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.     

玫瑰微球菌中treZ基因在毕赤酵母中的表达研究

首次将玫瑰微球菌中麦芽寡糖基海藻糖水解酶(MTHase)treZ基因序列连接到表达载体pPICZαA中,通过电转化法将构建好的表达载体分别转入巴斯德毕赤酵母GS115和KM71菌株中.利用含有Zeocin的YPD平板筛选到阳性转化子,并经PCR、SDS-PAGE电泳以及Western blot最终验证海藻糖水解酶基因treZ已经整合到巴斯德毕赤酵母的基因组上,并且得到了预期的表达.     

来源于玫瑰微球菌的麦芽寡糖基海藻糖水解酶基因在大肠杆菌中的融合表达

设计引物克隆玫瑰微球菌QS412中麦芽寡糖基海藻糖水解酶(MTHase)的基因treZ,通过与pET-28a( )载体相连,转化入宿主菌E.coli BL21,进行发酵诱导。通过SDS-PAGE检测到外源基因在大肠杆菌中有很高的MTHase表达量,但大部分都以不溶性包含体形式存在。对菌体超声破碎全菌液检测酶活,结果显示了水解酶酶活。这是来源于微球菌属的麦芽寡糖基海藻糖水解酶首次获得基因克隆和活性表达,为进一步提高酶活、增大海藻糖产量奠定了基础。     

嗜酸热硫化叶菌麦芽寡粉基海藻糖合酶基因的克隆和表达

The gene of MTSase (maltooligosyltrehalose synthase) from Sulfolobus acidocaldarius ATCC49426 was amplified by PCR. The primers were designed according to the published sequence of homologous gene from Sulfolobus acidocaldarius ATCC33909. This gene was inserted into the plasmid pBV220 and the resultant recombinant plasmid pBV220-GT was transformed to E. coli DH5 alpha. The activity of recombinant enzyme was about 10 u/g(wet cell). In order to improve the expression level of target protein, some nucleotides in the 3' and 5' of the gene were modified to optimize the second structure of mRNA by PCR amplification using the new primers devised according to the biosoftware GOLDKEY2.0. As a result, the activity of recombinant enzyme increase to 19.8 u/g(wet cell). Then, the helping plasmid pUBS520 which carried the gene encoding the tRNA of rare codons AGG and AGA was transformed to the recombinant strain. But it took little effect.     

海藻糖合酶产生菌筛选、鉴定及目的基因克隆

目的:为筛选出一株产海藻糖合酶的菌株,并以此菌的全DNA为模板,克隆出产海藻糖合酶的目的基因片段。方法:实验过程中采用了常规筛选菌种、快速提取细菌全基因、显微镜观察菌种、热启动PCR技术、电泳纯化回收基因片段、EcoRⅠ和HindⅢ双酶切鉴定目的基因片段等方法。结果:在电镜下可观察到有芽孢、杆菌;菌株16S rRNA基因扩增产物共计1490个碱基;PCR方法扩增出阳性克隆大约1700bp的基因片段。结论:通过生理、形态、结构特征分析及16S rRNA基因全序列比较得出结论:筛选到一株短小芽孢杆菌;PCR扩增出阳性克隆片段,全长1722bp,为实验所要的编码海藻糖合酶的基因片段。     

嗜酸热硫化叶菌麦芽寡糖基海藻糖合酶基因的克隆和表达

麦芽寡糖基海藻糖合酶( Maltodigosyltrehalose synthase;MTSase)是近年来在一些微生物中发现的新型分子内糖苷转移酶,能将淀粉或淀粉部分水解物(大于3个葡糖基)的糖链还原末端的α-1,4糖苷键转化为α-1,1糖苷键,生成具海藻糖末端结构的产物[1],如下所示: Gn为聚合度n(n＞3)的麦芽寡糖,Gn-2-T为麦芽寡糖基海藻糖,-T为海藻糖基.     

Overproduction of thymidine by recombinant Brevibacterium helvolum amplified with thymidine monophosphate phosphohydrolase gene from bacteriophage PBS2

A microbial fermentation process could be used to produce thymidine biologically but many of the enzymes related to nucleotide biosynthesis are highly regulated. To overcome the complex regulation steps, an analogue mutant of Brevibacterium helvolum resistant to fluorouracil, hydroxyurea, and trimethoprim was constructed. This mutant accumulated 380 mg thymidine 1(-1) in 16 h in shake-flask culture. However, the accumulation of thymidine monophosphate (TMP) inside the cells suggested a low activity of nucleotidase which degrades TMP to thymidine. This limitation was overcome by cloning the TMP phosphohydrolase (TMPase) gene of the unusual bacteriophage, PBS2. As a result, TMP in recombinant cells decreased from 230 micromol g(-1) cell to 20 micromol g(-1) cell with accumulation of 500 mg thymidine l(-1) in the medium.     

Purification and Characterization of a Trehalose Synthase from the Basidiomycete Grifola frondosa

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from d-glucose and α-d-glucose 1-phosphate (α-d-glucose 1-P) was detected in a basidiomycete, Grifola frondosa. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to d-glucose and α-d-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35°C and 35 to 37.5°C, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only α-d-glucose 1-P served as a donor substrate with d-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the α-d-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with d-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5°C, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO₄ to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.Trehalose (1-α-d-glucopyranosyl-α-d-glucopyranoside) is a nonreducing disaccharide with an α,α-1,1 glycosidic linkage and is widely distributed in plants, insects, fungi, yeast, and bacteria (7). Due to the absence of reducing ends in trehalose, it is highly resistant to heat, pH, and Maillard’s reaction (24). In trehalose-producing organisms, this compound may serve as an energy reserve, a buffer against stresses such as desiccation and freezing, and a protein stabilizer (5, 7, 26, 31, 32). If trehalose can be produced economically, then it has potential commercial applications as a sweetener, a food stabilizer, and an additive in cosmetics and pharmaceuticals (6, 25). Recently, trehalose production through fermentation of yeast (17) and Corynebacterium (30), enzymatic processes from starch (18, 34) and maltose (19, 22, 23, 33), and extraction from transformed plants (10) has been reported.Our approach to trehalose production is to use an enzymatic process to produce trehalose from sucrose, one of the least expensive sugars. Since sucrose is efficiently converted to α-d-glucose 1-phosphate (α-d-glucose 1-P) and fructose by sucrose phosphorylase (SPase), we screened microorganisms for an enzyme that converts α-d-glucose 1-P to trehalose on the assumption that the combination of the putative trehalose synthase (TSase) and SPase would convert sucrose into trehalose. Although similar enzyme activities have been reported in the basidiomycete Flammulina velutipes (11) and in the yeast Pichia fermentans (27), these enzymes have not been well characterized.Our objectives were (i) to screen microorganisms, primarily fungi, for TSase activity; (ii) to purify and characterize the TSase; (iii) to identify the enzymatic process by which trehalose is produced from sucrose; and (iv) to identify an enzymatic process for production of trehalose from sucrose in which the fructose component is also converted to trehalose.     

Enzymatic Synthesis of Trehalose from Maltose

Synthesis of trehalose from maltose by a coupled enzyme system with trehalose Phosphorylase and maltose Phosphorylase has been studied. Trehalose Phosphorylase was partially purified from Euglena gracilis and maltose Phosphorylase was obtained from Lactobacillus brevis. The optimum pH of the reaction was 6.5~7.0 and the reaction rate was faster in the rection mixture containing a low concentration of phosphate. The final ratio of conversion (the ratio of trehalose to maltose) in the pH range between 6.0 and 8.0 was about 60%.

Immobilized maltose and trehalose Phosphorylase in κ-carageenan could be used without any appreciable loss of activity for batch reactions at least 10 times.     

海藻糖合酶在毕赤酵母表面的展示

海藻糖合酶能够利用麦芽糖一步法转化生产海藻糖,其底物专一性较高,该酶体系生产工艺简单,不受底物麦芽糖浓度的影响,是工业生产海藻糖的首选。为获得具有生产海藻糖合酶能力的毕赤酵母表面展示载体,实验以筛选的Pseudomonas putide P06海藻糖合酶基因为模板,PCR扩增得到海藻糖合酶基因(tres,2064 bp),连接至pPICZαA质粒中,获得重组质粒pPICZαA-tres。以来自酿酒酵母的共价连接细胞壁的Pir系列蛋白的Pir1p成熟肽蛋白作为毕赤酵母表面展示的锚定蛋白,利用PCR技术扩增得到pir1p(847 bp),连接至重组质粒pPICZαA-tres中,获得重组质粒pPICZαA-tres-pir1p。将重组质粒电击转入毕赤酵母GS115中,利用α-factor信号肽将蛋白引导分泌至细胞壁展示于毕赤酵母表面。通过Zeocin抗性筛选,挑选出阳性克隆子并摇瓶发酵。发酵产物经离心、破碎并使用昆布多糖酶水解,洗脱,结果显示,SDS-聚丙烯酰胺凝胶电泳分析可见明显融合蛋白条带,表明海藻糖合酶已成功地锚定在毕赤酵母。将重组毕赤酵母使用pH 7.5的缓冲液清洗并重悬,与底物浓度为30%的麦芽糖在30℃~60℃水浴条件下作用2 h,反应产物利用HPLC检测,能够检测到酶学活性。在优化后的条件pH 7.5,50℃,表面展示海藻糖合酶酶活达到300.65 U/g。40℃~50℃酶活较稳定,保温60 min,残留酶活相对活力达75%以上;最适反应pH值为7.5,并在碱性环境下稳定。     

大肠杆菌海藻糖合成酶基因的克隆和表达

利用Ｍｕ转座子细胞内克隆了大肠杆菌海藻糖合成酶 ｏｔｓＢＡ基因,克隆频率为１．４５ ｘ １０(－３)／ Ｋａｎ(ｒ)转导子。经遗传互补、酶切和部分序列分析表明ｏｔｓＢＡ基因位于克隆质粒。亚克隆 ２．８７ｋｂ ＤＮＡ片段至不同拷贝数表达质粒并分别转化大肠杆菌ｏｔｓＢＡ基因缺失株,转化株恢复 在０．５ｍｏｌ／Ｌ ＮａＣｌ培养基上生长的功能,高渗透压诱导实验表明,转化株能够合成克隆基因 产物海藻糖,但合成量不受克隆质粒拷贝数影响。海藻糖良好的抗高渗能力可能在农作物育 种方面发挥重要作用。为构建含有海藻糖合成酶基因的植物表达载体,并在农杆菌的介导下 转入植物,赋予其抗高渗、耐干旱能力奠定了重要的研究基础。     

Identification and Characterization of a Novel Trehalose Synthase Gene Derived from Saline-Alkali Soil Metagenomes

A novel trehalose synthase (TreS) gene was identified from a metagenomic library of saline-alkali soil by a simple activity-based screening system. Sequence analysis revealed that TreS encodes a protein of 552 amino acids, with a deduced molecular weight of 63.3 kDa. After being overexpressed in Escherichia coli and purified, the enzymatic properties of TreS were investigated. The recombinant TreS displayed its optimal activity at pH 9.0 and 45 °C, and the addition of most common metal ions (1 or 30 mM) had no inhibition effect on the enzymatic activity evidently, except for the divalent metal ions Zn²⁺ and Hg²⁺. Kinetic analysis showed that the recombinant TreS had a 4.1-fold higher catalytic efficientcy (Kcat/K _m) for maltose than for trehalose. The maximum conversion rate of maltose into trehalose by the TreS was reached more than 78% at a relatively high maltose concentration (30%), making it a good candidate in the large-scale production of trehalsoe after further study. In addition, five amino acid residues, His172, Asp201, Glu251, His318 and Asp319, were shown to be conserved in the TreS, which were also important for glycosyl hydrolase family 13 enzyme catalysis.     

多酶复配合成海藻糖及其分离提取的研究

以大米淀粉为原料,多酶复配制备海藻糖。确定了实验室条件下多酶复配生产海藻糖的最佳条件:以15%(m/V)大米淀粉为底物,催化温度45℃、pH 6. 0、DE值16、α/β-CGTase加量为1. 4U/ml、催化28h后糖化处理12h,海藻糖转化率由双酶法催化的50%提高至73%。在底物浓度为25%(m/V)时,海藻糖产量最高达到182. 5g/L,随后对高浓度海藻糖进行分离提取,分别考察了活性炭脱色、离交分离、浓缩结晶等对海藻糖提取效率的响。     

利用定点突变分析海藻糖合酶的功能

我们通过对来自红色亚栖热菌(Meiothermus ruber) CBS-01中的海藻糖合酶(Trehalose synthase)序列比对及三维模型构建, 我们构建了D200G/H165R, R227C, R392A三个定点突变体, 检测其对麦芽糖及海藻糖的转化能力。结果发现: 在50°C时, D200G/H165R、R392A基本失去其原有活性, 而R227C产生海藻糖的能力降低。37°C时, D200G/H165R失去转化能力, 而R392A及R227C保有部分能力。因此我们推测, R392位点可能是维持酶的结构及热稳定性的关键位点, 而D200位点在反应过程中也起重要作用。     

对一株藤黄微球菌合成海藻糖能力的鉴定

报道了一株藤黄微球菌 (Micrococcusluteus)具有产酶能力 ,可以淀粉糊精为底物合成海藻糖 ,从还原糖含量变化、纸层析和高效阴离子交换 脉冲安培法检测几方面对酶反应予以证实。     

Isolation and Identification of a Thermophilic Strain Producing Trehalose Synthase from Geothermal Water in China

A slightly thermophilic strain, CBS-01, producing trehalose synthase (TreS), was isolated from geothermal water in this study. According to the phenotypic characteristics and phylogenetic analysis of the 16s rRNA gene sequence, it was identified as Meiothermus ruber. The trehalose synthase gene of Meiothermus ruber CBS-01 was cloned by polymerase chain reaction and sequenced. The TreS gene consisted of 2,895 nucleotides, which specified a 964-amino-acid protein. This novel TreS catalyzed reversible interconversion of maltose and trehalose.     

玫瑰链霉菌海藻糖合成酶基因克隆、表达及功能鉴定

目的:克隆玫瑰链霉菌海藻糖合成酶基因(Srt)使其在大肠杆菌XL10-Gold中高效表达,并对重组酶的酶学特性进行研究。方法:利用PCR技术从玫瑰链霉菌中克隆到一段长1 704bp的海藻糖合成酶基因(Srt),构建重组表达质粒pSE380-Srt-treS,将其转化大肠杆菌XL10-Gold中诱导表达,对重组纯酶进行SDS-PAGE分析及酶学特性测定。结果:SDS-PAGE显示在65kDa处有明显单一蛋白条带。该酶可催化麦芽糖和海藻糖之间的可逆反应,海藻糖得率达82%,且含有很低的副产物葡萄糖(5%左右)。最适反应温度和pH分别为30℃、7.5,Cu2+、Zn2+和Tris能明显抑制酶活力。该酶还可催化蔗糖生成一种无龋齿,适合糖尿病患者食用的糖类-海藻酮糖。结论:成功克隆表达了一个海藻糖合成酶基因,该酶转化率高,副产物较少,为工业酶法生产海藻糖奠定基础。     

Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli

Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h). No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose.     

Enzymatic Reactions by Five Chalcone Synthase Homologs from Hop (Humulus lupulus L.)

The enzyme activities encoded in five cDNAs for chalcone synthase (CHS) homologs from hop were investigated. Only valerophenone synthase (VPS) and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone forming activity. Narigenin-chalcone production by VPS was much lower than by CHS_H1. Therefore, it is highly possible that flavonoid depends mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS and CHS_H1.     

灰树花海藻糖合成酶基因的克隆及其在大肠杆菌中的表达

海藻糖主要作用是作为生物体的结构组分、以及保护生物膜和保护蛋白质。在灰树花中 ,海藻糖在干重中所占比例最高可达到 1 5 %～ 1 7% ,说明灰树花合成海藻糖的能力很强。将灰树花海藻糖合成酶基因克隆 ,并在大肠杆菌表达系统里表达。表达量为 1 90mg L。通过活性测定 ,证明在大肠杆菌中表达的海藻糖合成酶具有酶活性 ,结合基因工程和酶工程方法 ,为合成海藻糖的研究提供了新的方向