腾冲嗜热杆菌热稳定海藻糖磷酸化酶的基因表达与酶的分子生物学性质研究

分离克隆了腾冲嗜热杆菌(Thermoanaerobacter tengcongensis)海藻糖磷酸化酶(TreP)的编码基因(treP), 该酶可催化以葡萄糖和α-1-磷酸葡萄糖为底物的海藻糖合成反应及其逆向的分解反应. 反向mRNA点杂交实验表明, 腾冲嗜热杆菌中treP基因在高盐胁迫条件下表达量增加, 而在海藻糖诱导条件下表达量降低. 将该基因导入不含TreP的大肠杆菌中进行诱导表达, SDS-PAGE表明, 异源表达的TreP分子量约为90 kD, 与预期值相同. 通过葡萄糖氧化酶法测定分解产物葡萄糖的产率表明: TreP催化海藻糖分解反应的最适温度是70℃, 最适pH值为7.0; 通过HPLC检测合成产物海藻糖的产率表明: TreP催化合成反应的最适温度为70℃, 最适pH值为6.0. 在最适反应条件下, 50 μg的TreP粗酶可催化25 mmol/L α-1-磷酸葡萄糖与葡萄糖在30 min合成11.6 mmol/L海藻糖; 而同量的酶在同样时间内仅能将250 mmol/L海藻糖分解生成1.5 mmol/L葡萄糖. 以上体内胁迫和诱导表达分析及体外酶学性质分析均证明该酶的主要功能是催化海藻糖的合成反应. 热稳定性实验表明, 该酶性质比较稳定, 在50℃下温育7 h还能保持77%以上的活性, 是一个有潜在工业用途的新的热稳定海藻糖合成酶.     

Sulfolobus acidocaldarius ATCC 33909麦芽寡糖基海藻糖合成酶在Bacillus subtilis中的重组表达和发酵优化

为实现Sulfolobus acidocaldarius ATCC 33909来源的麦芽寡糖基海藻糖合成酶(MTSase)基因tre Y在枯草芽孢杆菌(Bacillus subtilis)中的重组表达,以质粒p ET-24a(+)-tre Y为模板PCR扩增得到目的基因,并与表达载体pHY300PLK连接,转入表达宿主Bacillus subtilis CCTCC M 2016536中,重组菌在TB培养基中培养48 h后MTSase酶活达到17.5 U/m L;在此基础上对重组菌发酵条件进行优化,通过单因素实验(氮源种类、氮源复配、氮源浓度、碳源种类、葡萄糖浓度、初始pH、诱导温度)和正交实验(氮源浓度、葡萄糖浓度、初始pH、诱导温度)确定其摇瓶发酵产酶的最适培养基和培养条件为:氮源(工业蛋白胨∶棉籽粉=3∶1)48.0 g/L、葡萄糖为10.0 g/L、培养基初始pH为7.0,最适培养温度为30℃;在此条件下,MTSase的酶活可达41.5 U/m L,是优化前的2.4倍。     

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。     

Escherichia coli str.K-12 substr.MG1655海藻糖酶Tre F的重组表达及性质研究

海藻糖酶(Trehalase)是一种海藻糖水解酶,能够特异性的将海藻糖分解为两分子的葡萄糖。为了将Escherichia coli str.K-12 substr.MG1655的海藻糖酶基因Tre F在E.coli BL21(DE3)中重组表达和应用,该研究通过PCR扩增获得E.coli str.K-12 substr.MG1655的海藻糖酶基因Tre F,构建了基因工程菌E.coli BL21(DE3)/p ET-24a(+)-Tre F。对重组菌进行摇瓶发酵,25℃,IPTG浓度为0.4 mmol/L时,摇瓶发酵诱导24 h时得到最高酶活为107 U/m L。进一步研究了海藻糖酶的酶学性质,发现该海藻糖酶的最适p H为7.0,最适温度为50℃;此外,将该酶应用于海藻糖的水解,起始海藻糖浓度为300 g/L,初始p H 7.0、反应温度30℃,加酶量为84 U/g,反应36 h,葡萄糖转化率可达98.4%。该研究是首次将E.coli str.K-12 substr.MG1655海藻糖酶基因Tre F在E.coli BL21(DE3)宿主中重组表达的报道。     

温特曲霉转富马酸为L-苹果酸的初步研究*

从大量霉菌中选育到一株具有较高富马酸酶活性的温特曲霉(Aspergillus wentii) A5-61。在摇瓶培养条件下,32℃ 96小时,产L-苹果酸达10.49g/100ml,对富马酸的转化率达90.80%。利用菌体细胞,进行酶转化试验,结果表明：1.6g湿菌体接入25ml含富马酸10.0%（用NaOH中和至pH7.0）的转化液中,35℃16～24小时,连续转化三次,分别产生L—苹果酸9.61g/100ml、9.73g/100ml、6.93g/100ml。对菌体整体细胞酶学性质的研究表明,其最适反应温度35℃,最适反应pH7.0,Cu2＋对该酶有明显的抑制作用,该酶的Km＝0.154mol/L,Vmax＝0.0571mol/L·h。     

改造大肠杆菌合成海藻糖途径以高效合成海藻糖

海藻糖是相容性溶质的一种,因其具有多种生物学功能,在食品、化妆品、药品以及器官移植等方面均有很广泛应用。然而近几年生产海藻糖主要集中在使用酶催化的方法,虽然这种方法的转化效率高,但是却存在着副产物的问题,难以得到高纯度的海藻糖产品,严重制约了海藻糖的应用。本文通过基因工程技术在大肠杆菌Escherichia coli中构建了海藻糖高效合成新途径,通过全细胞催化合成海藻糖。利用PCR技术在哈氏噬纤维菌Cytophaga hutchinsonii中克隆获得海藻糖双功能合成酶基因(tpsp),采用E.coli pTac-HisA高效表达载体,实现海藻糖双功能合成酶基因(tpsp)高效表达,利用高效表达菌株进行全细胞催化,将葡萄糖高效转化为海藻糖。结果表明C.hutchinsonii海藻糖合成酶基因(tpsp)在E.coli中成功实现表达,该酶能够在胞内将葡萄糖高效转化为海藻糖,并将其转运到胞外,实现海藻糖的高效率合成,海藻糖的产量提高到1.2 g/L,相对转化率为21%。当将此高产菌株在发酵罐中进行转化时,海藻糖的产量达到13.3 g/L,葡萄糖的相对转化率达到48.6%。采用C.hutchinsonii海藻糖合成酶基因高效表达并且应用于海藻糖全细胞合成催化在国内外尚属首次报道,海藻糖的转化率及产率都已达到文献报道最高水平,本研究为开拓海藻糖生产新技术奠定了基础。     

硫酸乙酰肝素3-O硫酸基转移酶5的表达、纯化及酶学性质研究

经过PCR克隆得到硫酸乙酰肝素3-O硫酸基转移酶5(3-OST-5)的基因,将其与大肠杆菌表达载体pET-15b连接后,在大肠杆菌BL21(DE3)中诱导表达,使用镍亲和层析柱纯化得到具有活性的3-OST-5。经测定纯化后的3-OST-5比活达到0.58 U/mg,是纯化前的5.27倍,回收率达80.4%。在此基础上,研究了该酶的酶学性质,酶反应的最适温度为35℃,稳定范围为20-40℃;最适pH为7.0,在pH7.0-9.0范围内稳定。在反应液中加入终浓度为1 mmol/L的K+、Ca2+、Ba2+对酶促反应有一定的促进作用。     

小菜蛾海藻糖酶酶学特性及四种杀虫剂对其活性的抑制作用

本试验以4龄小菜蛾Plutella xylostella(L.)体内海藻糖酶为研究对象,研究了其酶学特性以及4种常用杀虫剂在离体条件下对其活性的影响。结果表明,小菜蛾体内海藻糖酶的最适反应pH为6.0,温度50℃,其米氏常数(Km)为(12.57±0.99)mmol/L,最大反应速度(Vm)为(0.775±0.04)mmol/(min·g pro)。在药剂浓度为40mg/L时,4种杀虫剂(丙溴磷、灭多威、仲丁威和多杀霉素)对4龄幼虫海藻糖酶活性均有不同程度的抑制作用,其抑制率分别为25.83%、20.19%、18.99%和18.62%;而且随着药剂浓度的增加,上述4种杀虫剂对小菜蛾海藻糖酶活性的抑制率也逐渐增大。     

嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化

通过化学方法合成嗜热网球菌(Dictyoglomus thermophilum)来源的纤维二糖差向异构酶基因ce,将其引入到载体pBSuL3-ce,构建重组质粒pBSuL3-ce并转化进枯草芽孢杆菌,发酵48h后测定胞内酶活为7. 5U/ml。酶学性质结果表明:该酶的最适pH为8. 5;最适温度为85℃,85℃的半衰期为120min。为降低发酵成本,对发酵培养基进行优化:以35g/L豆粕粉为氮源、5g/L甘油为碳源时,酶活力最高可达12. 3U/ml。依据摇瓶优化的条件在3L发酵罐中扩大培养,胞内酶活达到56U/ml,比摇瓶培养酶活提高了8倍。利用发酵所得酶制备乳果糖,在乳糖浓度为400g/L、反应温度为85℃、初始pH 8. 5、加酶量为20U/ml的条件下,乳果糖转化率可达51%。     

固定化天冬氨酸酶制备(R)-3-氨基丁酸

从芽孢杆菌Bacillus sp.YM55-1基因组中克隆得到天冬氨酸酶基因,以pET-28a(+)为载体构建天冬氨酸酶基因的表达载体pET-28a(+)-Asp,将天冬氨酸酶基因进行定点突变,在E.coli BL21(DE3)系统中实现了天冬氨酸酶的异源表达。利用重组的天冬氨酸酶,以氨水、(NH_4)_2SO_4为辅料,将底物巴豆酸转化为(R)-3-氨基丁酸。将天冬氨酸酶的工程菌制备成固定化细胞,通过反应条件的优化研究,提高底物的转化率。结果表明:天冬氨酸酶最适pH为9.0,最适反应温度为40℃。在此反应条件下,加入30 g/L固定化细胞,转化22 h,(R)-3-氨基丁酸质量浓度达到220 g/L,对映体过量值e.e._s≥99.95%,底物转化率达到98%,固定化细胞重复使用次数不低于24次。     

Gene expression and molecular characterization of a thermostable trehalose phosphorylase fromThermoanaerobacter tengcongensis

A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from α-glucose-1-phosphate (α-Glc-1-P) and glucose, was cloned fromThermoanaerobacter tengcongensis and successfully expressed inEscherichia coli. The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70°C (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual activity after incubation at 50°C for 7 h. Under the optimum reaction conditions, 50 μg crude enzyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-Glc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L trehalose is degraded within the same time period. Dot blotting revealed that thetreP gene inT. tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of theT. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for industrial production of trehalose.     

羟基乙腈水解酶菌株的筛选及其催化特性

以羟基乙腈为唯一氮源, 从土壤中筛选到一株腈水解酶产生菌CCZU-12, 经形态观察生理生化实验和16S rDNA序列分析, 鉴定该菌为假单胞菌属(Pseudomonas sp.)。对菌株CCZU-12产腈水解酶的培养条件及催化反应条件进行优化, 最适产酶培养条件为: 碳源为10 g/L乙酸钠, 氮源为5 g/L酵母粉, 金属离子为1.0 mmol/L Mg2+, 培养温度30 °C, pH值7.0, 接种量4%, 装液量50 mL/250 mL; 最适催化反应温度35 °C, pH值7.0, 反应120 h, 羟基乙腈转化率达到98.9%。     

Tagatose production by immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant in a packed-bed bioreactor

Using immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant (Gali 152), we found that the galactose isomerization reaction was maximal at 70 degrees C and pH 7.0. Manganese ion enhanced galactose isomerization to tagatose. The immobilized cells were most stable at 60 degrees C and pH 7.0. The cell and substrate concentrations and dilution rate were optimal at 34 g/L, 300 g/L, and 0.05 h(-1), respectively. Under the optimum conditions, the immobilized cell reactor with Mn2+ produced an average of 59 g/L tagatose with a productivity of 2.9 g/L.h and a conversion yield of 19.5% for the first 20 days. The operational stability of immobilized cells with Mn2+ was demonstrated, and their half-life for tagatose production was 34 days. Tagatose production was compared for free and immobilized enzymes and free and immobilized cells using the same mass of cells. Immobilized cells produced the highest tagatose concentration, indicating that cell immobilization was more efficient for tagatose production than enzyme immobilization.     

High production of D-tagatose by the addition of boric acid

An L-arabinose isomerase mutant enzyme from Geobacillus thermodenitrificans was used to catalyze the isomerization of D-galactose to D-tagatose with boric acid. Maximum production of D-tagatose occurred at pH 8.5-9.0, 60 degrees C, and 0.4 molar ratio of boric acid to D-galactose, and the production increased with increasing enzyme concentration. Under the optimum conditions, the enzyme (10.8 units/mL) converted 300 g/L D-galactose to 230 g/L D-tagatose for 20 h with a yield of 77% (w/w); the production and conversion yield with boric acid were 1.5-fold and 24% higher than without boric acid, respectively. In 24 h, the enzyme produced 370 g/L D-tagatose from 500 g/L D-galactose with boric acid, corresponding to a conversion yield of 74% (w/w) and a production rate of 15.4 g/L.h. The production and yield of D-tagatose obtained in this study are unprecedented.     

Saturation mutagenesis and self-inducible expression of trehalose synthase in Bacillus subtilis

Trehalose is a nonreducing disaccharide synthesized by trehalose synthase (TreS), which catalyzes the reversible interconversion of maltose and trehalose. We aimed to enhance the catalytic conversion of maltose to trehalose by saturation mutagenesis, and constructed a self-inducible TreS expression system by generating a robust Bacillus subtilis recombinant. We found that the conversion yield and enzymatic activity of TreS was enhanced by saturation mutations, especially by the combination of V407M and K490L mutations. At the same time, these saturation mutations were contributing to reducing by-products in the reaction. Compared to WT TreS, the conversion yield of maltose to trehalose was increased by 11.9%, and the k_cat/K_m toward trehalose was 1.33 times higher in the reaction catalyzed by treS^V407M-K490L. treS^V407M-K490L expression was further observed in the recombinant B. subtilis W800N(Δσ^F) under the influence of P_srfA, P_cry3Aa, and P_srfA-cry3Aa promoters without an inducer. It was shown that P_srfA-cry3Aa was evidently a stronger promoter for treS^V407M-K490L expression, with the intracellular enzymatic activity of recombinant treS^V407M-K490L being over 5,800 U/g at 35 hr in TB medium. These results suggested the combination of two mutations, V407M and K490L, was conducive for the production of trehalose. In addition, the self-inducible TreS^V407M/K490L mutant in the B. subtilis host provides a low-cost choice for the industrial production of endotoxin-free trehalose with high yields.     

Enzymatic processes for the purification of trehalose

A dual‐enzyme process aiming at facilitating the purification of trehalose from maltose is reported in this study. Enzymatic conversion of maltose to trehalose usually leads to the presence of significant amount of glucose, by‐product of the reaction, and unreacted maltose. To facilitate the separation of trehalose from glucose and unreacted maltose, sequential conversion of maltose to glucose and glucose to gluconic acid under the catalysis of glucoamylase and glucose oxidase, respectively, is studied. This study focuses on the hydrolysis of maltose with immobilized glucoamylase on Eupergit® C and CM Sepharose. CM Sepharose exhibited a higher protein adsorption capacity, 49.35 ± 1.43 mg/g, and was thus selected as carrier for the immobilization of glucoamylase. The optimal reaction temperature and reaction pH of the immobilized glucoamylase for maltose hydrolysis were identified as 40°C and 4.0, respectively. Under such conditions, the unreacted maltose in the product stream of trehalose synthase‐catalyzed reaction was completely converted to glucose within 35 min, without detectable trehalose degradation. The conversion of maltose to glucose could be maintained at 0.92 even after 80 cycles in repeated‐batch operations. It was also demonstrated that glucose thus generated could be readily oxidized into gluconic acid, which can be easily separated from trehalose. We thus believe the proposed process of maltose hydrolysis with immobilized glucoamylase, in conjunction with trehalose synthase‐catalyzed isomerization and glucose oxidase‐catalyzed oxidation, is promising for the production and purification of trehalose on industrial scales. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Partial purification and properties of a highly specific trehalose phosphate phosphatase from Mycobacterium smegmatis

A specific trehalose phosphate phosphatase was purified approximately 50-fold from Mycobacterium smegmatis. The enzyme had a pH optimum of about 7.0 and was stimulated by Mg(2+). The optimum concentration of Mg(2+) was about 1.5 x 10(-3)m. Of other divalent cations tested, only Co(2+) showed some activity. The K(m) for trehalose phosphate was found to be about 1.5 x 10(-3)m. The enzyme showed slight activity toward mannose-6-P and fructose-6-P but was inactive on a large number of other phosphorylated compounds. Citrate was a competitive inhibitor of the enzyme both with respect to trehalose phosphate concentration and Mg(2+) concentration. This inhibition appears to be due to chelation of Mg(2+) by this compound. Ethylenediaminetetraacetic acid and NaF were also inhibitors of the enzyme, but these inhibitions were noncompetitive.     

Overexpression and characterization of a thermostable trehalose synthase from Meiothermus ruber

A thermostable trehalose synthase (TreS) gene from Meiothermus ruber CBS-01 was cloned and overexpressed in Escherichia coli. The purified recombinant TreS could utilize maltose to produce trehalose, and showed an optimum pH and temperature of 6.5 and 50°C, respectively. Kinetic analysis showed that the enzyme had a twofold higher catalytic efficiency (k _cat/K _m) for maltose than for trehalose, indicating maltose as the preferred substrate. The TreS also had a weak hydrolytic property with glucose as the byproduct, and glucose was a strong competitive inhibitor of the enzyme. The maximum production of trehalose by the enzyme reached 65% at 20°C. The most importantly the enzyme could maintain very high activity (above 90%) at pH 4.0–8.0 and 60°C 5 h. These results provided that the stable TreS was suitable for the industrial production of trehalose from maltose in a one-step reaction.