相似的单糖组成, 均为葡萄糖、木糖、半乳糖、甘露糖; 表观分子量分别为2.79×105、2.26×105; 均不含核酸、蛋白质等物质, 是非硫酸化多糖; 有较高的热稳定性, 其降解温度在245oC左右。但在微观结构上, 两者存在一定差别。     

发状念珠藻胞外多糖的纯化与性质分析

采用DEAE阴离子交换层析和Sephadex G100凝胶层析对液体悬浮培养发状念珠藻胞外多糖进行纯化, 得到两个组分NFPS1和NFPS2。对组分NFPS2进行理化性质分析, 并与野生发状念珠藻多糖NFPS0的性质进行对比。结果表明二者具有相似的单糖组成, 均为葡萄糖、木糖、半乳糖、甘露糖; 表观分子量分别为2.79×105、2.26×105; 均不含核酸、蛋白质等物质, 是非硫酸化多糖; 有较高的热稳定性, 其降解温度在245oC左右。但在微观结构上, 两者存在一定差别。     

Identification of novel smORFs and microprotein acting in response to rehydration of Nostoc flagelliforme

Nostoc flagelliforme, a terrestrial cyanobacterium spread throughout arid and semi-arid areas, has been long known for its outstanding adaptability to extremely dry conditions. This microorganism is able to recover biological activities within hours after months of anhydrobiosis state, attracting investigation through proteomic analysis. Except for canonical proteome, microproteins encoded by small ORFs (smORFs) have recently been regarded as indispensable participants in metabolic processes. However, the involvement of smORFs in N. flagelliforme remains unknown. Here we first constructed a smORF database in N. flagelliforme using bioinformatic prediction, resulting in 6072 novel smORFs. Then LS-MS/MS analysis was applied to identify expression patterns of microproteins and seek smORFs and their encoded microprotein playing a role during rehydration. In total, 18 novel microproteins were mined based on a smORF searching strategy combined with three proteomic assays, of which five were annotated as ribosomal proteins, one as RNA polymerase subunit, and one as acetohydroxy acid isomeroreductase. We also suggested the possible functions of smORFs according to their expression pattern and discovered two neighboring and homologous smORFs. All these results will expand our knowledge of smORFs-encoded microproteins and their relation to the stress response of extremophilic microorganisms.     

发菜藻蓝蛋白基因克隆及原核表达

发菜（Nostoc flagelliforme）是一种陆生固氮蓝藻,具有重要的经济和生态价值。运用双向电泳技术、MALDI-TOF-TOF/MS鉴定和数据库检索,获得藻蓝蛋白部分氨基酸序列并设计简并性引物,克隆藻蓝蛋白基因并研究其表达。结果表明,发菜藻蓝蛋白α和β亚基两个基因的编码序列及两者之间的间隔序列全长为1097bp,编码β亚基和α亚基的基因序列全长分别为519bp和489bp,β亚基基因序列位于α亚基基因序列上游,两者之间通过89bp的基因片段连接,GenBank登录号为GU549478,并对推译的α和β亚基三维结构进行了预测。将藻蓝蛋白的α和β亚基基因在大肠杆菌中表达,获得了符合预期的外源重组蛋白。研究结果为进一步研究发菜藻蓝蛋白的分子结构及生物学功能奠定了基础。     

干旱胁迫条件下发菜Ferritin差异表达与基因克隆

发菜(Nostoc flagelliforme)是一种陆生固氮蓝藻,具有强烈的旱生生态适应性.运用双向电泳技术、凝胶图像分析、MALDI-TOF-TOF/MS质谱鉴定和数据库检索,发现发菜Ferritin在干旱胁迫条件下表达量逐渐降低.根据鉴定的Ferritin已知氨基酸序列设计简并性引物克隆该基因,获得了长度为540 bp的DNA,GenBank登陆号为HM854287.序列比较显示该基因具有较高的保守性,蛋白质二级结构主要由α螺旋和随机卷曲构成.RT-PCR分析表明,Ferritin mRNA在干旱胁迫条件下表达量逐渐降低,与Ferritin的表达趋势一致.将Ferritin基因在大肠杆菌中表达,获得符合预期的外源重组蛋白(22.4 kD).实验结果可为进一步研究发菜耐旱的分子机理及探讨发菜对极端干旱环境的适应和保护机制奠定基础.     

念珠藻发菜的水分生理特性

对野生风干发菜水分生理指标进行了测定与分析,结果表明:风干含水量为7.3%～11.2%,饱和含水量为616.0%～1258.4%,相对含水量1%左右,水分饱和亏为99%左右,水势为-8.2MPa;最大吸水速率为8.029gH2O·g-1·min-1,饱和吸水后其体积膨大10～14倍,平均吸胀率为1286.9%,其中伸长率为23.5%,增粗率为235.0%;导水力极弱;干燥发菜具有超强的吸水力,能在相对湿度>28.4%的空气中吸取水分;发菜的保水力随环境而变,在阴暗或弱光照的环境中具有较强的保水力.另外,探讨了不同种源发菜的水分生理指标与环境因子的关系.     

发菜细胞培养物对盐胁迫的响应

用不同浓度(0、0.1、0.2、0.4 m o l.L-1)的N aC l处理BG 110培养的发菜细胞,结果显示,发菜光合速率与叶绿素荧光强度随N aC l浓度的升高先增加后降低,当N aC l浓度为0.1 m o l.L-1时光合速率与叶绿素荧光具有最大值,表明发菜细胞培养物能耐受一定浓度的盐胁迫.以BG 110+0.4 m o l.L-1N aC l为对照,在BG 11+0.4m o l.L-1N aC l的胁迫实验中,光合速率与叶绿素荧光强度下降较慢;丙二醛、脯氨酸含量较低;类胡萝卜素含量较高,表明在培养液中添加外源硝酸盐后可以缓解N aC l对发菜细胞培养物的生理胁迫效应,增强其抗盐性.     

发菜培养条件的研究(简报)

在Ｃａ＾２＋、１０ｍｇ．Ｌ＾－１ＮＡＡ、１０ｍｇ．Ｌ＾－１ＧＡ作用下,干湿节律为干１ｄ、湿１ｄ发菜生长有较大的促进作用,生长１４ｄ发菜灌体最高增长率为３５％．     

干燥和复吸水条件下发菜Mn-CAT差异表达与基因克隆

运用双向凝胶电泳技术、MALDI-TOF-TOF/MS质谱鉴定和数据库检索,分析发菜锰过氧化氢酶(Mn-CAT)在干燥和复吸水后的差异表达水平,根据Mn-CAT鉴定的已知氨基酸序列设计简并引物克隆该基因,并研究其原核表达.结果表明:干燥发菜复吸水后Mn-CAT表达量明显高于干燥状态下的表达量.使用简并引物克隆获得长度为693 bp的Mn-CAT基因(GenBank登录号为GU549477).将Mn-CAT基因在大肠杆菌中表达,获得1个约26 kD的外源蛋白,经Western blotting验证,该外源蛋白为Mn-CAT.研究结果为进一步研究发菜耐旱的分子机理、探讨发菜对极端干旱环境的适应机制奠定了基础.