疏绵状嗜热丝孢菌脂肪酶在毕赤酵母中的高效表达和性质鉴定

通过密码子优化改造编码sn-1,3位置专一性疏绵状嗜热丝孢菌脂肪酶(TLL)基因,同时通过重叠PCR技术合成该基因,并将其克隆到巴斯德毕赤酵母的表达载体p PIC9K中。结果表明,TLL脂肪酶被高效胞外表达,在摇瓶中发酵168 h得到的发酵液的上清脂肪酶表达量达到0.1 mg/m L,对应的p NPP水解酶活达到312.72U/m L。与对应的原始编码基因重组菌酶活(179.42 U/m L)相比,密码子优化后酶活提高74.29%,表明密码子优化策略成功提高了TLL在巴斯德毕赤酵母中的重组表达。比较毕赤酵母来源的重组TLL与商业化的米曲霉酶学性质发现它们具有相似的最适p H和温度耐受范围。另外,它们在有机溶剂耐受性、表面活性剂和金属离子效应以及位置选择性方面均较为相似,表明毕赤酵母来源的重组TLL同样具有商业化应用的潜力。     

常压室温等离子诱变选育L-天冬酰胺酶高产重组菌

以Bacillus subtilis WB600为宿主,构建了能分泌表达L-ASNase的重组菌,并通过常压室温等离子体诱变进一步提高了重组菌的产酶量。重组Bacillus subtilis WB600(p MA5-wap Aans Z)发酵30 h,胞外酶活达到37.2 U/m L,表明L-ASNase在信号肽wap A介导下能分泌至胞外。在功率120 W、气流量10 L/min、诱变时间40 s的诱变操作条件下,对重组菌进行了等离子体诱变。突变株的酶活最高达48.4 U/m L,较诱变前提高30%。上述结果表明,常压室温等离子体诱变能有效提高重组菌产L-ASNase的酶活.研究结果为L-ASNase的工业化生产提供了高效的生产菌株。     

洋葱伯克霍尔德氏菌Lu10-1脂肪酶的异源表达

通过PCR扩增出洋葱伯克霍尔德氏菌Lu10-1脂肪酶基因,将基因片段分别克隆到大肠杆菌、毕赤酵母和枯草杆菌的表达载体,转入表达菌株中。结果表明,重组脂肪酶在大肠杆菌中主要以包涵体形式表达,在毕赤酵母中未有表达,而在枯草杆菌中实现了胞外分泌表达,测得发酵上清液酶活为13.8 U/m L。对重组枯草杆菌发酵条件进行了摇瓶初步优化和3 L的反应器分批培养。当以TB为出发培养基,初始p H 6.5,温度为37℃时,在3 L的发酵罐上最终酶活达到34.5 U/m L,是野生菌表达量的4.2倍。     

两阶段温度控制策略以及助剂促进淀粉分支酶的胞外表达

为了促进重组淀粉分支酶在枯草芽孢杆菌(Bacillus subtilis WB600)中的胞外表达,对重组淀粉分支酶在摇瓶水平上进行了发酵优化。首先,以胞外酶活力为指标,先后考察了初始培养基、p H、培养温度和培养时间4种因素对胞外表达的影响;然后,通过两阶段温度策略和助剂的添加进一步提高胞外表达水平。结果表明,当发酵培养基为TB、p H为7.5时,25℃培养24 h后胞外酶活力达到19.9 U/m L;同时,菌体生长最适温度和酶分泌的最适温度是不一致的,通过两阶段温度控制策略(即0~12 h控制温度30℃,12 h后温度调至25℃),胞外酶活力与单一温度条件下的最高水平(25℃)相比提高了1.4倍,达到了47.1 U/m L;在此基础上,进一步添加0.05%的吐温-80,将胞外酶活进一步提高40%,达到65.8 U/m L。     

嗜酸芽孢杆菌普鲁兰酶在大肠杆菌中的表达及发酵优化

为了在3L发酵罐水平上提高重组菌E.coli BL 21(DE3)/pET20b(+)-BapulA的普鲁兰酶的表达量和胞外分泌,将嗜酸芽孢杆菌普鲁兰酶(Bacillus acidopullulyticus)在大肠杆菌中进行重组表达,并在3L发酵罐水平上对重组菌进行发酵优化,考察了发酵条件对重组酶表达的影响。重组菌发酵的最佳条件为:发酵前期菌体生长温度和pH分别为30℃和7.0,当菌体浓度OD_(600)达到50时,发酵温度降低至25℃,此时调节pH为6.2,同时开始流加乳糖[0.4g/(L·h)]进行诱导;在发酵过程中,当菌体浓度OD_(600)依次达到15,45,75时,分别加入浓度为1.5g/L的甘氨酸,当菌体OD600为105时,再加入浓度为3g/L的甘氨酸。发酵结束后普鲁兰酶的胞外酶活达659.0U/mL,最高总酶活达1 910.1U/mL,与摇瓶的初始总酶活(41.1U/mL)和胞外酶活(6.5U/mL)相比,分别提高了45.4倍和100倍。     

纤维微菌β-1,3-葡聚糖酶的克隆表达及对灵芝细胞壁的水解作用

该文旨在研究一种酶解灵芝(Ganoderma lucidum)菌丝体从而提高其胞内蛋白提取效率的方法。通过PCR方法扩增获得纤维微菌(Cellulosimicrobium sp.)CICIM6906的β-1,3-葡聚糖酶编码基因，该基因命名为bgl6906并与表达载体连接，构建重组质粒pET28a-bgl6906,重组质粒转化大肠杆菌BL21(DE3),获得重组大肠杆菌BL21(DE3)/pET28a-bgl6906。重组菌在TB培养基中进行诱导表达，表达产物在自身信号肽引导下大部分分泌到细胞外。以茯苓多糖为底物时，重组酶BGL6906纯酶比酶活力为567.8 U/mg,最适pH为5.5,最适温度为50℃。重组菌在15 L发酵罐中发酵72 h胞外酶活力达到67 U/mL。重组酶BGL6906与果胶酶交替水解灵芝菌丝体培养物，可以有效水解细胞壁，获得部分原生质体。进一步辅助短时超声波破碎可以大幅度提高灵芝胞内产物的释放。     

一种耐酸热普鲁兰酶生产菌种的分子构建及发酵研究

将长野芽胞杆菌的普鲁兰酶基因经密码子优化后,组建了人工合成的二联启动子Pga2,并将它克隆到枯草芽胞杆菌穿梭质粒p MK4-BPB以及自杀质粒p GE-BPB中;经转化和筛选获得了中性蛋白酶基因npr E被敲除的普鲁兰酶生产菌株CH-1;该重组菌在基础培养基中所产普鲁兰酶的酶活达到30.3 U/m L;经过对培养基组分及发酵条件(培养温度、起始p H,起始接种量等)进行优化,确定了发酵的最适碳源为45 g/L的蔗糖,氮源为60 g/L的麸皮+豆粕时,设定初始培养基的p H为6.2,在培养温度为32℃时进行发酵,CH-1发酵产重组普鲁兰酶酶活高达268 U/m L。     

谷氨酸脱羧酶在枯草芽孢杆菌中的表达及应用

为了研究在枯草芽孢杆菌发酵过程中添加相对廉价的辅酶前体对谷氨酸脱羧酶(GAD)酶活力和催化效率的影响,将来源于Escherichia coli的谷氨酸脱羧酶基因gadB,通过构建重组质粒p HY300PLK-gadB,在枯草芽孢杆菌中重组表达,得到重组菌B. subtilis/pHY300PLKgadB。研究了分别在重组菌发酵过程中添加辅酶磷酸吡哆醛(PLP)、辅酶前体吡哆醛(PL)和吡哆醇(PN)对GAD酶活力的影响。当设置摇床转速200 r/min,发酵温度33℃,发酵培养基初始p H 7.0时,在发酵过程中分别添加PLP、PL、PN至终浓度0.5 mmol/L,诱导48 h后发酵液中总酶活分别达到25.40、28.14、15.55 U/mL,是对照总酶活的1.55、1.72、0.95倍。基于以上结果,进一步研究了发酵过程中PL的添加浓度对重组菌生长情况及GAD表达的影响。结果表明,发酵液中GAD总酶活随着PL浓度的增加而增加,当PL浓度为0.1 mmol/L时,总酶活最高达到28.28 U/mL。利用重组菌全细胞制备γ-氨基丁酸(GABA),结果表明,最适转化pH为5.0,最适转化温度为40℃,发酵过程中添加PL的重组菌(简称"GAD-PL")和发酵过程中不添加任何辅酶及辅酶前体的重组菌(简称"GAD-0")的最适加菌量(以酶活力单位计)分别是40 U/g(以谷氨酸计)和50 U/g(以谷氨酸计),当谷氨酸最终质量浓度达到400 g/L时,得到的GABA质量浓度分别为275.60 g/L和273.61 g/L。     

产碱性淀粉酶重组枯草芽孢杆菌的构建与发酵优化

碱性淀粉酶催化淀粉在碱性环境中高效降解,广泛应用于纺织退浆、洗涤、制药等领域。通过信号肽筛选,将来源于嗜碱单胞菌Alkalimonas amylolytica的碱性淀粉酶基因于枯草芽孢杆菌Bacillus subtilis WB600实现分泌表达,并优化了重组菌产酶的发酵条件。在最佳信号肽ywb N介导下,重组B.subtilis发酵51 h,胞外Amy K酶活最高达146.86 U/m L,且能在发酵上清液中检测到与Amy K理论相对分子质量(60 k Da)一致的蛋白质条带。通过单因素实验优化了发酵培养基(糊精32 g/L,蛋白胨12 g/L,酵母粉24 g/L,KH_2PO_42.32 g/L,K2HPO_4·3H_2O 16.43 g/L),并在发酵24 h后添加0.2 g/d L Na_2CO_3调节发酵液p H,发酵72 h胞外Amy K酶活达640.33 U/m L,较优化前提高了336%,是目前已报道重组B.subtilis产碱性淀粉酶的最高水平。研究结果为Amy K发酵生产的工业化提供了数据支撑。     

共表达HAC1基因对重组毕赤酵母分泌表达葡萄糖氧化酶的影响

通过增强不可折叠蛋白质响应(UPR)信号途径以及研究不同培养温度下的影响,来提高重组葡萄糖氧化酶在毕赤酵母中的分泌表达。影响毕赤酵母外源蛋白表达的因素,主要为内质网中的折叠速率以及不可折叠蛋白积累造成的胞内胁迫压力。通过共表达HAC1基因对不可折叠蛋白信号通路进行调控,摇瓶中改造菌株PP-G-HAC1胞外酶活达到161 U/m L,相比于原始重组葡萄糖氧化酶菌株提高了34%。进一步研究不同温度下过量表达HAC1基因对菌株的生长和分泌外源蛋白的影响,菌株PP-G-HAC1在3 L发酵罐中28℃培养,酶活达到1 008 U/m L,胞外重组蛋白质达到14.43 g/L,相比原始菌株在相同条件下提高了3.12倍。     

葡萄糖异构酶的研究——酶的分离提纯

葡萄糖异构酶细胞经丙酮处理,缓冲液抽提。DEAE—纤维素离子交挟柱层析和Sephadex G—200凝胶柱层析等方法得到分离井纯化。该法与超声破碎法相比,比活提高3.3倍,活力收率为74%,得到了高纯度异构酶,经Sephadex G—200凝胶柱层析获得较高收率的电泳纯制品。     

A New Regenerable Immobilized Glucose Isomerase

A new, immobilized glucose isomerase high productivity has been developed for isomerization of glucose into high fructose syrups. Soluble glucose isomerase from Streptomyces rubiginosus is highly purified and electrostatically adsorbed onto a granular DEAE-cellulose comprised of fibrous DEAE-cellulose, food grade polystyrene and titania. The same enzyme on fibrous DEAE-cellulose has been in commercial use in the United States since 1968. Because the soluble enzyme is electrostatically adsorbed to the carrier, the immobilized enzyme can be regenerated after use and the carrier reloaded with fresh soluble enzyme. Regeneration and reimmobilization can be repeated several times thus providing a minimum cost enzyme-carrier system. The enzyme is tightly bound to the carrier during isomerization. The immobilized isomerase is compatible with bisulfite in the substrate sufficient to stabilize the enzyme and provide a degree of protection against infection at low isomerization temperature. High purity and high specific activity of the soluble enzyme make possible high activity levels for the immobilized enzyme with potencies of up to 1500 IGIU/g. There occurs essentially no elution of color from the immobilized enzyme during start up of isomerization. Due to the combination of low compressibility of the particles and high immobilized activity, productivities greater than 9 metric tons of 42% fructose syrup solids Perkg of immobilized enzyme are being achieved on a commercial scale.     

Effect of Gamma Irradiation on Whole-cell Glucose Isomerase. Part 2. Properties of Irradiated Whole-cell Glucose Isomerase

Several properties of Actinoplanes missouriensis and Streptomyces olivaceus glucose isomerase have been studied after irradiation the enzyme of the dose of 10 kGy in dry state. The temperature at which the Actinoplanes missouriensis cells show the highest activity decreased by at least five centigrades. Other investigated enzymatic properties have been found to show no significant differences after irradiation.     

Determination of Functional Groups in Glucose Isomerase

Following the introduction and widespread application of immobilized glucose isomerase for the industrial production of isosyrup (HFCS), the most significant challenge facing experts is to solve the problem of increasing the fructose content. In addition to well-known procedures another approach may be the changing of the equilibrium of the enzymatic reaction by modifying the active centre of the enzyme. Histidyl-, ϵ-amino-, arginyl-, tyrosyl- and tryptophyl- amino acid side chains in the active centre have been modified by means of selective protein reagents. The results were evaluated by kinetic methods and the role of functional groups of amino side chains has been determined. A new hypothesis is proposed for the mechanism of enzymatic reactions.     

固定化葡萄糖异构酶的研究 Ⅰ 游动放线菌(Actinoplanes missouriensis)-Ac-81-2葡萄糖异构酶的活力测定

测定游动放线菌胞内的葡萄糖异构酶时,无论在新鲜菌体、干菌体或用戊二醛直接交联的固定化菌体中,找不到酶量与反应生成物间的直线范围。但是在新鲜菌体中培养液量的倒数与反应速度的倒数呈直线关系。在干菌体或固定化菌体中则是所测酶量的对数与反应生成物的对数呈直线关系;酶量的对数与酶活力(单位/克)的对数呈直线关系。据此,对测定反应生成物果糖的半胱氨酸一咔唑反应作了修改和补充。     

Immobilized Glucose Isomerase Use in Mirin Production

The conversion of glucose in mirin (an alcoholic seasoning) to fructose using immobilized glucose isomerase (IGI) was studied in order to produce sweetened mirin without chill haze. The initial conversion velocity (C_m/t_m) with IGI was affected by temperature, pH, and ethanol concentration. This reaction was first order, for which the temperature coefficient (Q₁₀) over the range 20–55°C was 2.0. The value of C_m/t_m was a maximum at pH 8.0, and decreased with an increase in ethanol. Conversions carried out in a continuous column reaction system had C_m/t_m values 36 times higher than that in a batch system. Mirin treated with IGI did not form chill haze.     

Effect of Gamma Irradiation on Whole-cell Glucose Isomerase

Gamma-rays induced inactivation of Actinoplanes missouriensis and Streptomyces olivaceus glucose isomerase has been studied. This enzyme exhibits high resistance against ionizing radiation. The D₃₇ value was found to be equal to 131 kGy for Actinoplanes missouriensis cells and 88 kGy for Streptomyces olivaceus cells when irradiated in the dry state in the presence of air. Mg²⁺ ions do not affect the radiosensitivity of the enzyme in cells, while the addition of Co²⁺ ions to the cell suspension increases its stability against ionizing radiation.     

纳米花固定化葡萄糖异构酶的制备及性能分析

该研究采用共沉淀法制备了葡萄糖异构酶（Glucose Isomerase,GI）纳米花,对固定化条件进行了优化,同时对纳米花固定化酶的形态特征以及酶学性质进行了探究。结果表明,40 μL酶液中加入9 mL、pH值7.4的PBS缓冲液后与30 μL CuSO4混合,在35 ℃条件下静置反应18 h,制得的纳米花固定化葡萄糖异构酶（Glucose Isomerase @ Nano flowers,GI@NFs）的酶活回收率高达183.06%。SEM表征结果显示GI@NFs有完整的纳米花结构,傅里叶红外光谱显示GI@NFs具有酶和PO43-的特征吸收,X-射线衍射结果进一步证明其载体为Cu3(PO4)2。酶学性质研究发现,GI@NFs的最适反应温度为60 ℃,比自由酶的提高了10 ℃;最适反应pH值为8,比游离酶的最适pH更高;GI@NFs的温度稳定性和pH稳定性均比自由酶的明显提高;固定化酶被循环使用8次,其酶活力仍保持最初活力的60.32%。实验结果表明,纳米花结构提高了葡萄糖异构酶的酶活,表现出较好的循环性能和稳定性,具有一定的应用价值。     

Glucose Isomerase and Its Behaviour under Hydrogenation Conditions

A survey is given of glucose isomerase, its sources, its mechanism of isomerization and its data and properties in three different immobilized forms. In addition, the effect of a number of parameters on the activity of immobilized glucose isomerase has been investigated, e. g. hydrogen pressure, Mg(II) and Ca(II), transition metal ions, borate and sugar alcohols. Immobilized glucose isomerase remains sufficiently active under hydrogenation conditions to maintain D -glucose and D -fructose in equilibrium. D -Glucitol, in contrast to D -mannitol, has some inhibiting effect on the enzyme action. The D -glucose/D -fructose equilibrium constant is independent of the total sugar concentration (between 0.2 – 2.2-M).     

微波处理对葡萄糖异构酶性质的影响

研究微波处理对葡萄糖异构酶酶活性、稳定性、动力学参数及构象的影响。结果表明：不同微波处理时间和微波处理功率对葡萄糖异构酶的活力都有着不同的影响。在70 ℃处理5 min条件下,当微波功率为300、400 W时,葡萄糖异构酶的相对酶活力分别增加14.23%、8.42%;而当微波功率为600、800 W时,葡萄糖异构酶的相对酶活力分别降低到95.69%、90.78%;微波处理也对葡萄糖异构酶的最适反应温度和动力学参数Km和Vmax有影响,但对其最适pH值几乎没有影响。紫外和荧光光谱研究表明微波处理将导致葡萄糖异构酶部分去折叠,分子中赖氨酸和色氨酸残基所处的微环境发生变化,葡萄糖异构酶的三级结构因此可能发生了改变,即微波处理可能通过改变葡萄糖异构酶的构象从而改变酶的性质。