不同处理方法对灵芝β-葡聚糖降解效果的比较研究

本研究采用酸法、碱法、酶法和微波法对灵芝β-葡聚糖进行降解,通过降解率、产物分子量变化、产物聚合度分布等指标比较了不同方法的降解效果。结果表明,微波法降解率高达94%,处理后产物的分子量明显降低,寡糖产物聚合度分布广。酶法降解率约为40%,寡糖产物中含有DP2-5的成分。酸法及碱法降解率低于20%,寡糖产物少。研究表明,与其他3种方法相比,微波法降解率高、产物丰富、操作条件易于控制,是一种简单、高效的降解灵芝β-葡聚糖、制备灵芝β-葡寡糖的方法。     

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。     

浙江省主要亚热带森林群落类型物种和谱系水平的α和β多样性比较

了解不同森林群落类型的物种和谱系水平的α和β多样性, 有助于指导森林经营和生物多样性保护。本研究比较了浙江省内不同地点主要森林类型(包括常绿阔叶林、常绿落叶阔叶混交林、落叶阔叶林和针阔叶混交林)的物种α多样性和谱系α多样性, 以及物种β多样性和谱系β多样性。研究表明, 该地区主要森林类型的物种和谱系α多样性均存在较大差异, 但控制了空间和地形因子的作用后, 差异几乎全部消失; 森林类型内部及相互间的物种和谱系β多样性均存在显著差异, 同种森林类型内部的物种和谱系β多样性分别小于不同森林类型之间的物种和谱系β多样性, 且在控制了空间和地形因子的作用后, 以上差异仍然显著。本研究表明影响亚热带主要森林群落类型物种和谱系水平的α和β多样性的因素存在差异: α多样性可能主要受到空间和地形因子等的影响, 而β多样性则可能受到森林类型的重要影响。     

人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。     

青弋江鱼类分类群和功能群的α和β多样性纵向梯度格局

确定溪流鱼类多样性的时空分布格局可为鱼类多样性保护与管理提供科学基础。尽管溪流鱼类分类群多样性的纵向梯度格局已有大量报道, 但以鱼类生物学特征为基础的功能多样性研究较少。本文基于2009-2010年4个季度对青弋江1-5级溪流共15个样点的调查数据, 利用形态特征数据和食性构建了鱼类复合功能群, 研究了不同级别溪流间鱼类分类群和功能群组成及多样性的异同, 着重探讨了鱼类分类群和功能群的α和β多样性沿溪流纵向梯度的变化规律。采集到的56种鱼类可分为4个营养功能群和5个运动功能群, 共计14个“营养-运动”复合功能群。双因素交互相似性分析结果显示, 鱼类分类群和功能群组成都随河流级别显著变化, 但季节动态不显著; 双因素方差分析后发现, 鱼类分类群和功能群α、β多样性都随河流级别显著变化, 但受季节影响不显著。经回归分析, 分类群和功能群α多样性与河流级别大小呈显著的线性正相关, 但最大分类群α多样性出现于4级河流, 最大功能群α多样性在4级和5级河流间一致; 分类群和功能群β多样性与河流级别大小呈显著的二项式关系, 呈U型分布。分类群β多样性的空间变化主要取决于物种周转, 而功能群β多样性主要由嵌套所驱动。本研究表明, 沿着“上游-下游”的纵向梯度, 河流鱼类的α和β多样性的空间变化规律不同, 分类群和功能群α多样性的空间格局基本一致, 但分类群(主要是物种周转)和功能群β多样性(主要是功能嵌套)的空间变化过程的驱动机制不同。     

黑河中游春玉米叶片水δD和δ18O的富集过程和影响因素

植物水的稳定同位素分馏过程是水在土壤-植物-大气连续体中循环的重要环节。以往研究由于叶片水¹⁸O同位素比值(δ¹⁸O _l,b)和氘(D)同位素比值(δD_l,b)(合称δ_l,b)实测数量少只能作为模型验证数据, 导致δ_l,b富集机制研究多集中于模型研究, 缺乏基于野外试验条件的δ_l,b富集的控制机制研究。叶片水δD_l,b和δ¹⁸O _l,b的富集程度(ΔD_l,b和Δ¹⁸O _l,b, 合称Δ_l,b)通常表示为δ_l,b与茎秆水D同位素比值(δD_x)和¹⁸O同位素比值(δ¹⁸O_x) (合称δ_x)之差, 即Δ_l,b = δ_l,b - δ_x。该研究以黑河中游沙漠绿洲春玉米(Zea mays)生态系统为研究对象, 重点采集和分析了季节和日尺度δ_l,b和δ_x数据, 配套开展了大气水汽δ¹⁸O和δD (合称δ_v)等辅助变量的原位连续观测, 探讨了季节和日尺度上的δ_l,b富集特征及其影响因素。结果表明: 叶片水δ_l,b和Δ_l,b的季节变化趋势不明显, 而受蒸腾作用影响表现出白天富集夜间贫化的单峰日变化特征。对于D来说, 无论季节尺度上还是日尺度上, 大气水汽δ_v和相对湿度是δD_l,b和ΔD_l,b的主要环境控制因素; 而对于¹⁸O来说, 无论季节尺度上还是日尺度上, 相对湿度是δ¹⁸O _l,b和Δ¹⁸O _l,b的主要环境控制因素。由于D和¹⁸O在热力学平衡分馏上有约8倍差异, 直接分析叶片水ΔD_l,b和Δ¹⁸O_l,b与影响因素的差异性, 有助于理解叶片水δD和δ¹⁸O富集过程以及对模型发展有一定的指导意义。     

提高源自Bacillus circulans 251的β-CGTase对麦芽糖亲和性及其在生产海藻糖中的应用 *

将B. circulans 251 β-CGTase应用于海藻糖制备,海藻糖转化率从50.4%提高至71.9%。为进一步提高底物的转化率,运用易错PCR-高通量筛选技术筛选对以麦芽糖为歧化反应受体的亲和性提高的B. circulans 251 β-CGTase突变体。利用低底物浓度的96孔板4,6-亚乙基-对硝基苯-α-D-麦芽七糖苷(EPS)显色法,最终筛选得到了一株对麦芽糖亲和性提高的突变体M234I。将野生型β-CGTase和突变体酶M234I进行蛋白质纯化,测定其酶学性质。结果表明,突变体的比活为345.25U/mg,野生型则为357.63U/mg;突变体M234I对麦芽糖的K_m为0.258 2mmol/L,仅为野生型(0.474 9mmol/L)的54.4%,对麦芽糖的亲和性显著提高;突变体的最适温度、最适pH较野生型未发生较大变化。以麦芽糊精(DE值16)为底物,将突变体M234I用于多酶复配体系生产海藻糖,酶反应结果表明海藻糖的转化率最高达74.9%,较野生型β-CGTase提高约3%。     

壳聚糖和ε-聚赖氨酸处理对双孢蘑菇贮藏过程中品质的影响

以延长双孢蘑菇货架期为目标,探讨壳聚糖和ε-聚赖氨酸处理对采后双孢蘑菇在4 ℃贮藏过程中生理特性、营养品质和贮藏特性的影响。结果表明：与对照组双孢蘑菇相比,壳聚糖与ε-聚赖氨酸6:4复配溶液处理能够有效抑制双孢蘑菇表面微生物的生长和多酚氧化酶活性的增加,保持双孢蘑菇子实体较高的L*值和硬度,延缓双孢蘑菇的质量损失和细胞膜透性的升高,减少双孢蘑菇子实体的腐烂,保持较高商品率。在4 ℃条件下,壳聚糖与ε-聚赖氨酸6:4复配溶液对双孢蘑菇的保鲜性能最优,能够有效保持双孢蘑菇的商品品质和延长其贮藏时间。     

AAV载体介导的蓬佩病模型小鼠体内基因治疗研究*

Objective: Pompe disease is a lysosomal glycogen storage disease caused by acid α-glucosidase (GAA) deficiency, which is characterized by glycogen accumulation in the heart, skeletal muscle, and central nervous system (CNS). AAV vector-mediated gene therapy is expected to be a breakthrough in the treatment of Pompe disease. In this study, AAV9 vector was used to mediate GAA gene transfer in Pompe disease model mice, and the changes of GAA protease activity, glycogen accumulation in tissues and pathological changes in mice after transgenic intervention were evaluated. Methods: Codon optimized GAA gene (coGAA) was carried by AAV9 vector, and the AAV vector was packaged by baculovirus production process. Adult Pompe model mice were given a single intravenous injection at the dose of 1.1×10¹³, 3.0×10¹³, 1.2×10¹⁴ vg/kg, and aged Pompe model mice were given a single intravenous injection at the dose of 3.0×10¹³ vg/kg. After reaching the end point of the experiment, the mice were euthanized, GAA protease activity was determined by fluorescence spectrophotometry, glycogen accumulation was observed by PAS staining, and pathological changes were detected by HE staining. Results: Five weeks after administration, GAA protein was widely expressed in all tissues of adult model mice, with higher expression levels in heart and liver, and lower expression levels in brain and spinal cord. After rAAV9-coGAA treatment, glycogen content in myocardium, skeletal muscle and brain decreased, and vacuolar degeneration in myocardium and skeletal muscle decreased significantly. After treatment, the tissue enzyme activity of the aged animals was significantly increased compared with that of the model mice. The vacuolar degeneration and inflammatory cell infiltration of the myocardium were decreased, but the pathological improvement of skeletal muscle was limited. Conclusion: A single intravenous injection of rAAV9-coGAA can enhance GAA enzyme activity, reduce glycogen accumulation and improve pathology in Pompe model mice. The therapeutic effect was dose-dependent, and the injection also had certain therapeutic effect on aged animals. This study laid a theoretical foundation for the clinical application of AAV9 mediated gene therapy via intravenous route in Pompe disease.     

形貌调控与镀层修饰结合制备口服疫苗载体*

口服疫苗因其具有接种方便、能产生黏膜免疫等优点而备受关注,但胃肠道屏障、酸性环境和蛋白酶等不利条件制约了口服疫苗免疫效果的发挥。为提升其免疫效果,将形貌调控与镀层修饰策略结合制备新型口服疫苗载体,具体为将溶剂蒸发法与快速膜乳化法结合制备聚乳酸-羟基乙酸共聚物(PLGA)杆状颗粒,并采用能够增强免疫反应的β-葡聚糖及具有更高降解pH的硫醇化修饰的羟丙基甲基纤维素苯二甲酸酯(T-HPMCP)对PLGA杆状颗粒镀层修饰。在制备PLGA杆状颗粒时,通过对外水相条件的摸索制备出了适合小肠上皮细胞摄取的长度在2~4 μm、宽度在1~2 μm的PLGA杆状颗粒。体外实验结果表明通过T-HPMCP修饰的疫苗载体在酸性环境下保持稳定有利于抗原活性保护,同时能够在pH≥7.4时分解而使抗原释放。细胞和动物实验结果表明其特殊的杆状形貌可实现较高的肠道上皮摄取速率及转运效率,并且β-葡聚糖的修饰能活化树突状细胞(DC),提升OVA特异性IgA和IgG抗体水平。综上,制备的镀层PLGA杆状颗粒作为口服疫苗载体可提升机体免疫应答,为口服疫苗的研究提供了新的材料和思路。     

奎尼酸生物合成的代谢工程

奎尼酸及其衍生物氢醌和苯醌等是一类重要的化工原料,可作为一些化学合成制剂和药物中间原料,且在食品和化学工业中有着广泛的应用。目前奎尼酸的制备方法有植物提取法、化学合成法、酶工程法和微生物发酵法,其中微生物发酵法是近年发展起来的一种十分经济有效的方法。在介绍奎尼酸的制备方法的基础上重点综述了应用代谢工程在生物合成奎尼酸基因工程菌的改造中的研究进展,其中涉及奎尼酸生物合成途径中相关基因及其酶的调控、中心代谢途径的改造和修饰等,并探讨了将来的发展前景。     

KEGG数据库在生物合成研究中的应用

KEGG(Kyoto Encyclopedia of Genes and Genomes)提供了一个操作平台,即以基因组信息(GENES)和化学物质信息(LIGAND)为构建模块,通过代谢网络(PATHWAY)将基因组和生物系统联系起来,然后根据功能等级进行归纳分类(BRITE)。KEGG还为各种组学研究提供相关软件,用于代谢途径重建、遗传分析和化合物比对。作为一个综合数据库,KEGG不仅指导生物燃料、药物和新材料等生物基化学品的合成,而且致力于研究日趋严重的环境问题。系统介绍了KEGG数据库的结构、功能及其相关工具的最新进展,并展望在生物合成中的应用前景。     

3-脱氢莽草酸生物合成的代谢工程

3-脱氢莽草酸,是芳香族氨基酸生物合成代谢途径中一种重要的中间产物,可作为一些化学合成制剂和药物中间原料。这样以无毒可再生物质为起始原料的合成方法与传统的有机合成化学制剂的方法相比,对环境更加有利。此外,它还是一种十分有效的抗氧化剂。工业上一般采用化学合成法和发酵法来生产3-脱氢莽草酸,随着代谢工程的兴起,使得更加理性改造菌株成为可能,这更加促进了发酵法的广泛应用。本文主要介绍了代谢工程在生物合成3-脱氢莽草酸生产菌改造中的应用情况,其中涉及3-脱氢莽草酸生物合成途径中相关基因及其酶的调控、中心代谢途径的改造和3-脱氢莽草酸合成支路的修饰等,并探讨了将来的发展前景。     

Modeling effect of GABAergic current in a basal ganglia computational model

Electrical high frequency stimulation (HFS) of deep brain regions is a method shown to be clinically effective in different types of movement and neurological disorders. In order to shed light on its mode of action a computational model of the basal ganglia network coupled the HFS as injection current into the cells of the subthalamic nucleus (STN). Its overall increased activity rendered a faithful transmission of sensorimotor input through thalamo-cortical relay cells possible. Our contribution uses this model by Rubin and Terman (J Comput Neurosci, 16, 211–223, 2004) as a starting point and integrates recent findings on the importance of the extracellular concentrations of the inhibiting neurotransmitter GABA. We are able to show in this computational study that besides electrical stimulation a high concentration of GABA and its resulting conductivity in STN cells is able to re-establish faithful thalamocortical relaying, which otherwise broke down in the simulated parkinsonian state.     

Pathway Length and Evolutionary Constraint in Amino Acid Biosynthesis

The evolutionary properties of a metabolic network may be determined by the topology of the network. One attribute of pathways that make up the network is the number of enzymatic steps between initial substrates and final products. To determine the effect of pathway length on evolutionary lability of pathway structure, we examined amino acid biosynthetic pathways across 48 sequenced organisms. We demonstrate that longer pathways exhibit lower rates of change in pathway structure than shorter pathways. This finding suggests that increasing complexity may increase constraint on evolutionary change. (Matthew T. Rutter and Rebecca A. Zufall) Both authors contributed equally to this work.     

苯乳酸生物合成的研究进展*

苯乳酸是一种天然无毒且具有广谱抑菌作用的新型生物防腐剂,可由多种不同的微生物代谢产生,大多数乳酸菌都能够代谢生成苯乳酸。苯乳酸应用广泛,不仅能够应用于食品行业抑制多种病原菌,还可以广泛应用于医药和化工业。对苯乳酸的特性和应用以及其生物合成进行了综述,以期对苯乳酸及其生物合成有较系统的了解。     

苯乳酸生物合成的研究进展*

苯乳酸是一种天然无毒且具有广谱抑菌作用的新型生物防腐剂,可由多种不同的微生物代谢产生,大多数乳酸菌都能够代谢生成苯乳酸。苯乳酸应用广泛,不仅能够应用于食品行业抑制多种病原菌,还可以广泛应用于医药和化工业。对苯乳酸的特性和应用以及其生物合成进行了综述,以期对苯乳酸及其生物合成有较系统的了解。     

Identification of a region in G protein γ subunits conserved across species but hypervariable among subunit isoforms

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: alpha, beta, and gamma. There are 12 known mammalian gamma subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain gamma(10) protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of gamma isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 gamma subunit sequences from four different species shows that the N-terminus of a given gamma subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of gamma is a functionally important part of the protein exhibiting substantial isoform-specific variation.     

Interactions and chaperone function of alphaA-crystallin with T5P gammaC-crystallin mutant

T5P gammaC-crystallin mutation is associated with Coppock-like cataract, one of the autosomal dominant congenital cataracts. It is not known why the abundant alpha-crystallin cannot prevent the mutation-related aggregation. Our previous studies indicate that the mutation changes conformation and reduces solubility and stability, but it is not known whether it is these events or the loss of interaction with other crystallins that causes the cataract. It is also not known whether the alpha-crystallin can protect T5P mutant as effectively from heat-induced aggregation as the wild-type (WT) gammaC-crystallin. To investigate the mechanism of interactions and chaperone function between alphaA- and gammaC-crystallin, human alphaA-crystallin and W9F mutant as well as WT gammaC-crystallin and T5P mutant were cloned. Interactions between alphaA- and gammaC-crystallin were studied with fluorescence resonance energy transfer (FRET), and chaperone activity was assessed by the suppression of heat-induced aggregation of substrate proteins. Conformational changes of substrate proteins were studied by spectroscopic measurements. The results indicate that the T5P mutant showed a slightly greater FRET than WT gammaC-crystallin with alphaA-crystallin, and alphaA-crystallin could effectively prevent both WT and T5P gammaC-crystallin from heat-induced aggregation. Spectroscopic measurements show that both alphaA-crystallin and gammaC-crystallin underwent only slight conformational change after chaperone binding. Together with previous results obtained with a two-hybrid system assay of interactions between alphaA- and gammaC-crystallin, the present FRET and chaperone results indicate that loss of interactions of T5P mutant with other crystallins may play a larger role than the protection afforded by chaperone-like activity in Coppock-like cataract.     

透明质酸生物合成途径及基因工程研究进展

透明质酸是由N-乙酰氨基葡萄糖和葡萄糖醛酸组成的双糖单位聚合而成的直链酸性黏多糖,已被广泛应用于药物、化妆品和食品添加剂。微生物发酵法是目前生产透明质酸最有效的方法。生物体内透明质酸的合成途径基本一致,均为Leloir途径。透明质酸合成操纵子由透明质酸合酶基因、尿苷二磷酸葡萄糖脱氢酶基因和尿苷二磷酸葡萄糖焦磷酸化酶基因组成,其表达受Cov S/CovR和Lux S等多种调控系统调控。随着分子生物学技术的迅速发展以及对透明质酸合成相关基因了解的不断深入,人们从提高透明质酸安全性、提高透明质酸产量和调控透明质酸分子质量三个方面出发,通过基因工程手段构建出了高产、安全、一定分子质量范围的透明质酸生产菌株。就有关透明质酸生物合成途径、合成相关基因表达调控及生产菌株分子生物学改造的策略与研究进展进行综述和展望。