黄蓝状菌几丁质酶E2纯化、性质及抗菌活性研究

黄蓝状菌在SMCS液体培养基中置于恒温摇床（200r／min，27．5C）上培养13天，诱导产生大量的几丁质酶。培养滤液经硫酸铵分级沉淀，DEAE--Sepharose FastFlow阴离子交换层析、Phenyl—Sepharose Fast Flow疏水层析、Sephacryl S-100分子筛层析得到了凝胶电泳（SDS—PAGE）谱带单一的几丁质酶E2，其分子量为32KD。其最适反应温度为50℃，最适pH值是5。Mn^2＋对其有明显的激活作用，Cu^2＋、FeCu^2＋对其有强烈的抑制作用。抗菌活性显示．其对供试病原菌具有明显的抑菌作用。     

小黑杨转几丁质酶基因及酶活性

This paper reports a successful transformation of chitinase gene into Populus simonii×P. nigra by Agrobacterium-mediated means. For reducing adventitious buds,the optimal concentration of kanamycin was 40 mg·L~ -1 . Up to 700 mg·L~ -1 of Cefazolin Sodium had no obvious effect on differentiation of leaves. About 20-day-old leaf disc explants were pre-cultured for 3-4 d,then immersed in Agrobacterium suspension for infection for 6-15 min,then co-cultured on non-selection culture medium for 3 d in the dark at ...     

3种白腐菌木质素降解酶的比较

对3种白腐菌——黄孢原毛平革菌、变色栓菌和木质层孔菌在恒温振荡培养条件下产漆酶、锰过氧化物酶的情况进行比较研究,同时还对此3种菌在培养过程中还原糖的变化作了研究.结果表明:3种白腐菌中变色栓菌产漆酶相对最高,其漆酶酶活最高达到136.5 U/L,产酶最高时间为第8天;而木质层孔菌产锰过氧化物酶相对最高,其最高酶活达到880.2 U/L,产酶最高时间为第8天;同时在培养过程中还原糖含量随培养时间的延长而逐渐降低.     

橡胶树胶乳中几丁质酶的研究进展

几丁质酶是一种分解N-乙酰氨基葡萄糖分子的糖苷酶。在高等植物中，几丁质酶虽然普遍存在，但迄今未发现其底物的存在。据研究，几丁质酶在植物抗病机制中起重要作用。近年来，通过对橡胶胶乳中几丁质酶的研究，认识到它不仅是橡胶树防御病原微生物的抗病系统的重要组成部分，而且还参加了胶乳的凝固过程，与橡胶树的排胶及产量具有密切的关系。文章概述了几丁质酶研究的现状及最新进展，介绍了橡胶树胶乳中几丁质酶的理化性质、生理学功能、在细胞中的定位及其调控。     

HearNPV几丁质酶对CypoGV的协同增效作用

利用大肠杆菌可溶性表达棉铃虫核型多角体病毒(HearNPV)几丁质酶,用表达粗产物与苹果蠹蛾颗粒体病毒(CypoGV)按不同比例混合喂食3龄苹果蠹蛾幼虫,测定其对CypoGV的增效作用。生测结果显示,表达的可溶性He-arNPVChiA在2.0μg/mL的添加水平时,LT50和LT90分别比对照缩短了13.7%和20.7%。研究结果说明HearN-PVChiA对CypoGV具有明显增效作用。     

几丁质酶及其在转基因植株内活力的测定及定位

介绍了几丁质酶的作用原理,植物中几丁质酶的分类及表达特点,采用荧光定量法测定转几丁质酶基因植株中几丁质酶的活性,对转基因植物的叶片进行体外包埋与切片,然后利用激光共聚焦显微镜扫描,以确定所转几丁质酶基因的表达部位。     

木材蓝变生物控制菌康氏木霉胞外几丁质酶的纯化及特性

本研究以木材蓝变生物控制菌康氏木霉(6111-ET)为供试菌株进行胞外几丁质酶纯化和特性的研究.将液态PDA培养基中的康氏木霉(Trichoderma koningii)置于恒温播床(200 r/min,28℃)培养10天.培养滤液经过硫酸铵分级沉淀、Q-HP强阴离子交换柱层析、Phenyl-Sepharose疏水层析...     

蜡梅几丁质酶基因的克隆与原核表达

在构建好蜡梅花cDNA文库并进行EST分析基础上,通过随机克隆测序,得到1个蜡梅几丁质酶的cDNA基因,命名为Cpchia(GenBank登录号:FJ749130).Cpchia cDNA全长为1 184 bp,开放阅读框为954 bp,编码317个氨基酸,其结构包括信号肽、几丁质结合域、可变交联区、催化区,无C端延伸区,为Class Ⅰ b型胞外几丁质酶,属于几丁质酶第19家族.将Cpchia克隆到原核表达载体pET-28a(+),在大肠杆菌BL21细胞中以包涵体形式表达融合蛋白,利用透析法获得复性蛋白,其几丁质酶活性经DNS法检测达到200 U·mL~(-1).酶活性和稳定性分析表明,试验条件下,pH 7.0有利于酶的稳定和活性发挥,40 ℃活性最高,在0 ℃低温下也有较高活性.上述结果说明,分离的Cpchia基因编码蛋白具有几丁质酶活性,而且可能与蜡梅花的抗寒性形成有关.     

来源于解酯菌的嗜热耐碱脂肪酶的表达纯化及其酶学性质研究

脂肪酶广泛应用于食品加工、生物柴油制备等领域。为了有效提高微生物脂肪酶的可利用度,将来源于解酯嗜热菌(Thermosyntropha lipolytica DSM 11003)的脂肪酶基因(tll2)克隆到大肠杆菌表达载体pET28a中,获得重组质粒pET28a TLL2,将pET28a TLL2转入大肠杆菌BL21(DE3)宿主菌中进行高效表达。通过热处理和镍柱亲和层析得到电泳纯的蛋白TlLip2,对其进行酶学性质表征。结果显示,TlLip2的亚基分子质量为57 ku,比酶活为31 U/mg,最适反应pH及温度分别为8.0和60℃。在60℃以下、pH 6.0~11.0范围内,TlLip2的稳定性较好,相对酶活均维持在60%以上。金属离子K+和Na+对TlLip2有较大激活作用,而Co^2+、Zn^2+、SDS和Tween 80对TlLip2酶活力抑制作用明显。TlLip2在极性小的有机溶剂中具有较好的耐受性。TlLip2对中等碳链长度的对硝基苯酯显示出更高的活性,其中以对硝基苯己酯为底物时,TlLip2的酶活最高;以对硝基苯棕榈酸酯为底物时,该酶的动力学常数Km为0.29 mmol/L,Vmax为2.28 mmol/(L·min^-1),kcat为6.19 s^-1。此外,将其应用到油酸乙酯的合成研究中,以油酸和乙醇为底物,研究TlLip2催化制备油酸乙酯的转化条件,结果表明在60℃、加酶量为100 U/g的TlLip2,催化摩尔比为1∶2的油酸和乙醇,12 h后油酸乙酯的转化率达到62.1%,表明TlLip2在催化脂肪酸酯化方面具有较大的应用潜能。     

优化彩绒革盖菌产漆酶条件及染料脱色研究

对一株漆酶高产白腐菌———彩绒革盖菌的培养和产酶条件,包括培养基初始pH值、金属离子Cu2 和Mn2 的含量,以及诱导剂的种类、添加量和添加时间等相关因素进行了优化。结果发现该菌种适宜在pH值3.5~5.7环境下生长并合成漆酶,培养基中添加缓冲液可以获得更高的酶活,但不加则有较高的酶产率;铜和锰可以显著提高漆酶活力,当以0.1 mmol/L 2,5-二甲基苯胺为诱导剂时,培养基中宜含0.008~0.08 mmol/L Cu2 ,0.01 mmol/L左右的Mn2 。在5种常用漆酶诱导剂中,2,5-二甲基苯胺的效果最佳,且应在菌种进入对数生长期时加入效果最好,其最适添加浓度为0.4 mmol/L,此时最适产酶Cu2 浓度为0.4 mmol/L。研究显示漆酶在30和40℃下保持6 h后残余酶活分别为原来的95%和80%;60℃条件下,该酶保温1 h后的残余活力仅为原来的14%。该酶的最适反应温度是65℃,最适反应pH值为2.4~3。对20种常用染料进行的脱色研究显示,在pH值5、40℃条件下,该粗漆酶液可以直接作用于13种工业染料。     

洋葱伯克霍尔德菌C23抗真菌活性多肽的分离纯化

为了深入研究洋葱伯克霍尔德菌C23(Burkholderia cepacia C23)菌株产生的抗真菌活性物质,以立枯丝核菌(Rhizoctonia solani)为指示菌进行生物活性跟踪,采用有机沉淀法、超滤法、Sephadex G25分子凝胶过滤法对活性物质进行分离纯化。结果表明,洋葱伯克霍尔德菌C23菌株产生的抗真菌活性物质为分子量5 000～10 000 Da的多肽类物质;建立了用超滤、分子凝胶过滤快速有效分离纯化伯克霍尔德菌C23菌株活性多肽的技术方法,为进一步制备单一的活性多肽成分奠定了基础。     

Identification of the antifungal compound,trans-cinnamic acid,produced by Photorhabdus luminescens,a potential biopesticide against pecan scab

Pecan scab (caused by Fusicladium effusum) limits the productivity of pecan in the southeastern USA. Alternatives to conventional fungicides should be biorational, of low environmental risk with a lower risk of fungicide resistance. Research showed that metabolites from the nematode symbiont Photorhabdus luminescens suppress pecan scab, but the bioactive molecules had not been identified. Extracts from P. luminescens were investigated using a bioactivity-directed fractionation approach to identify the constituent(s) responsible for the activity. High throughput antifungal bioautography assays against Colletotrichum gloeosporioides, C. fragariae, and C. acutatum were used to guide the fractionation. One of the metabolites was purified and identified as trans-cinnamic acid (TCA) using silica gel chromatography followed by semi-preparative high-performance liquid chromatography. In vitro tests confirmed toxicity of TCA to C. gloeosporioides, C. fragariae, and C. acutatum at 10 and 100 μg mL^?1 using fungal bioautography inhibition screening plates. The antimycotic activity of TCA was tested in vitro against F. effusum. Zone of inhibition tests, and tests with TCA incorporated into agar showed TCA toxicity to F. effusum at concentration 148–200+ μg mL^?1. Further tests incorporating TCA into liquid media demonstrated that TCA arrested all growth of F. effusum at a concentration even as low as 64 μg mL^?1. Naturally occurring antimicrobial products might offer an alternative to disease control in crops, helping in minimizing the risk of fungicide resistance, while also minimizing any negative impact on the environment. Additional research is warranted to determine the potential to use TCA as a suppressive agent for pecan scab and other diseases.     

松材线虫纤维素酶的分离纯化及性质研究

以浙江省松材线虫病疫区马尾松样品中分离得到的松材线虫为研究对象,对其分泌物中纤维素酶的活力和酶学性质进行分析,并与试验筛选出的伴生真菌所分泌的纤维素酶进行对比。试验结果证明,两者在酶促反应最适温度、酶促反应最适pH值、金属离子对酶活力影响方面均有明显差异。     

Antibacterial and antifungal activity of pinosylvin, a constituent of pine

The antibacterial and antifungal activities of pinosylvin (3,5-dihydroxy-trans-stilbene), a constituent of pine, were studied and compared with those of resveratrol (3,5,4'-trihydroxy-trans-stilbene). Pinosylvin exhibited more potent growth inhibitory activity against Candida albicans and Saccharomyces cerevisiae.     

Purification of a lectin-like antifungal protein from the medicinal herb, Withania somnifera

A 30 KDa monomeric acidic lectin-like protein was purified from the leaves of an important medicinal herb, Withania somnifera (L.) Dunal (Solanaceae), by a series of gel filtration and affinity chromatography methods. The inhibitory concentration of the protein ranged from 7 microg to 11 microg against major phytopathogens under in vitro conditions. The peptide sequence showed similarity to concanavalin A like lectin from Canavalia ensiformis and caused distinct cell wall adhesion of the protein treated hyphae under SEM. Further, the antifungal activity of the protein was compared with standard lectins like concanavalin A, phytohemagglutinin and wheat germ agglutinin.     

拮抗细菌Bs-03菌株鉴定及抑菌活性测定

在土壤有益微生物的分离、筛选中获得一株拮抗细菌Bs-03,抑菌谱测定结果表明该菌对供试的多种植物病原真菌具有较强的抑菌活性,表现较为广谱的抑菌活性。通过对菌株的培养性状、形态特征、生理生化特性等试验项目,初步鉴定为枯草芽孢杆菌(Bacillus subtilis)。采用平板对峙培养法测定Bs-03菌株抑菌活性,结果表明Bs-03对供试的14种植物病原真菌,除花生白绢病菌外,都具有抑菌活性。其中对核桃炭疽病菌、苹果轮纹病菌、黄瓜灰霉病菌、冬枣黑斑病菌效果最好,与其他处理在P0.05水平上具有显著性差异。该菌具有较好的开发应用前景。     

Glucanohydrolase enzyme activity in embryos of Scots,Corsican and lodgepole pines infected in vitro with Heterobasidion annosum

Embryos of Scots pine (Pinus sylvestris), Corsican pine (P. nigra var. maritima) and Lodgepole pine (P. contorta) were infected in vitro with Heterobasidion annosum and the activities of 1,3-β-glucanase, chitinase and β-glucosidase were monitored. Both the infected and control embryos of the three pine species showed the presence of the enzymes. Statistical analysis showed no significant increase (P > 0.05) in the levels of 1,3-β-glucanase in infected embryos for all the three pine species. However, there was a significant increase in levels of chitinase in infected embryos of P. contorta and P. nigra var. maritima and also in levels of β-glucosidase in infected embryos of all three of the pine species. The study has therefore revealed that constitutive levels of glucanohydrolase enzymes exist in the embryos of the three pine species and that the induction of chitinase and β-glucosidase was a factor in the response of the embryos to stimulation by H. annosum infection.     

Dimensional stability and strength properties of particleboard produced by a closed-press system

The purpose of this study was to reveal the effects of various levels of mat-moisture content (m.m.c.) and the closed-press system for making single- or three-layer particleboard on the density profile, thickness swelling, specific moduli of elasticity (MOE) and rupture (MOR) and internal bond strength. Internal gas pressure was measured in an enclosed frame; and the larger the m.m.c., the higher the internal gas pressure became. When rising water vapor (steam) struck particles, it plasticized them and cured the adhesive, resulting in improved interparticle contact. The vertical density gradient in the three-layer board was larger than that in the single-layer board. As for thickness swelling by cold-water soaking, the single-layer boards were less affected than the three-layer boards and showed good dimensional stability with increased m.m.c. The open-system boards swelled more than the closed-system boards. The closed-system single-layer board made at high m.m.c. returned nearly to the prime thickness by air-drying after cold-water soaking. Specific MOE and MOR were larger at 15% or 10% m.m.c. than those at other m.m.c. Considerable reductions of specific MOR and MOE of the closed-system three-layer board were observed at 20% or 25% m.m.c.Part of this report was presented at the 45th annual meeting of the Japan Wood Research Society, Tokyo, April 1995 and at the 48th annual meeting of the Japan Wood Research Society, Shizuoka, April 1998