产气肠杆菌菌体内核苷磷酸化酶的诱导表达

目的:阿糖腺苷(Ara-A)是一种广谱抗病毒药物,临床上用于治疗多种病毒性疾病.同时也是合成阿糖腺苷单磷酸(Ara-AMP)的重要原料.本课题旨在寻找一种高效酶法生产嘌呤类阿糖核苷的方法.方法:以产气肠杆菌完整细胞为酶源,研究产气肠杆菌菌体培养条件对核苷磷酸化酶的影响及其诱导性.结果:胸苷磷酸化酶、尿苷磷酸化酶和嘌呤核苷磷酸化酶均可被多种核苷、核苷酸甚至碱基诱导.胞苷或胞苷酸的添加量为15-20mmol/L,诱导时间在0-8小时均可.经胞苷和胞苷酸诱导的菌体可使酶反应时间缩短6倍,大大提高了反应效率.经诱导的菌体,在反应后仍保持较高的核苷磷酸化酶活力;而未经诱导的菌体,一次反应后即丧失大量的酶活力.结论:核苷磷酸化酶的活性可以通过诱导而提高,以此优化阿糖腺苷的生产.     

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.     

两种耐热磷酸化酶的构建及高效催化合成红景天苷

目的:使用表达耐热蔗糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Spase和耐热纤维二糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Cpase,发酵培养后粗酶液作为催化剂,以价格低廉的蔗糖为原料合成红景天苷。方法:分别构建耐热蔗糖磷酸化酶和耐热纤维二糖磷酸化酶大肠杆菌重组菌,然后将重组菌、蔗糖、酪醇和磷酸混合,得到反应混合物,使反应混合物在45℃下转化,而产生红景天苷。结果:在耐热蔗糖磷酸化酶酶液1200 U/L、耐热纤维二糖磷酸化酶酶液500 U/L、蔗糖110 g/L、酪醇30 g/L和磷酸50 m M的浓度下,反应条件为pH 7.0、温度45℃、转速50转/分、反应时间32小时后,红景天苷浓度达到23.7 g/L。结论:本研究使用蔗糖磷酸化酶和纤维二糖磷酸化酶联合催化的工艺,成功地高收率合成了红景天苷。同时,本研究构建的耐热磷酸化酶酶活高,处理简单,为拓展糖苷类似物的合成提供了一种新的方法。     

应用乙酰短杆菌酶法合成2'-脱氧腺苷

胸苷和腺嘌呤在乙酰短杆菌作用下转化成胸腺嘧啶和目标产物2’-脱氧腺苷。50mL的三角瓶装液10mL,40mmol/L的底物,50mmol/L的磷酸盐缓冲液(pH7)进行反应,5%游离乙酰短杆菌在55℃下,经1h转化率就可达到65.6%。     

响应面分析法优化重组大肠杆菌生物合成谷胱甘肽的条件

通过响应面分析法和典型性分析得出重组大肠杆菌酶法合成谷胱甘肽的最优条件:菌体量249 mg/mL,磷酸钾缓冲液145 mmol/L,MgCl243 mmol/L和ATP 34 mmol/L,预测谷胱甘肽最大量为16.50 mmol/L。验证性实验证明在优化条件下,重组大肠杆菌酶法合成谷胱甘肽达16.42 mmol/L。响应面分析还表明,在重组大肠杆菌酶法合成谷胱甘肽各因素中,MgCl2和ATP,以及菌体量与磷酸钾缓冲液之间的交互作用较显著。     

重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷

利用重组大肠杆菌Escherichia coli Rosetta(DE3)/pET-SPase发酵生产蔗糖磷酸化酶(EC 2.4.1.7,Sucrose phosphorylase,SPase)。收集的菌体经高压破碎后离心得到粗酶液,通过镍NTA亲和层析、超滤除盐后得到电泳纯的SPase,纯化后的SPase的比酶活是原来的2.1倍,酶活回收率达到82.7%。经SDS-PAGE电泳测定,重组SPase的分子量约为59 kDa。该酶在不高于37℃,pH 6.0~6.7的条件下比较稳定,最适催化温度与最适催化pH分别为37℃,pH 6.7,该酶对蔗糖的米氏常数(Km)为7.3 mmol/L,最大反应速率(Vmax)为0.2μmol/(min.mg)。此外文中还以蔗糖和氢醌为底物,利用重组SPase催化合成α-熊果苷。其最佳反应条件为:20%蔗糖,200 U/mL的酶液,1.6%氢醌,pH 6.0~6.5,25℃,反应21 h。α-熊果苷的摩尔产率为78.3%,α-熊果苷的产量为31 g/L。     

ATP合成菌株的构建及用于联合生产S-腺苷甲硫氨酸

为降低S-腺苷甲硫氨酸的生产成本,构建了同时表达腺苷激酶、腺苷酸激酶和乙酸激酶3种酶的重组大肠杆菌菌株用于ATP的合成,并对ATP的转化条件进行了优化,优化后的反应体系为:腺苷30 mmol/L,乙酰磷酸二锂盐135 mmol/L,硫酸镁5 mmol/L,硼砂50 mmol/L,菌体2 g/L(湿重),反应液初始pH7.5,反应温度为35℃,反应时间为3 h,反应转化率可以达到99%以上。按照上述反应体系进行5 L放大,反应结束后再投入65 mmol/L D,L-甲硫氨酸和50 g/L(湿重)表达腺苷甲硫氨酸合成酶的重组大肠杆菌菌体,并补加15 mmol/L硫酸镁,转化18 h S-腺苷甲硫酸浓度能达到8.7 g/L,转化率达到72.5%。     

蔗糖磷酸化酶全细胞催化AA-2G的条件优化

目的:使用表达蔗糖磷酸化酶(EC 2.4.1.7,Sucrose phosphorylase,SPase)的大肠杆菌重组工程菌E.coli BL21/pET-spase,作为全细胞催化剂,合成2-O-D-吡喃葡糖基-L-抗坏血酸(Ascorbic acid 2-glucoside,AA-2G)。通过反应条件的优化研究,提高AA-2G的收率。方法:分别考察菌体量、缓冲液pH、蔗糖浓度、维生素C浓度、反应时间和温度对AA-2G合成反应的影响,再组合上述最佳条件进行反应。AA-2G的产量使用高效液相色谱法进行定量。结果:最佳反应条件为:菌体量15 mg/mL,缓冲液pH 4.5,蔗糖浓度100 g/L,维生素C浓度175 g/L,反应时间20 h,温度37℃。在此条件下,AA-2G产量达到了35.7 g/L。结论:以蔗糖为底物,使用SPase合成AA-2G的研究报道较少。本研究通过优化此方法的反应条件,让AA-2G的产量得到了大幅提高。同时本研究中成功地采用了大肠杆菌工程菌作为全细胞催化剂,这比传统的使用粗酶液的方法更省时省力,有良好的应用潜力。     

重组大肠杆菌全细胞催化合成莱鲍迪苷D

莱鲍迪苷D(Rebaudioside D,RD)是一种稀有具有高甜度的甜菊糖苷类化合物。本文实现了重组大肠杆菌全细胞催化莱鲍迪苷A(Rebaudioside A,RA)合成RD。以水稻c DNA为模板,扩增得到葡萄糖基转移酶基因eugt11,构建了重组菌株E.coli BL21(p ETDuet-eugt11),并成功表达了重组蛋白6His-EUGT11。通过Ni柱亲和层析纯化并在体外酶催化反应表征了其催化活性。将重组菌BL21(p ETDuet-eugt11)应用于催化合成RD研究。探讨了反应体系pH、温度、柠檬酸钠浓度、菌体密度、二价金属离子、二甲苯体积分数、UDPG添加浓度对反应效率的影响。单因素考察结果显示,在菌体密度0.16 g湿细胞/m L反应液,底物RA浓度为1.0 mmol/L,pH 8.0,60 mmol/L柠檬酸钠,1%二甲苯,0.1 mmol/L Zn Cl2,12.0 mmol/L UDPG,反应温度42℃,反应时间24 h的条件下,RD产量为123.6 mg/L(约0.1 mmol/L)。     

α-环糊精葡萄糖基转移酶催化合成α-熊果苷

以对苯二酚和麦芽糊精为底物,通过α-环糊精葡萄糖基转移酶和淀粉葡萄糖苷酶的两步酶法反应体系催化合成α-熊果苷。优化后的催化条件:以葡萄糖当量(DE)值为8%~10%的麦芽糊精作为供体底物,麦芽糊精60g/L,对苯二酚150 mmol/L,缓冲液p H 6.0,在40℃下反应24 h。在此反应条件下,α-熊果苷的产量为3.17 g/L,对苯二酚转化率为7.77%。通过萃取法对α-熊果苷进行了初步分离,再经高效液相色谱-电喷雾串联质谱技术进行了结构测定,确定产物为α-熊果苷。     

Enzymatic synthesis of amylose

Amylose is a linear polymer of α-1,4-linked glucose and is expected to be used in various industries as a functional biomaterial. However, pure amylose is currently not available for industrial purposes, since the separation of natural amylose from amylopectin is difficult. It is known that amylose has been synthesized using various enzymes. Glucan phosphorylase, together with its substrate, glucose-1-phosphate, is the most suitable system for the production of amylose since the molecular size of amylose can be controlled precisely. However, the problem with this system is that glucose-1-phosphate is too expensive for industrial purposes. This review summarizes our work on the enzymatic synthesis of essentially linear amylose, together with recent progress in the production of synthetic amylose using sucrose or cellobiose through the combined actions of phosphorylases.     

Characterization of Foam- and Emulsion-stabilizing Functions of Enzymatically Modified Proteins with Surfactancy

A papain-catalyzed reaction involving covalent incorporation of l-leucine n-alkyl ester is available for producing an enzymatically modified protein (EMP) with surfactancy [Agric. Biol. Chem., 45, 1621 (1981)]. In the present work we used gelatin as a starting material and incorporated l-leucine n-hexyl ester to produce a whippable EMP and l-leucine n-dodecyl ester to produce an emulsifiable EMP. A foam system formed with the whippable EMP was much stabler than that formed with sodium dodecylsulfate. The emulsifiable EMP also gave a much stabler oil-in-water emulsion than Tween-80 did. The stability of the emulsion formed with EMP was not affected by the presence of NaCl at a very high concentration. The observed foam and emulsion stabilities were well explained by the data for decreased mobility of the involved water protons. These results may indicate that EMP molecules, when arranged at the air/water or oil/water interface, can bind a part of the water to form thick barriers which prevent the air or oil particles from coalescing.     

Isolation and Structure of New Isocoumarins

Besides reticulol, the strain MD611-C6 produced two compounds which inhibited cyclic nucleotide phosphodiesterases [EC 3.1.4. C.] These substances were isolated and their structures were elucidated to be 8-hydroxy-6, 7-dimethoxy-3-hydroxymethyIisocoumarin (II) and 6, 8-dihyroxy-7-methoxy-3-hydroxymethylisocoumarin (III). Concentrations of II and III for 50% inhibition of cAMP phosphodiesterase were 3.97 × 10^?4m and 1.26 × 10^?8m, respectively.     

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

Cellodextrins are linear β‐1,4‐gluco‐oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP‐controlled, bottom‐up synthesis from expedient substrates is desired for their bulk production. Here, a three‐enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short‐chain (soluble) cellodextrins (DP range 3–6). The cascade reaction involves iterative β‐1,4‐glucosylation of glucose from α‐glucose 1‐phosphate (αGlc1‐P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1‐P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L^?1 is obtained under near‐complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)^?1. Through a simple two‐step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.     

纤维二糖磷酸化酶和纤维寡糖磷酸化酶的研究与应用

自然界中一些厌氧的纤维素降解菌能够产生纤维二糖磷酸化酶(Cellobiose Phosphorylase,CBP)和纤维寡糖磷酸化酶(Cellodextrin phosphorylase,CDP)磷酸化裂解纤维二糖和纤维寡糖。CBP和CDP属于糖苷水解酶94家族(Glycoside Hydrolase Family 94,GH94),对β-1,4糖苷键高度专一。目前已广泛研究了不同来源的CBP和CDP的功能性质和催化机理,并通过蛋白质晶体结构分析揭示了二者采用不同聚合度纤维寡糖作为底物的结构基础。因CBP和CDP可催化独特的磷酸化裂解反应和逆向合成反应,已有多篇报道显示CBP和CDP在生产实践上的应用,主要在构建直接利用纤维寡糖的工程酵母菌、构建酶法纤维素转淀粉体系和酶法合成特殊糖类等3个方面。由于对CBP和CDP的持续关注,在此对二者的相关研究进行综述,并提出今后的研究重点。     

Amino acid anthranilamide derivatives as a new class of glycogen phosphorylase inhibitors

A series of amino acid anthranilamide derivatives identified from a high-throughput screening campaign as novel, potent, and glucose-sensitive inhibitors of human liver glycogen phosphorylase a are described. A solid-phase synthesis using Wang resin was also developed which provided efficient access to a variety of analogues, and resulted in the identification of key structure–activity relationships, and the discovery of a potent exemplar (IC₅₀ = 80 nM). The SAR scope, synthetic strategy, and in vitro results for this series are presented herein.     

Correlation between the Antitumor Activity of Schizophyllan and Its Triple Helix

trans-3-Methylthioacrylamide (3-MTAA-NH₂) was isolated as colorless needles from the culture broth of Streptomyces sioyaensis, a siomycin-producer. This substance is considered to be not only a new metabolite from methionine but also a new substance. The isolation and identification of 3-MTAA-NH₂, as well as the cultural conditions for production, were investigated. A variety of other Streptomyces also produced 3-MTAA-NH₂ from methionine.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Determination of glycogen and enzymes of glycogen metabolism in human hair follicles

The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles.The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.