利用激光微束穿刺法获得抗黄萎病转基因棉花的研究

通过激光微束穿刺法 ,将 β 1,3 葡聚糖酶及几丁质酶基因双价植物表达载体 pBLGC导入棉花幼胚。转化一代棉花幼苗用蘸根法进行抗黄萎病筛选 ,将存活的苗移入病圃进行了卡那霉素(Kan 1% )抗性测定。结果表明 ,在移栽的 2 9株幼苗中 ,有 9株表现出明显的Kan抗性 ,对 9株抗Kan植株进行了PCR检测 ,结果显示 ,其中 7株表现为阳性。病圃中的T1代转基因植株在经历了黄萎病发病高峰期后 ,7株PCR阳性植株表现明显抗病 ,并已正常开花结铃 ,其他T1代植株及对照植株全部因后期发病死亡。这初步证明外源基因已整合到棉花基因组中 ,且使转基因植株对黄萎病表现出一定的抗性。     

腐乳发酵中毛霉菌丝的自溶及其影响因素研究

对腐乳发酵过程中甲壳素酶、脱乙酰甲壳质酶、壳多糖酶和N.乙酰氨基葡萄糖酶进行研究,旨在探索腐乳毛霉后发酵菌丝自溶机理.在保持腐乳发酵所需各种酶活力的同时,达到后期腐乳成熟及毛霉菌丝充分自溶,以提高腐乳品质.结果表明:甲壳素酶是影响毛霉菌丝自溶的关键酶;温度是影响菌丝自溶的主要凼素;香辛料(砂仁)对几丁质酶活力影响较大.通过正交试验确定:控制适宜的香辛料比例,温度30℃,初始pH6.5,初始酒精度10°,初始NaCl浓度11%并添加0.2%几丁质进行腐乳发酵.70d完全成熟,腐乳毛霉菌丝长度比对照低4.1倍.     

Chitinibacter sp. GC72的筛选鉴定及其几丁质降解产物研究

为向几丁质酶资源的开发利用提供研究基础,作者筛选获得了一株具有高产几丁质酶活性的细菌。经形态、生理生化及分子生物学研究手段鉴定表明该菌属奈瑟菌科,Chitinibacter属,命名为Chitinibacter sp.GC72。经1L发酵体系检测,其粗酶液酶活可达2835U/g。利用薄层层析法、液相色谱法和质谱法对其酶解几丁质产物进行了分析鉴定,结果表明产物主要为N-乙酰-D-氨基葡萄糖。最后利用该酶进行了多次酶解实验,证实投入3.44U酶量经27h转化可降解200mg几丁质至159mg N-乙酰-D-氨基葡萄糖,收率为79.6%。     

Chitinolyticbacter meiyuanensis SYBC-H1几丁质酶的性质及在虾皮水解中的作用

以一株产几丁质酶细菌Chitinolyticbacter meiyuanensisSYBC-H1为材料,对几丁质酶进行了分离纯化及酶学性质研究。通过硫酸铵沉淀、DEAE-cellulose阴离子交换介质和SephadexG-100分子筛层析柱处理其所产几丁质酶,获取纯度为95%以上的几丁质酶。酶学性质表明,该酶的最适温度为40℃,最适pH值为6.5,在50℃以下较为稳定,在pH值为5.0时最稳定,β-巯基乙醇有利于防止半胱氨酸氧化而增加几丁质酶稳定性,EDTA可增加该酶稳定性,Na+,K+对几丁质酶有明显的激活作用,Zn2+,Mn2+,Fe2+,Fe3+对几丁质酶有较大的抑制作用。SYBC-H1几丁质酶可把虾皮水解为N-乙酰氨基葡萄糖,水解率为21.8%。     

几丁质酶生产菌的诱变育种及摇瓶发酵条件优化

从本实验室分离筛选的产几丁质酶菌株TS30出发,经紫外线(UV)+LiCl复合诱变处理,获得一株遗传性稳定的高活性几丁质酶变异株TUC13。采用正交设计试验法对TUC13发酵培养基和发酵条件进行了优化,采用经实验改进后培养基和培养条件,变异株TUC13单位发酵液几丁质酶活性提高了236.5%。     

The Chitinolytic Activities of Streptomyces sp. TH-11

Chitin is an abundant biopolymer composed of units of N-acetyl-D-glucosamine linked by β-1,4 glycosidic bonds. Chitin is the main component of the shells of mollusks, the cell wall of fungi and yeast and of the exoskeleton of crustaceans and insects. The degradation of chitin is catalyzed by chitinases that occur in a wide range of organisms. Among them, the chitinases from microorganisms are extremely important for the degradation and recycling of the carbon and nitrogen trapped in the large amount of insoluble chitin in nature. Streptomyces sp. TH-11 was isolated from the sediment of the Tou-Chien River, Taiwan. The chitinolytic enzyme activities were detected using a rapid in-gel detection method from the cell-free preparation of the culture medium of TH-11. The chitinolytic enzyme activity during prolonged liquid culturing was also analyzed by direct measurement of the chitin consumption. Decomposition of the exoskeleton of shrimps was demonstrated using electron microscopy and atomic force microscopy.     

Insertion of a Loop Structure into the “Loopless” GH19 Chitinase from Bryum coronatum

Chitinases belonging to the GH19 family have diverse loop structure arrangements. A GH19 chitinase from rye seeds (RSC-c) has a full set of (six) loop structures that form an extended binding cleft from -4 to +4 (“loopful”), while that from moss (BcChi-A) lacks several loops and forms a shortened binding cleft from -2 to +2 (“loopless”). We herein inserted a loop involved in sugar residue binding at subsites +3 and +4 of RSC-c (Loop-II) into BcChi-A (BcChi-A+L-II), and the thermal stability and enzymatic activity of BcChi-A+L-II were then characterized and compared with those of BcChi-A. The transition temperature of thermal unfolding decreased from 77.2 ˚C (BcChi-A) to 63.3 ˚C (BcChi-A+L-II) by insertion of Loop-II. Enzymatic activities toward the chitin tetramer (GlcNAc)₄ and the polymeric substrate glycol chitin were also suppressed by the Loop-II insertion to 12 and 9 %, respectively. The Loop-II inserted into BcChi-A was found to be markedly flexible and disadvantageous for protein stability and enzymatic activity.     

Preparation of antibacterial chito‐oligosaccharide by altering the degree of deacetylation of β‐chitosan in a Trichoderma harzianum chitinase‐hydrolysing process

BACKGROUND: Chito‐oligosaccharide (COS) is generally known to possess many specific biological functions, especially antibacterial activity, depending on its size. To prepare a specific size range of COS, however, has proved difficult. The aim of this study was to establish a method for preparing a specific size range of antibacterially active COS by adjusting the degree of deacetylation (DD) of β‐chitosan in a Trichoderma harzianum chitinase‐hydrolysing process. RESULTS: The molecular weight spectrum, elucidated by viscosity‐average molecular weight, high‐performance liquid chromatography and thin layer chromatography, of COS in chitosan hydrolysate was significantly related to the DD of its original chitosan. Compared with the original form, COS produced at 90% DD showed superior activity against most Gram‐negative bacteria tested, with a minimum inhibition concentration (MIC) ranging from 55 ± 27 to 200 ± 122 µ g mL^?1. Conversely, most Gram‐positive strains tested were less sensitive to COS (MIC > 880 ± 438 µ g mL^?1) than to its original form. Among the Gram‐positive strains, Staphylococcus xylosus was the only exception in that it showed a high susceptibility to COS and had an MIC as low as 45 ± 11 µ g mL^?1. CONCLUSION: The results indicate that the production of a specific size range of COS product is possible by altering the DD of chitosan in the chitinase‐catalysed process. To produce various sizes of COS for versatile biological functions, as seen in this study to inhibit various types of bacteria, is made possible in this established process. Copyright © 2008 Society of Chemical Industry     

单胞菌产几丁质酶的酶学性质研究

对从海水中分离得到的嗜水气单胞菌所产的几丁质酶的酶学性质进行研究.其最适pH为7.0左右,最适反应温度为45 ℃左右.在40 ℃以下有良好的热稳定性,12 h仍有50%残余酶活.Mg2 对酶有激活作用,Cu2 和Al3 对酶有明显的抑制作用.该酶对底物的亲和力很大,水解几丁质的Km值为29.15 g/L.