产壳聚糖酶菌株的筛选、鉴定及酶学特性分析

【目的】利用筛选培养基,从福建沿海潮间带泥样中分离筛选产壳聚糖酶的菌株,并研究菌株的产酶特性。【方法】通过形态学观察,结合26S rDNA序列进行分类鉴定,采用DNS法测定酶活力。【结果】筛选得到产壳聚糖酶的菌株KQ-1002与草酸青霉(Penicillium oxalicum)的同源性为99%,并初步鉴定为青霉属的一种。发酵培养的最适温度为30°C,最适碳源为1.0%水溶性壳聚糖,最适氮源为1.87%(NH4)2SO4,最适pH为6.0。该菌株液体发酵培养72 h产壳聚糖酶活性最高,经优化后最高产酶量为18 U/mL。纯化后的壳聚糖酶经SDS-PAGE分析其分子量约40 kD。酶促反应最适pH为5.0,最适反应温度为55°C,Km值为1.293 g/L。在离子浓度为1.0×10 3mol/L时,金属离子Cu2+、Hg2+、Ag+对酶的活性均有强烈的抑制作用。壳聚糖酶对不同底物及脱乙酰度的壳聚糖具有不同的降解作用。【结论】筛选获得产壳聚糖酶的真菌菌株KQ-1002的壳聚糖酶活力经优化后提高了约7倍,是一株具有研究和应用潜力的产壳聚糖酶菌株。     

丝孢菌Monodictys asperospera (Cooke & Massee) Ellis漆酶的分离纯化及其酶学性质

本研究首次发现Monodictyx asperospera(Cooke&Massee)Ellis具有较好的产漆酶能力.粗酶液经硫酸铵盐析、DEAE-纤维素层析及丙烯葡聚糖凝胶S-300层析纯化,纯化倍数为8.1,回收率为5.7%.漆酶分子量约为77kD,最适反应温度为55℃,最适反应pH6.0,以丁香醛连氮为底物时Km为0.163mm0l/L,Vmax为0.194 mmol(L·min),含糖量为18.14%,Cu2+对漆酶有明显抑制作用.     

烟叶果胶质分解菌的选育

从烟叶叶面分离到产果胶酶真菌 18株 ,在此基础上 ,进行液体培养 ,测定酶活 ,得到一株酶活性较高的黄曲霉DPE - 0 0 5。对该菌株产酶培养基的碳、氮源进行正交法研究 ,正交实验的结果表明 ,影响该菌产酶活性的因素依次为A(麸皮 ) >B(乳糖 ) >C(果胶 ) >D(硫酸铵 ) ,其最佳组合为A2B3C3D1。最适产酶条件为 :麸皮 4 0 % ,果胶 0 3% ,乳糖 1 5 % ,(NH4 ) 2 SO4 0 5 % ,KH2 PO4 0 2 5 % ,MgSO4 ·7H2 O 0 0 5 % ,NaNO30 0 2 % ,FeSO4 ·7H2 O 0 0 0 1% ,起始pH 6 0 ,在 2 8℃摇床培养 7d产酶量达到最高。以玉溪B3F烟叶为材料 ,施加DPE - 0 0 5菌株所产酶液 ,在 5 0℃贮存 12h。经化学成分检测 ,结果表明 ,果胶质降低了 18 15 %。     

甲壳素酶Chisb的定向进化及生物转化合成几丁寡糖

甲壳素酶具有广泛的工业应用前景,如可将虾壳、蟹壳和其他甲壳废物降解成以几丁寡糖为主的高附加值产品,但野生型甲壳素酶催化效率低,大大限制了几丁寡糖的生产。笔者在前期研究中表达了一个具有较高效催化效率的甲壳素酶Chisb,并对其酶学性质进行了初步研究。为进一步提高甲壳素酶Chisb的催化效率,以R13NprB-C-SP-H为亲本,采用易错PCR(Error-pronePCR)技术构建随机突变体文库,对甲壳素酶Chisb进行定向进化。经过96孔板初筛和摇瓶复筛,获得了两个催化效率进一步提高的突变体C43D和E336R。对突变体的酶学性质进行分析, C43D和E336R的最适催化温度为55℃, C43D的最适pH为5.0,E336R的最适pH为9.0;其催化效率相比对照分别提高了1.35倍和1.57倍;而E336R和C43D催化产几丁寡糖的含量分别为2.53 g/L和2.06 g/L,相比对照(0.89 g/L)分别提高了2.84倍和2.31倍;底物转化率分别为84.3%和68.7%,相比对照(29.7%)分别提高了54.6%和39%。研究表明,通过易错PCR引入随机突变的方法能够有效提高甲壳素酶Chisb的催化效率。上述研究获得的催化效率提高的正向突变体及其酶学性质分析对生物转化合成几丁寡糖具有重要研究意义和应用价值。     

拟南芥阿拉伯糖-5-磷酸异构酶的原核表达、纯化及酶催化特性

阿拉伯糖-5-磷酸异构酶(Kds D)是2-酮-3-脱氧辛糖酸(KDO)生物合成途径的第一个关键限速酶,通过无缝克隆技术将拟南芥Kds D基因构建至原核表达载体p ET-HTT,经过IPTG诱导,在大肠杆菌BL21(DE3)中获得了大量重组蛋白的可溶性表达;表达产物经Ni-NTA亲和层析和分子筛层析(SEC)方法进行酶蛋白的分离纯化步骤,得到纯度85%以上的高纯度酶;分子筛层析结果发现纯化后的目的蛋白Kds D在溶液中主要以多聚体、二聚体和单体形式存在,这同微生物来源Kds D酶在溶液中以四聚体形式存在很大差异;进一步使用Western blotting和MALDI-TOF MASS技术对纯化的蛋白进行鉴定;测定了拟南芥Kds D酶学性质,证明该酶催化反应的最适p H值为8.0,最适作用温度为37℃,各种金属离子在低浓度均对酶活性存在不同程度的抑制作用,其中以Co~(2+)、Cd~(2+)对酶活性的抑制作用最强,而5 mmol/L金属螯合剂EDTA对酶有激活作用。此外,以阿拉伯糖-5-磷酸(A5P)为底物时,拟南芥Kds D酶动力学常数Vmax和Km值分别为0.18 mmol/(L·min)、0.16 mmol/L,比较发现该酶与底物的亲和性高于大肠杆菌Kds D。以上研究结果为Kds D蛋白结构与功能及其在新型抗生素研制领域中的工业化应用奠定了基础。     

假交替单胞菌LJ1菌株产褐藻胶裂解酶的培养条件优化及酶学性质

[目的]为了优化Lj1菌株的培养条件使之产生高活性的胞外褐藻胶裂解酶.[方法]通过富集培养技术从海带筛选到一株褐藻胶裂解酶产生菌Lj1,依据表型特征、脂肪酸组成分析及16S rRNA基因序列分析对该菌株进行鉴定.通过单因子和正交试验对Lj1菌株产胞外褐藻胶裂解酶的培养条件进行了优化.[结果]Lj1菌株属于假交替单胞菌属(Pseudoalteromonas).该菌株产酶的最佳培养基组成为:褐藻胶3g/L、(NH4)2SO43 g/L、NaCl 20 g/L、KH2PO4 0.1 g/L、CaCl2 0.1 g/L;最佳培养条件为:250 mL三角烧瓶中装液量25 mL、接种量3%、摇瓶转速150 r/min、pH7.5、培养温度为28℃、培养时间为24 h.LJl菌株所产褐藻胶裂解酶的最适温度为40℃,最适pH7.6,最适NaCl浓度为0.3 mol/L.1 mol/1.金属离子Mg2+对酶活力有明显的促进作用,而C02+和Zn2+对酶活力有较强的抑制作用.[结论]LJ1菌株是Pseudoalteromonas新的胞外褐藻胶裂解酶产生菌,在最佳培养条件下,该菌株的酶活力提高了66%.     

产壳聚糖酶菌株的发酵条件优化

方法:通过单因子实验,对保存的1株产壳聚糖酶的菌株C001进行发酵产酶条件优化,确定了最适产酶培养基组分.结果:温度30℃,发酵时间18h,pH5.5,接种量5%优化发酵条件后,产壳聚糖酶活力增长了37.4%.     

黑色葡萄状穗霉S607耐碱性纤维素酶发酵条件的研究

研究了耐碱性纤维素酶生产菌株黑色葡萄状穗霉S6 0 7(StachybotrysatraS6 0 7)的产酶条件。结果表明 ,S6 0 7的最适生长和产酶 pH为 7.0 ,最适产酶温度为 2 8℃ ,摇床最适转速为 180r/min。在以 2 .0 %纤维素粉和 2 .5 %的麸皮作为碳源时产酶最高 ,添加含 (NH4 ) 2 SO4 、尿素和蛋白胨的复合氮源对生长和产酶有明显的促进作用。在最适培养条件下 ,发酵周期为 4～ 5d ,发酵液中CMC酶活为 2 .12IU /mL ,FP酶活为 1.0 3IU /mL ,β 葡萄糖苷酶活为 0 .86IU /mL。     

Eupergit C250L固定化D-海因酶及其催化性质

D-海因酶是海因酶法制备D-氨基酸的关键酶。利用Burkholderic cepecia1003菌发酵产酶,所得海因酶纯化后,以Eupergit C250L为载体进行共价固定化。分别考察了酶液蛋白浓度、固定化时间对蛋白固定量和酶活回收率的影响以及固定化前后海因酶催化性质的变化。结果表明:较高的酶液蛋白浓度和较长的固定化时间均有助于改善海因酶的固定化效果;固定化可显著提高海因酶的最适作用温度,但对其最适作用pH影响不大;固定化后海因酶对D,L-BH和MH的米氏常数均有较大幅度的降低。固定化酶反应器的实验表明:40℃下,底物(D,L-BH)1.0 g.L-1,体积流速1.0 mL.min-1,经21 h转化,产物N-Phe质量浓度可达0.47 g.L-1,转化率达43.21%。     

变色栓菌锰过氧化物酶同工酶的纯化及其性质研究

变色栓菌(Trametes versicolor)胞外产酶培养液经硫酸铵沉淀、DEAE-cellulose DE52离子交换柱层析后,获得两个活性组分D1和D2,其中活性组分D2经Phenyl SepharoseTM6Fast Flow疏水层析后,所得样品MnP1经SDS-PAGE检测已达到电泳纯。活性组分D1经Phenyl SepharoseTM6Fast Flow疏水层析、Sephacryl S-200HR凝胶过滤层析后,所得样品MnP2经SDS-PAGE检测已达到电泳纯。两种同工酶MnP1及MnP2,各自的比活力为579.09、425.00U/mg;纯化倍数为17.51、12.85;活力回收率为6.17%、2.47%。由SDS-PAGE法测得MnP1及MnP2的表观分子量分别为46.3kD、43.0kD。两种同工酶催化DMP(2,6-二甲氧基酚)氧化反应的最适pH值及最适反应温度有所不同,最适pH值分别为pH5.8、pH6.2,最适反应温度分别为60℃、65℃。在45℃以下,pH4.0~7.0之间,MnP1及MnP2的稳定性好。DMP为最佳酶促反应底物,以DMP为底物的Km分别为13.43μmol/L、12.45μmol/L。在无Mn2 存在的条件下,酶促反应几乎不发生。EDTA在较高浓度时抑制酶的活性,DTT在所试浓度下都完全抑制酶的活性。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.     

Composition and diversity of bird communities in Swedish farmland–forest mosaic landscapes

CapsuleThe amount of forest (at local and landscape scales) and occurrence of residual habitats at the local scale are shown to be the major factors influencing bird community composition in farmland–forest landscapes in central Sweden.

Aims To investigate the importance of local habitat and landscape structure for breeding birds in farmland–forest landscapes in central Sweden.

Methods Breeding birds were censused at 292 points. A detailed habitat mapping was made within 300 m of the points. Within a 300–600 m radius only two major habitats (forests and arable fields) were identified.

Results Cluster analyses of bird communities identified three site types that also differed in habitat composition: (i) partially forested sites in forested landscapes; (ii) heterogeneous sites with residual habitats in mosaic landscapes; and (iii) field-dominated farmland sites in open landscapes. A total of 19 of 25 farmland bird species (restricted to farmland or using both farmland and forest) had the highest abundance in farmland sites with mosaics of forest and farmland, while only six farmland species had the highest abundance in field-dominated sites. The bird community changed from being dominated by farmland species to being dominated by forest species (common in forest landscapes without farmland) at small proportions (10–20%) of forest at the local scale. A major difference in habitat composition between heterogeneous and field-dominated sites was the occurrence of different residual habitats (e.g. shrubby areas and seminatural grasslands). These habitats seemed to influence bird community composition more than land-use, despite covering <10% of the area. Seminatural grasslands were important for bird community composition and species-richness, but grazing seemed to be less important. Among different land-use types, cereal crops were the least preferred fields. Set-asides with tall vegetation and short rotation coppices were positively associated with species-richness of farmland birds.

Conclusion In general, the composition of the landscape was important for bird community composition, although amount and distribution of forests, occurrence of residual habitats and land-use of fields at the local scale had the strongest influence on bird community composition. The possible implications of these patterns for managing farmland–forest landscapes are discussed.     

Chemical constituents from the fruits of Rhus typhina L. and their chemotaxonomic significance

This work describes the isolation and characterization of twenty-nine compounds from the fruits of Rhus typhina L., including eleven flavonoids (1–11), eleven phenols (12–22), two pentacyclic triterpenes (23–24), two organic acids (25–26), one lumichrome (27), one courmarin (28) and one pyrimidine (29) on the basis of their spectroscopic data. Compounds apigenin (1), daidzein (4), orobol (5), 3′, 5, 5′, 7-tetrahydroxyflavanone (6), naringenin (7), butein (8), (-)-catechin (9), quercetin-3-O-α-L-(3″-O-galloyl)-rhamnoside (11), 2-hydroxybenzoic acid (13), 4-hydroxybenzaldehyde (14), vanillin (15), methyl 3,4-dihydroxybenzoate (16), 3,5-dihydroxybenzamide (18), tyrosol (19), caffeic acid (20), 3-(2,4,6-trihydroxyphenyl)-1-(4-hydroxyphenyl)-propan-1-one (21), phlorizin (22), friedelin (23), oleanolic acid (24), 4,4-dimethyl-heptanedioic acid (25), anthranilic acid (26), lumichrome (27), scoparone (28) and uracil (29) have not been recorded before in this plant. This is the first report on the occurrence of compounds 4–7, 9, 11, 13–14, 16, 18–21, 25–29 from the genus Rhus. Moreover, the chemotaxonomic significance of these isolated compounds was also summarized.