The plasmodesmata-associated β-1,3-glucanase gene GhPdBG regulates fiber development in cotton

Trichomes are specialized structures that originate from epidermal cells of organs in higher plants.The cotton fiber is a unique single-celled trichome that elongates from the seed coat epidermis.Cotton(Gossypium hirsutum)fibers and trichomes are models for cell differentiation.In an attempt to elucidate the intercellular factors that regulate fiber and trichome cell development,we identified a plasmodesmal β-1,3-glucanase gene(designated GhPdBG)controlling the opening and closing of plasmodesmata in cotton fibers.Structural and evolutionary analysis showed haplotypic variation in the promoter region of the GhPdBG gene among 352 cotton accessions,but high conservation in the coding region.GhPdBG was expressed predominantly in cotton fibers and localized to plasmodesmata(PD).Expression patterns of PdBG that corresponded to PD permeability were apparent during fiber development in G.hirsutum and G.barbadense.The PdBG-mediated opening-closure of PD appears to be involved in fiber development and may account for the contrasting fiber traits of these two species.Ectopic expression of GhPdBG revealed that it functions in regulating fiber and trichome length and/or density by modulating plasmodesmatal permeability.This finding suggests that plasmodesmal targeting of GhPdBG,as a switch of intercellular channels,regulates single-celled fiber and trichome development in cotton.

Partitioning and purification of extracellular β-1,3-1,4-glucanase in aqueous two-phase systems

The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene glycol （PEG）/MgSO4） was examined with regard to the effects of PEG molecular weight （MW） and concentration, MgSO4 concentration, pH and NaC1 concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly, pH had little effect on β-glucanase or proteases partition but affected a-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.

Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution

In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.

黑曲霉内切β-1,3(4)-葡聚糖酶的基因克隆与酶学特性分析

基于黑曲霉CBS 513.88的基因组信息,以黑曲霉CICIM F0510为出发菌株,提取其总RNA,反转录得到c DNA,成功将其3个内切β-1,3(4)-葡聚糖酶的基因(en3g A、en3g B、en3g C)在毕赤酵母中进行了克隆与表达。获得了3个重组菌GS115(p PIC-en3g A)、GS115(p PIC-en3g B)和GS115(p PIC-en3g C)。在摇瓶水平上,这株重组菌的酶活分别为1.3、8.5和10.1 U/m L。重组酶En3g A、En3g B和En3g C的最适作用温度分别为65、50和55°C;最适作用p H分别为5.0、5.0和3.5。此外,3种重组酶对羧甲基纤维素钠(sodium carboxymethylcellulose,CMC-Na)和凝结多糖都具有典型的内切水解作用。这3个重组β-1,3(4)-葡聚糖酶具有良好的温度和p H稳定性,可为其在啤酒制造以及饲料加工过程中的应用奠定基础。

拟青霉β-1,3(4)-葡聚糖酶同源建模及共价固定化

采用同源建模的方法构建拟青霉β-1,3(4)-葡聚糖酶的三维结构。通过对其活性位点及表面氨基酸残基侧链的分析,利用氨基载体Sepabeads EC-HA共价固定化葡聚糖酶,优化固定化条件,比较固定化酶与游离酶的酶学参数。结果表明:m(酶粉):m(载体)=1.2:1、温度40～45℃、固定化时间8h,固定化效果最好。蛋白结合率可达91.7%,酶活回收率达87.6%,固定化酶最适温度、热稳定性、pH值稳定性和批次使用稳定性均得到明显提高。

微波促进的1,3-偶极环加成4β-1,2,3-三唑鬼臼毒素的合成

在微波作用的无溶剂条件下 ,由 4β 叠鬼臼毒素与炔烃反应 ,合成了 8个含有 1,2 ,3 三唑的鬼臼毒素衍生物 ,并用IR ,1HNMR ,MS确定了这些化合物的结构 .该反应操作简单 ,产率较高 ,副产物少 ,同时具有原子经济性 .

源于Paecilomyces sp.FLH30内切β-1,3(4)葡聚糖酶基因在毕赤酵母中的表达

【目的】β-1，3（4）-葡聚糖酶是一种重要的工业用酶，广泛地应用于饲料业、酿造业及纺织业。【方法】以来源于Paecilomyces sp．FLH30的葡聚糖酶基因为研究对象。据毕赤酵母GS115对密码子的简并性和偏爱性，对来源于淡紫拟青霉（Paecilomyces lilacinus）β-1，3（4）-葡聚糖酶基因进行了优化，通过全基因技术合成了全长基因GLUnm并构建重组酵母表达载体pPIC9K—GLUnm，用SacI线性化重组质粒pPIC9K—GLUnm，电击转化至毕赤酵母GS115中进行表达。【结果】通过表型筛选，遗传霉素抗性筛选，经PCR鉴定表明，葡聚糖酶基因整合至酵母染色体DNA中。在试管中经甲醇诱导表达，并测定葡聚糖酶酶活性。在试管水平表达的酶活性是0．68IU／mL，即可以认为在摇瓶表达水平的初始酶活性为0．68IU／mL，将此菌株在摇瓶水平表达，酶活性在表达84h为最高，是11．26IU／mL。【结论】重组β-1，3（4）-葡聚糖酶的最适pH为5．0，最适反应温度为50℃，在pH5．0～8．0，温度37—50℃的条件下较稳定。

Improving the Heat Resistance ofβ-1,4 Glucanase by Introducing Disulfide Bonds

Each possible pair of residues inβ-1,4 glucanase for disulfide formation was assessed using online websites,and four pairs L28C-S256C,Q41C-P278C,S122C-N163C and A184C-A215C were selected.Accordingly,four recombinant plasmids pET28a(+)EccslH28,pET28a(+)EccslH41,pET28a(+)EccslH122 and pET28a(+)EccslH184 were prepared and transformed into E.coli to express the recombinant enzymes.Then analysis on enzymatic properties showed that T50 of the recombinant enzymes was increased from 10 min for EccslHt2 to 90 min for EccslH28 and 40 min for EccslH41 at 70℃,while their optimum pH value and pH stability were not affected,which proved that the introduction of disulfide bond improved the thermal stability ofβ-1,4 glucanase.

Changes in Protein Content and Chitinase and β-1,3- glucanase Activities of Rice with Blast Resistance Induced by Ag-antibiotic 702

[Objective] The paper was to explore the effect of rice blast resistance induced by Ag-antibiotics 702 on protein content and chitlnase and β-1,3-glucanase activities in rice. [ Method] At the fourth leaf stage of Luliangyou 996, 15 μg/mL Ag-antibiotic 702 was sprayed, while Validamycin and distilled wa- ter were sprayed as positive control and negative control, respectively. All treatments were inoculated with spore fluid of Magnaporthe grisea at 48 h post spraying, and the rice inoculated with only distilled water was used as blank control. The enzymes activities （endochitinase, exochitinase and β-1,3-glucanase） and total pro- tein content in rice leaves were determined every 24 h within 168 h post spraying. [ Result] Compared with the blank control, the rice inoculated with spore fluid of M. grisea could significantly increase the total protein content and the activities of β-1,3-glucanase and chitinase. The induction effect of Ag-antibiotics 702 exceeded that of Validamycin treatment. And the changes in activities of β-1,3-glucanase and chitinase had obvious synchronicity. [ Conclusion] Ag-antibiotic 702 can significantly improve the total protein content and the activities of β-1,3-glucanase and chitinase, thus enhancing the resistance to rice blast.

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase （GALl） and phosphoglycerate kinase 1 （PGK1） promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and ex- pressed under the control of the GALl promoter, α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was en- hanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.

一锅法合成4-[4,6-二(2,4-二甲苯基)-1,3,5-三嗪-2-基]-1,3-苯二醇

以三聚氯氰为起始原料,与间二甲苯和间苯二酚在氯化铝和浓盐酸的催化作用下经两次傅克反应一锅法合成了4-[4,6-二(2,4-二甲苯基)-1,3,5-三嗪-2-基]-1,3-苯二醇,产物结构经^(1)H NMR、^(13)C NMR和ESI-MS进行了确证。采用单因素实验考察了催化剂种类、助催化剂用量、反应温度和反应时间对该反应的影响。实验结果表明,较佳的反应条件为:以氯化铝为催化剂,浓盐酸用量为氯化铝质量的6%、第一步反应温度为5℃,第二步反应温度为80℃,4-[4,6-二(2,4-二甲苯基)-1,3,5-三嗪-2-基]-1,3-苯二醇的收率可达91.5%,纯度为99.3%。

催化邻羟基苯基取代对亚甲基醌与酮亚胺环加成反应合成二氢-1,3-苯并噁嗪化合物

发展了邻羟基苯基取代对亚甲基醌与酮亚胺在磷酸催化体系中的[4+2]环加成反应,以高产率和优良的非对映选择性得到了一系列二氢-1,3-苯并噁嗪化合物.当向反应体系加入催化量的B(C_(6)F_(5))_(3)时,产物的产率得到保持且发生非对映选择性翻转.

手性毛细管气相色谱法拆分4-氯甲基-2,2-二甲基-1,3-二氧戊环对映异构体

手性化合物具有独特的生物活性,其在药物治疗中发挥着关键作用。为准确测定对映异构体中不同构型的纯度,建立手性化合物对映体分离方法十分关键。4-氯甲基-2,2-二甲基-1,3-二氧戊环是一种重要的手性医药中间体,本文建立了一种手性毛细管气相色谱拆分测定4-氯甲基-2,2-二甲基-1,3-二氧戊环对映异构体的方法。采用固定相为环糊精衍生物的色谱柱Rt-bDEXse进行分离,并以氢火焰离子化检测器进行检测。经过优化,得到最优色谱条件:线速度为70 cm/s;升温程序为初始柱温70℃保持1 min,以2.0℃/min的速率升温到150℃;溶样溶剂为甲醇。实验结果表明,在该条件下,(R)-4-氯甲基-2,2-二甲基-1,3-二氧戊环和(S)-4-氯甲基-2,2-二甲基-1,3-二氧戊环可在10 min内快速分离,两者的分离度远大于1.5,在0.5~50.0 mg/L的范围内线性关系良好,线性相关系数均大于0.998,(R)-和(S)-4-氯甲基-2,2-二甲基-1,3-二氧戊环的检出限分别为0.07和0.08 mg/L,定量限分别为0.22 mg/L和0.25 mg/L。以甲醇作为空白样品,进行0.5、2.0、10.0 mg/L 3个不同水平的加标回收试验,(R)-和(S)-4-氯甲基-2,2-二甲基-1,3-二氧戊环的回收率分别为94.0%~99.1%和96.0%~98.8%,相对标准偏差(RSD)分别为1.26%~4.87%和1.51%~4.46%。该方法可为手性医药中间体4-氯甲基-2,2-二甲基-1,3-二氧戊环对映异构体的拆分提供参考。

2,2-二甲基-4-羟基-1,3-苯并二噁茂的合成研究

以2-甲氧基丙烯代替2,2-二甲氧基丙烷为原料合成了2,2-二甲基-4-羟基-1,3-苯并二噁茂。研究了影响反应收率的因素,优化条件为:以二甲苯为溶剂,m(焦性没食子酸)∶m(二甲苯)=1.0∶5.0,n(焦性没食子酸)∶n(2-甲氧基丙烯)=1.00∶1.20,2-甲氧基丙烯滴加温度135℃,滴加时间6 h。优化条件下产物纯度99.2%,收率88.5%,其结构经GC-MS、^(1)H NMR确认。该工艺转化率高、收率高、“三废”少,具有工业化应用前景。

拟青霉内切β-1,3(4)-葡聚糖酶基因克隆、表达及重组酶性质

采用改造的酿酒酵母表达载体pYES2-GSL构建了拟青霉Paecilomyces sp.FLH30表达cDNA文库,刚果红平板法筛到一内切β-1,3(4)-葡聚糖酶基因(PsBg16A),全长cDNA为1 217 bp,完整开放阅读框(ORF)951 bp,编码316个氨基酸,属于糖苷水解酶16家族。去信号肽的PsBg16A克隆入表达载体pET28a(+)并在E.coli BL21成功表达,经16°C 20%乳糖诱导24 h,酶活达482 U/mL。重组PsBg16A可水解大麦葡聚糖、地衣多糖和昆布多糖,显示PsBg16A为典型的β-1,3(4)-葡聚糖酶(EC 3.2.1.6),重组酶最适pH和温度分别为7.0和65°C,对大麦葡聚糖、地衣多糖和昆布多糖的Km分别为3.12、4.86和10.32 g/L。

2-[5-(3,4,5-三甲氧基苯基)-1,3,4-噻二唑-2-基硫代]-1-(2,3,4-三甲氧基)苯乙酮肟醚合成与抗烟草花叶病毒活性研究

合成了4个2-[5-(3,4,5-三甲氧基苯基)-1,3,4-噻二唑-2-基硫代]-1-(2,3,4-三甲氧)苯乙酮肟醚新化合物,并经过元素分析、红外光谱、核磁共振氢谱对其结构进行了表征.初步生物活性测试表明,化合物具有一定抗烟草花叶病毒活性.

噁唑、苯并噁唑及1，3，4-噁二唑硫醚的合成及抗肿瘤活性研究

设计并以噁唑、苯并噁唑及1,3,4-噁二唑硫醇与卤代烃反应合成了14个硫醚类杂环化合物,其中13个化合物未见文献报道.目标化合物的结构均通过1H NMR谱、MS和元素分析进行了表征.采用MTT[3-(4,5-dimethylthiagol-2-yl)- 2,5-diphenyltetrogolium bromide]法对14个目标化合物进行了体外抗肿瘤活性测定,结果表明:在10-5mol/L浓度下,10个化合物对肿瘤细胞具有抑制活性.

沙质微泡菌β-1,3(4)-葡聚糖酶的克隆表达及酶学性质

【背景】β-葡聚糖是自然界中广泛存在的非淀粉多糖,是谷类植物细胞壁的主要成分。β-葡聚糖酶能够水解β-葡聚糖生成低聚合度的寡糖,在食品、饲料、造纸等领域发挥着重要的作用。【目的】从海洋细菌沙质微泡菌(Microbulbifer arenaceous)中克隆到一个β-1,3(4)-葡聚糖酶基因,在大肠杆菌中可溶表达,研究其相关酶学性质。【方法】以沙质微泡菌(Microbulbifer arenaceous)基因组DNA为模板,克隆一个β-1,3(4)-葡聚糖酶基因(MaGlu16A),构建重组表达载体p ET-28a-MaGlu16A并在大肠杆菌BL21(DE3)中诱导表达,通过Ni-NTA亲和层析纯化后进行酶学性质研究。【结果】MaGlu16A的最适pH和最适温度分别为pH 6.0和40°C,在pH 5.0-10.5和35°C以下稳定。对EDTA具有较高的抵抗性,在1 mmol/L和10 mmol/L EDTA浓度下仍保持99.3%和82.5%的酶活力。该酶能够有效水解可得然多糖、昆布多糖、大麦葡聚糖、地衣多糖、燕麦葡聚糖和酵母葡聚糖,水解产物主要为葡萄糖、二糖、三糖和四糖。【结论】海洋细菌沙质微泡菌(Microbulbiferarenaceous)来源β-1,3(4)-葡聚糖酶的克隆表达及酶学性质的测定为β-葡聚糖酶的挖掘及β-葡寡糖的制备奠定了基础。

1,3-二甲基-5-甲硫基-4-苯腙基羰基吡唑的合成及抑菌活性

Eight derivatives of 1,3-dimethyl-5-methylthio-4-phenylhydrazonocarboxyl pyrazole were synthesized from 1,3-dimethyl-5-methylthio-4-hydrazinocarboxyl pyrazole. All compounds synthesized were identified by 1H NMR and elemental analysis. Some of them exhibited certain growth inhibition action to Phoma asparagi, Alternaria solani, Gibberella zeae, Physalospora piricola, Colletotrichum lagenarium as tested in vitro at dosage of 50 mg/L.