蔗糖异构酶PalI在解脂耶氏酵母中的表面展示

【背景】蔗糖异构酶(PalI)生物转化蔗糖是目前生产异麦芽酮糖的主要方法,但在生产过程中存在的蔗糖异构酶转化蔗糖副产物比例较高、游离酶需要分离纯化等问题限制了异麦芽酮糖工业生产的应用。【目的】构建蔗糖异构酶PalI在解脂耶氏酵母(Yarrowia lipolytica) Po1g中的表面展示菌株,以降低蔗糖异构酶转化蔗糖的副产物比例及其纯化成本。【方法】为获得具有生产PalI能力的Y.lipolytica Po1g表面展示菌株,通过重叠延伸PCR将克雷伯氏菌(Klebsiella singaporensis)LX3的PalI的编码基因PalI与全基因合成的来自Y.lipolytica细胞壁的锚定蛋白Pir1融合,转入Y.lipolytica Po1g中构建表面展示菌株。利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)比色定糖法测定表面展示的PalI酶活力并对其酶学性质进行探究,通过高效液相色谱法分析其转化蔗糖的产物。【结果】构建了蔗糖异构酶表面展示菌株Pir1-PalI/Po1g,经DNS法测得展示在Y. lipolytica Po1g表面的Pal...     

阴沟肠杆菌GX-3的中性蔗糖酶在大肠杆菌中的表达及其特性鉴定

【目的】在阴沟肠杆菌(Enterobacter cloacae)GX-3中发现了一个编码中性p H范围内起作用的蔗糖酶基因,在大肠杆菌(Escherichia coli)中进行克隆表达及纯化,并研究该蔗糖酶的酶学性质,为蔗糖酶的应用研究及开发利用奠定基础。【方法】通过查找Gen Bank数据库中来自阴沟肠杆菌同属中的编码蔗糖酶的基因序列,对这些序列进行比对分析设计简并引物扩增保守区;利用PCR扩增目的基因,以p QE30为表达载体构建重组质粒;镍亲和层析纯化重组蛋白,对重组酶的酶学性质进行详细研究。【结果】一个编码蔗糖酶的基因(Einv)被从阴沟肠杆菌GX-3中发现和克隆。生物化学特性鉴定表明这个蔗糖酶的活性最适温度为40℃和最适p H为6.5。凝胶渗透色谱法分析显示Einv是以二聚体的形式存在。Einv这个蔗糖酶在1170 mmol/L的蔗糖条件下仍保留有80%以上的水解活性。而在高浓度的蔗糖条件下,Einv只有水解活性而没有转糖基的副反应发生。它能够水解棉子糖、蔗果三糖、蔗果四糖、蔗果五糖和水苏糖。【结论】获得的Einv是一个在中性p H范围内起作用的β-呋喃果糖苷酶,只有水解功能而没有转糖基功能。这些特性表明这个中性蔗糖酶在食品工业具有重要的应用价值。     

黑曲霉QU10果糖基转移酶的克隆表达

微生物果糖基转移酶能够以蔗糖为底物产生低聚果糖。为获得更多新酶资源,通过PCR法成功地克隆出黑曲霉QU10的果糖基转移酶基因(Gen Bank Accession No.KF699529),基因片段长度为1 941 bp,包含一个54 bp的内含子。进一步利用RT-PCR法克隆了果糖基转移酶的c DNA,其编码628个氨基酸。将所得片段定向克隆到p ET-22b、p GAPZA及p GAPZαA载体,并转化至大肠杆菌或毕赤酵母中,通过筛选获得果糖基转移酶表达活力高的转化子。利用α信号肽的毕赤酵母转化子获得最高果糖基转移酶胞外酶活力为431 U/m L,是原始菌株酶活力的35倍。此毕赤酵母重组酶为同源二聚体,半天然PAGE表观分子量约200 k Da。以蔗糖为底物,果糖基转移酶在p H 5.0、45℃下反应4 h,酶解产物中主要是蔗果三糖和四糖,蔗果寡糖最高可占总质量的58%。结果表明,果糖基转移酶酵母工程菌具有很高的转果糖基的能力,而且表达活力高,具有潜在的工业应用价值。     

菊芋蔗糖代谢相关产物与关键酶基因对高温的响应

蔗糖是一类重要的碳水化合物,其代谢与植物生长发育及抵抗胁迫等有密切的关系。蔗糖合成酶(SUS)、蔗糖磷酸合成酶(SPS)与蔗糖转化酶(INV)是参与蔗糖代谢的三类关键酶。本研究依据转录组测序数据,从能源植物菊芋中鉴定了2个SUS、2个SPS和7个INV基因(GenBank No:MK386943-53)。生物信息学分析表明,菊芋SUS、SPS和INV的氨基酸序列与其他物种具有较高的相似性,均属于亲水性蛋白。在25、30°C处理10、15、20 d的菊芋幼苗叶片中,这三种基因家族成员呈现不同的表达模式;除可溶性总糖含量减少外,果糖、蔗糖、蔗果三糖等含量没有发生明显变化。表明高温下幼苗蔗糖代谢关键酶基因发生了响应,蔗糖代谢处于平衡状态,显示了菊芋对高温的良好耐受性。     

黑曲霉QU10果糖基转移酶的克隆表达（英文）

微生物果糖基转移酶能够以蔗糖为底物产生低聚果糖。为获得更多新酶资源,通过PCR法成功地克隆出黑曲霉QU10的果糖基转移酶基因(Gen Bank Accession No.KF699529),基因片段长度为1 941 bp,包含一个54 bp的内含子。进一步利用RT-PCR法克隆了果糖基转移酶的c DNA,其编码628个氨基酸。将所得片段定向克隆到p ET-22b、p GAPZA及p GAPZαA载体,并转化至大肠杆菌或毕赤酵母中,通过筛选获得果糖基转移酶表达活力高的转化子。利用α信号肽的毕赤酵母转化子获得最高果糖基转移酶胞外酶活力为431 U/m L,是原始菌株酶活力的35倍。此毕赤酵母重组酶为同源二聚体,半天然PAGE表观分子量约200 k Da。以蔗糖为底物,果糖基转移酶在p H 5.0、45℃下反应4 h,酶解产物中主要是蔗果三糖和四糖,蔗果寡糖最高可占总质量的58%。结果表明,果糖基转移酶酵母工程菌具有很高的转果糖基的能力,而且表达活力高,具有潜在的工业应用价值。     

青春双岐杆菌中β-呋喃果糖苷酶的提纯和一些性质

双岐杆菌是革兰氏阳性、不产芽孢的厌氧菌,主要存在于人和一些动物的肠道中,具有阻止有害细菌滋生和感染的作用。双岐杆菌能选择性水解低聚果糖,而哺乳动物的消化酶不能水解低聚果糖。 微生物中的β—呋喃果糖苷酶可分为两类,一类为蔗糖酶,它能水解蔗糖但不水解菊糖;另一类为外菊糖酶(β-果糖苷酶),它不仅水解蔗糖也能水解菊糖,它们对蔗糖和菊糖的活性比相差甚大。有些青春双岐杆菌提取物水解1-蔗果三糖的速度比蔗糖和菊糖快,并能从1-蔗果三糖产生果糖和蔗糖。青春双岐杆菌G-1菌株的提取物对1-蔗果三糖的活性很高,当底物浓度各为5mM时,它对1-蔗果三     

以蔗糖为底物利用重组大肠杆菌合成甘露醇

【目的】异型发酵乳酸菌可利用胞内产生的甘露醇脱氢酶将果糖高效转化为甘露醇,但果糖作为底物相对昂贵,不利于工业化生产。为了降低生产成本,必须选择廉价的底物。蔗糖相对便宜,并且大量存在于自然界中,能够被重组大肠杆菌利用产生甘露醇。蔗糖水解酶(Sucrose hydrolase)和甘露醇脱氢酶(Mannitol dehydrogenase)是发酵生产甘露醇中催化蔗糖转化成甘露醇的关键酶,构建蔗糖水解酶和甘露醇脱氢酶共表达菌株并进行相关研究是本文的主旨。【方法】利用PCR方法分别从植物乳杆菌(Lactobacillus plantarum)和布氏乳杆菌(Lactobacillus buchneri)基因组DNA中获得sac A和mdh基因,得到大小分别为1 502 bp和1 032 bp的目的基因,经序列分析后将其连接到表达载体p ET-28a(+)上,得到重组表达载体p ET28a-sac A-mdh。将重组质粒转化到大肠杆菌BL21(DE3)中,并用SDS-PAGE分析目的蛋白的表达情况并测定其酶活。【结果】SDS-PAGE显示表达蛋白的大小亚基分子量分别为55.1 k D和37.8 k D,与预期分子量一致,实现sac A和mdh基因的表达。蔗糖水解酶和甘露醇脱氢酶酶活分别为25.78 U/m L和14.56 U/m L。对重组菌株BL21(DE3)/p ET28a-sac A-mdh进行发酵条件优化,甘露醇质量浓度达到45.19 g/L,总糖转化率为37.66%。【结论】与乳酸菌利用蔗糖发酵生产甘露醇相比,产量提高了6倍,且具有发酵周期短、稳定性高等优点,菌株的成功构建为甘露醇工业化生产奠定了基础。     

固定化蔗糖酶水解蜂蜜蔗糖的研究

用复合破壁方法从酵母提取蔗糖酶,用海藻酸钙凝胶包埋、戊二醛交联方法制备固定化蔗糖酶,并在40℃下进行脱水处理。对自然酶和固定化酶的酶学性质进行了系统研究。自然酶和固定化酶的最适底物浓度为10％,最适反应时间是120分钟,最适pH是4.0,最适反应温度自然酶是50℃,固定化酶60℃。果糖对自然酶和固定化酶有很强的抑制作用,在果糖和葡萄糖并存情况下抑制作用降低。用固定化蔗糖酶反复水解蜂蜜蔗糖40批,蜂蜜中蔗糖含量由10％下降为5％以下,固定化蔗糖酶仍保持75％水解酶活力。     

蔗糖富集环境中蔗糖磷酸化酶的酶学性质研究与改造

【背景】蔗糖富集土壤是蔗糖磷酸化酶的重要来源之一。此酶能以较廉价的蔗糖为底物进行转葡萄糖基反应,改善受体底物的理化性质,因而具有重要的应用价值。【目的】研究蔗糖富集土壤中蔗糖磷酸化酶的酶学性质和转糖苷活性,为其更优质的改造提供材料和理论基础。【方法】将宏基因组中获得的蔗糖磷酸化酶基因克隆到表达载体上构建重组大肠杆菌,诱导表达并进行镍亲和层析纯化蛋白。以蔗糖为底物测量重组酶的基本酶学性质,研究其对糖类底物的转糖苷活性。通过底物通道分析,利用反向PCR技术对其第155位点进行饱和突变,并测定突变体基本酶学性质和转糖苷活性。【结果】纯化的酶蛋白分子量大小约为56 kD,活性状态时以三聚体形式存在。在以蔗糖为底物时,最适温度和最适pH值分别为55°C和6.5;Km和Vmax值分别为23.1±2.4 mmol/L和407.9±8.5μmol/(mg·min),对第155位点进行了定点饱和突变,获得了部分酶学性质或转糖苷活性改善的突变体。【结论】宏基因组中蔗糖磷酸化酶的研究丰富了酶学数据,并通过分子改造获得了部分性质更优的突变体,为转糖苷活性关键氨基酸的研究和该酶的工业应用奠定了基础。     

光合碳在叶片淀粉和蔗糖间分配的调节

叶片光合作用中产生的三碳糖在淀粉和蔗糖之间的分配受许多因素控制,蔗糖形成速率是决定性因素。蔗糖形成的调节酶是果糖1,6—二磷酸酯酶(F1,6P_2ase)和磷酸蔗糖合成酶(SPS),调节作用是通过无机磷(Pi)、磷酸二羟丙酮(DHAP)、磷酸己糖(己糖—P)、果糖1,6—二磷酸(F1,6P_2)和果糖2,6—二磷酸(F2,6P_2)之间的复杂的调节关系进行的。其中F2,6P_2起着关键作用,它以极低的浓度调节生糖和酵解作用,既参与蔗糖合成又参与反馈抑制。     

Isolation of sucrose: sucrose 1-fructosyltransferase (1-SST) from barley (Hordeum vulgare)

The enzyme sucrose: sucrose 1-fructosyltransferase was partially purified from barley leaf growth zones. Four steps (ammonium sulphate precipitation and polyethylene glycol precipitation, followed by chromatography on Concanavalin A-sepharose and hydroxylapatite) yielded a 35-fold purification. The resulting preparation of 1-SST which still contained a number of different activities related to fructan metabolism, was subjected to preparative isoelectric focusing, and sections of the gel were analysed individually for 1-SST and related activities, using sucrose and 1-kestose as substrates. This procedure yielded a 196-fold purification and revealed the presence of two isozymes of 1-SST with pI values of 4.93 and 4.99, as determined by analytical isoelectric focusing of the corresponding fractions. Both isozymes produced glucose and 1-kestose when incubated with sucrose. In addition, small amounts of 6-kestose and tetrasaccharides were formed. In particular, one of the two 1-SST isozymes yielded fructose when incubated with 1-kestose, indicating that it also acts as a fructan exohydrolase. The other isozyme exhibited less fructan exohydrolase activity. Nystose was also degraded by the fructan exohydrolase activity but less than 1-kestose, whereas 6-kestose was not a substrate for the enzyme. Incubation of both 1-SSTs with different concentrations of sucrose showed that the enzyme was not saturated even at 500 mM. As for the barley sucrose: fructan 6-fructosyltransferase, both isozymes of 1-SST yielded two polypeptide bands of molecular weight 50 and 22 kDa upon sodium dodecylsulphate polyacrylamide gel electrophoresis, suggesting their close relationship to invertase (composed of two subunits of similar size), as previously reported for other plants.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne)

A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.     

Purification and Characterization of the Enzymes of Fructan Biosynthesis in Tubers of Helianthus tuberosus Colombia (II. Purification of Sucrose:Sucrose 1-Fructosyltransferase and Reconstitution of Fructan Synthesis in Vitro with Purified Sucrose:Sucrose 1-Fructosyltransferase and Fructan:Fructan 1-Fructosyltransferase)

Sucrose:sucrose 1-fructosyltransferase (1-SST), an enzyme involved in fructan biosynthesis, was purified to homogeneity from tubers of Helianthus tuberosus that were harvested in the accumulation phase. Gel filtration under native conditions predicted a molecular mass of about 67 kD. Electrophoresis or gel filtration under denaturing conditions yielded a 27- and a 55-kD fragment. 1-SST preferentially catalyzed the conversion of sucrose into the trisaccharide 1-kestose (GF2). Other reactions catalyzed by 1-SST at a lower rate were self-transfructosylations with GF2 and 1,1-nystose (GF3) as substrates yielding GF3 and 1,1,1-fructosylnystose, respectively, as products. 1-SST also catalyzed the removal of the terminal fructosyl unit from both GF2 and GF3, which resulted in the release of sucrose and GF2, respectively, and free Fru. The purified enzyme did not display [beta]-fructosidase activity. An enzyme mixture of purified 1-SST and fructan:fructan 1-fructosyltransferase, both isolated from tubers, was able to synthesize fructans up to a degree of polymerization of at least 13 with sucrose as a sole substrate.     

Production of 1-Kestose in Transgenic Yeast Expressing a Fructosyltransferase from Aspergillus foetidus

Sucrose-inducible secretory sucrose:sucrose 1-fructosyltransferase (1-SST) from Aspergillus foetidus has been purified and subjected to N-terminal amino acid sequence determination. The enzyme is extensively glycosylated, and the active form is probably represented by a dimer of identical subunits with an apparent molecular mass of 180 kDa as judged from mobility in seminative acrylamide gels. The enzyme catalyzes fructosyl transfer from sucrose to sucrose producing glucose and 1-kestose. Oligosaccharides with a higher degree of polymerization are not obtained with sucrose as the substrate. The cDNA encoding the A. foetidus 1-SST has been cloned and sequenced. Sequence homology was found to be highest to levanases, but no hydrolytic activity was observed when levan was incubated with the enzyme. Expression of the cloned gene in an invertase-deficient mutant of Saccharomyces cerevisiae resulted in 1-kestose production, with 6-kestose and neokestose being side products of the reaction. Products were well distinguishable from those formed by yeast transformants expressing a cytosolic invertase.     

Mutational analysis of the active center of plant fructosyltransferases: Festuca 1-SST and barley 6-SFT

The active center of the glycoside hydrolase family 32 contains the three characteristic motifs (N/S)DPNG, RDP, and EC. We replaced the N-terminal region including the (N/S)DPNG motif of barley 6-SFT (sucrose:fructan 6-fructosyltransferase) by the corresponding region of Festuca 1-SST (sucrose:sucrose 1-fructosyltransferase). The chimeric enzyme, expressed in Pichia, retained the specificity of 6-SFT. Attempts to replace a larger piece at the N-terminus including also the RDP motif failed. A point mutation introduced in the RDP motif of 1-SST abolished enzymatic activity. Interestingly, point mutations of the EC-motif resulted in an enzyme which had lost the capability to form 1-kestose and glucose from sucrose but still accepted 1-kestose, producing fructose and sucrose as well as nystose.     

Fructan Synthesis in Excised Barley Leaves (Identification of Two Sucrose-Sucrose Fructosyltransferases Induced by Light and Their Separation from Constitutive Invertases)

Excised leaves of barley (Hordeum vulgare L.) exposed to continuous light accumulate large amounts of soluble carbohydrates. Carbohydrates were analyzed in deionized extracts by high-pressure liquid chromatography on an anion exchange column coupled with pulsed amperometric detection. During the first few hours of illumination, the main sugar to accumulate was sucrose. The levels of glucose and fructans (oligofructosylsucroses) increased later. The trisaccharide 1-kestose (1-kestotriose) predominated initially among the fructans. Later, 6-kestose (6-kestotriose) and tetra- and pentasaccharides accumulated also. Total extracts from barley leaves were chromatographed on a MonoQ column, and each fraction was assayed for enzymes of interest by incubation with 200 mM sucrose for 3 h, followed by carbohydrate analysis. Freshly excised leaves yielded two peaks of invertase, characterized by formation of fructose and glucose, but had almost no trisaccharide-forming activities. In leaves exposed to continuous light, two new enzyme activities appeared that generated fructan-related trisaccharides and glucose from sucrose. One of them was a sucrose-sucrose fructosyl-1-transferase (1-SST), producing 1-kestose exclusively: the peak fractions of this activity contained almost no invertase. The other was a sucrose-sucrose fructosyl-6-transferase (6-SST), producing 6-kestose. It comigrated with one of the constitutive invertases on MonoQ but was separated from it by subsequent chromatography on alkyl Superose. Nevertheless, the preparation retained invertase activity, suggesting that this enzyme may act both as fructosidase and fructosyltransferase. When incubated with 1-kestose in addition to sucrose, this enzyme formed less 6-kestose but instead produced large amounts of the tetrasaccharide bifurcose (1&6-kestotetraose), the main fructan tetrasaccharide accumulating in vivo. These results suggest that two inducible enzymes, 1-SST and 6-SST, act in concert to initiate fructan accumulation in barley leaves.