粗糙脉孢菌C2H2转录因子家族基因敲除突变体产纤维素酶筛选分析

丝状真菌的木质纤维素水解酶基因的表达很大程度上受到转录水平的调控,对纤维素酶系调控转录因子的研究对提高纤维素酶的产量具有重要意义。为了挖掘纤维素酶表达新调控基因,本研究对粗糙脉孢菌锌指转录因子C₂H₂家族的57株基因敲除突变株在以2%结晶纤维素为唯一碳源的培养条件下进行产纤维素酶水平分析筛选。通过测定发酵液的蛋白、内切β-1,4-葡聚糖酶酶活、外切纤维素酶酶活、β-葡萄糖苷酶酶活、木聚糖酶酶活及生物量,发现突变株L-38、L-85、L-40、L-64、L-99、L-07、L-86在蛋白水平及内切β-1,4-葡聚糖酶酶活水平均比野生型有25%-77%不等的显著提高,而突变株L-87、L-06在蛋白水平及内切β-1,4-葡聚糖酶酶活水平均比野生型有65%-80%不等的显著降低。这些纤维素酶新调控基因的获得为后续相关表达调控的研究提供了新素材。     

两种新型L-苏氨酸醛缩酶的鉴定及活性检测方法

为了实现重要医药中间体β-羟基-α-氨基酸的生物酶法合成,挖掘验证新型的L-苏氨酸醛缩酶。以pET-28a(+)作为表达载体,通过蛋白表达纯化、薄层层析色谱(TLC)和高效液相色谱(HPLC)技术分析L-苏氨酸醛缩酶及其催化产物的性质。基于4-氨基-3-肼基-5-巯基-1,2,4-三氮唑(Purpald)显色试剂开发检测醛缩酶的新方法。Streptomyces coelicolor SCO1844(天蓝色链霉菌,S.coelicolor SCO1844)和Streptomyces xinghaiensis SFR7A(星海链霉菌,S.xinghaiensis SFR7A)来源的醛缩酶被证明能够成功地合成β-羟基-α-氨基酸,且均为L-苏氨酸醛缩酶,实现了以苯甲醛和甘氨酸为底物合成l-threo/erythron-苯基丝氨酸的醇醛缩合反应。开发的可视化活性检测方法可以实现醛缩酶的快速鉴定和高通量筛选。两种新型L-苏氨酸醛缩酶的鉴定以及活性检测方法的开发,不仅丰富了生物法合成β-羟基-α-氨基酸的酶库,也为下一步对L-苏氨酸醛缩酶进行分子改造提高其催化活性和选择性奠定了研究基础。     

脑血管内皮细胞敲除Prmt5导致脑血管损伤及星形胶质细胞活化*

目的: 研究蛋白质精氨酸甲基转移酶5(protein arginine methyltransferase 5,Prmt5)在小鼠脑血管发育、稳态维持中的功能,并考察脑血管内皮细胞特异性敲除Prmt5后对中枢神经系统的影响。方法: 利用脑血管内皮细胞特异性表达SP-A-Cre转基因小鼠和Prmt5条件基因打靶小鼠交配,构建脑血管内皮细胞特异性Prmt5敲除小鼠。利用H-E染色、免疫荧光染色、激光散斑成像、Sulfo-NHS-Biotin染料灌注等方法评价脑血管内皮细胞特异性Prmt5敲除小鼠脑血管结构、脑血流量、血脑屏障渗透性等;利用实时定量PCR进一步检测补体C1q(complement C1q,C1q)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin 1β,IL-1β)等细胞因子的表达水平。通过免疫荧光、Western blot等检测胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、S100钙结合蛋白β(S100 calcium-binding protein β protein,S100β)和补体C3(complement C3,C3)的表达,检测小鼠皮层、丘脑和小脑中星形胶质细胞活化水平。结果: 脑血管内皮细胞特异性敲除Prmt5导致血管损伤, C1q、TNF-α和IL-1β等炎症因子表达水平上调,活化星形胶质细胞比例明显增加。结论: 脑血管内皮细胞中Prmt5在小鼠脑血管稳态维持中发挥了重要功能。     

壳聚糖固定化真菌漆酶及其用于处理酚类污染物的研究

Trametessp. AH282在液体培养条件下经邻甲苯胺诱导能有效合成漆酶同工酶A。以壳聚糖为载体,戊二醛为交联剂进行了漆酶A的固定化研究,确定酶固定化适宜条件为：0.1g壳聚糖与15 mL 5%戊二醛交联8 h后,加入30.0U酶固定12h。在此条件下获得的固定化漆酶催化能力为176.4U/g载体,酶活回收率58.5%。与游离酶相比,固定化漆酶与作用底物愈创木酚的亲和力降低,但固定化酶的稳定性有明显改善。固定化漆酶的最适温度为55℃,比游离酶提高5℃;70℃条件下保温8 h,固定化酶保留酶活56.5%,而在相同条件下游离酶酶活明显下降。使用固定化漆酶反应装置进行酚类化合物转化实验,连续进行12批次操作,固定化酶酶活仍保持60%以上,漆酶使用效率明显提高。     

副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 *

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei)TK1501 β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86.63kDa和675.56U/mg。最适作用温度和pH分别为30℃和6.5。 Mg ²⁺和Ca ²⁺对β-葡糖苷酶酶活抑制作用最小,Cu ²⁺几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的K_m和V_max分别为1.44mmol/L和58.32mmol/(L·s),催化系数k_cat为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501 β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。     

地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T. reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50 ℃,在Fe³⁺ (≤ 4 mmol/L)和Cu²⁺ (0-10 mmol/L)存在条件下酶活稳定。     

氨基载体共价结合固定化海洋假丝酵母脂肪酶

共价结合法是重要的工业酶固定化方法,利用稳定的共价键固定化工业酶,在载体和酶间形成多点共价连接,可以制备稳定性较好的固定化酶,更具有实际应用价值。利用氨基载体共价结合固定化海洋假丝酵母脂肪酶,采用较为廉价的戊二醛进行辅助交联,通过单因素和正交试验,确定最佳固定化条件为:25℃、pH5. 0、0. 1%戊二醛、0. 25g载体、交联0. 5h、固定化1h、加酶量为800U,最终得到的固定化酶酶活达到83. 01U/g。固定化脂肪酶的最适pH较游离酶向碱性方向偏移,最适反应温度提高10℃,固定化酶的热稳定性和酸碱稳定性比游离酶好且重复使用性和储存稳定性明显优于游离酶。同时发现交联剂是制备固定化脂肪酶的重要因素,因此探索新型交联剂对于固定化效果的提高具有重要意义,为海洋假丝酵母脂肪酶的固定化工艺技术和工业应用奠定了良好基础。     

高分子膜载体固定化木瓜蛋白酶

在浸润条件下,以0.5%(v/v)戊二醛交联的高分子膜尼龙载体固定化木瓜蛋白酶。对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数。结果表明,4℃、pH6.0条件下,将膜载体浸润于2mg/mL酶液中5h,固定化酶活为303.4U/g。固定化酶最适反应pH为6.0～7.0,最适反应温度为65℃。其pH稳定性、热稳定性均比游离酶高。     

紫陀螺菌化学成分及其抑制肿瘤细胞增殖活性的研究

以紫陀螺菌为对象,研究其子实体的化学成分及其抑制肿瘤细胞增殖活性。采用溶剂提取、柱层析和高效液相色谱等方法分离纯化化学成分,通过核磁共振和质谱技术鉴定单体化合物结构,运用结晶紫法评价单体化合物抑制肿瘤细胞增殖活性。从乙酸乙酯提取物中共分离鉴定6个单体化合物,分别为(22E,24R)-麦角甾-5,7,22-三烯-3β-醇(1)、3β,5α-二羟基-(22E,24R)-麦角甾-7,22-二烯-6-酮(2)、(22E,24R)-麦角甾-7,22-二烯-3β,5α,6β-三醇(3)、吲哚-3-甲酸甲酯(4)、4,4-二甲基-1,7-庚二酸(5)和(8E,10E)-12羰基十八碳-8,10-二烯酸(6),其中化合物1为主要成分,相对含量为23.8%。活性测试结果表明3对人乳腺癌细胞株MCF-7 细胞、人胰腺癌细胞株PANC-1细胞和人乳腺癌细胞株MDA-MB-231细胞具有微弱的细胞增殖抑制活性。本研究首次报道了紫陀螺菌化学成分,对深入挖掘其在健康领域中的开发价值具有重要意义。     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

吸附-交联法固定D-泛解酸内酯水解酶的研究

选择6种吸附树脂和离子交换树脂对D-泛解酸内酯水解酶进行固定化,筛选出了固定化效果较好的大孔弱碱性丙烯酸系阴离子交换树脂D-380为载体,用先吸附后交联的方法固定化。通过实验对固定化条件进行了优化,得出最佳的固定化条件为：加酶量6U／g树脂、吸附pH7．5、吸附时间4h、吸附温度30℃、交联剂戊二醛终浓度0．1％、交联时间2h。实验表明在此条件下制得的固定化酶有很好的稳定性：固定化酶在连续20次的底物水解反应后,剩余酶活达到71％。当温度达到80℃时游离酶几乎失去酶活,而固定化酶剩余酶活为60％以上。游离酶的pH稳定性范围为pH7～8,而固定化酶为pH6．5～8．5。     

Immobilization of β-galactosidase onto functionalized graphene nano-sheets using response surface methodology and its analytical applications

Background

β-Galactosidase is a vital enzyme with diverse application in molecular biology and industries. It was covalently attached onto functionalized graphene nano-sheets for various analytical applications based on lactose reduction.

Methodology/Principal Findings

Response surface methodology based on Box-Behnken design of experiment was used for determination of optimal immobilization conditions, which resulted in 84.2% immobilization efficiency. Native and immobilized functionalized graphene was characterized with the help of transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Functionalized graphene sheets decorated with islands of immobilized enzyme were evidently visualized under both transmission and scanning electron microscopy after immobilization. FTIR spectra provided insight on various chemical interactions and bonding, involved during and after immobilization. Optimum temperature and energy of activation (E_a) remains unchanged whereas optimum pH and K_m were changed after immobilization. Increased thermal stability of enzyme was observed after conjugating the enzyme with functionalized graphene.

Significance

Immobilized β-galactosidase showed excellent reusability with a retention of more than 92% enzymatic activity after 10 reuses and an ideal performance at broad ranges of industrial environment.     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

Study on Remediation-improvement of 2,4-Dichlorophenol Contaminated Soil by Organic Fertilizer Immobilized Laccase

ABSTRACT

In this paper, laccase is immobilized by the cross-linking method, using organic fertilizer as a carrier and glutaraldehyde as a cross-linking agent. Here, the optimal conditions of laccase immobilization were explored and the optimal operating conditions and stabilities of free laccase and immobilized laccase were also studied. Then, free laccase and immobilized laccase were applied to the soil remediation. Meanwhile, the effect of soil improvement treated with immobilized laccase was studied through ecological evaluation. The results showed that the optimal conditions for laccase immobilization were: the volume fraction of glutaraldehyde was 5%, the amount of enzyme added was 15 mL, and the immobilization time was 6 h. Under the same conditions, thermal stability and acid-base stability of immobilized laccase were better than free laccase. Under the optimal conditions, using laccase to treat 2,4-dichlorophenol in the soil, it was found that the free laccase group degraded 44.4% within 5 days, while the immobilized laccase group degraded 58.6%. Although both the degradation trends and route are the same, the degradation effect of the latter is obviously better. Ecological evaluation showed that organic fertilizer carrier had an impact on soil physical and chemical properties and soil enzymes, playing a positive role in soil ecological security and improving the soil.     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

壳聚糖固定化琼脂酶的研究

采用壳聚糖微球对琼脂酶进行固定化,在单因素实验的基础上用正交试验法确定最佳固定化工艺。结果表明:在戊二醛体积分数为2.5%,交联时间为6 h,加酶量为15 mL,固定时间为3 h时固定酶的活力最高;固定化酶的最适反应温度及最适pH分别为50℃和8.5,高于游离酶;同时其热稳定性及操作稳定性均高于游离酶。     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.