【作者】 张中；

【导师】 黄勃； 樊美珍；

【作者基本信息】 安徽农业大学 ， 微生物学， 2006， 硕士

【摘要】 本研究测定了28株粉棒束孢(Isaria farinosa)菌株的几丁质酶的活性,从中筛选出高产几丁质酶菌株RCEF0622,优化了该菌株的产酶条件,纯化了几丁质酶并测定了其纯酶的性质。还对高产蛋白酶的粉棒束孢菌株RCEF0685进行了几丁质酶基因的克隆研究。在实验中首次采用了先将菌种接种于铺有玻璃纸的完全培养基上培养,待其生长至对数生长前期时,再转移至以胶体几丁质为唯一碳源的贫瘠培养基中继续诱导培养。研究了pH值、装液量、接种量、温度等单因子对RCEF0622菌株的产几丁质酶影响。在此基础上,通过正交试验,对该菌株的产酶条件进行了综合研究,结果表明最佳产酶条件为胶体几丁质浓度为2%,培养基初始pH值6.0,接种量6%,装液量15ml(100ml烧瓶),温度19℃,培养时间36h。RCEF0622菌株摇瓶培养工艺优化结果表明:优化后的工艺与原有工艺相比,几丁质酶活的产量提高了55.61%,而蛋白质的产量也增加了55.34%,其酶比活并没有提高,基本上是维持在同一水平上。通过检测,初步推断该酶属于几丁质内切酶。在酶纯化过程中,通过两次硫酸铵沉淀试验,以上清液酶比活最小,沉淀比活力最大和酶得率较高为依据,确定出80%为最佳硫酸铵饱和度浓度。确定在pH8.0时,DEAE-Sepharose Fast Flow对几丁质酶的吸附性最好,由此选择了pH8.0的缓冲液作为纯化过程中的最佳pH值。粉棒束孢RCEF0622菌株发酵液经(NH4)2SO4分级沉淀、DEAE-Sepharose F F柱层析和Sephadex G-100柱层析后,纯化得到单一的几丁质酶。经聚丙酰胺凝胶电泳检测,为单一带。通过以上步骤纯化后,比活由粗酶液154.2提高至1950,提纯12.65倍,回收率为5.9%。用SDS-PAGE测得纯化后的几丁质酶分子量是38.5kD。该几丁质酶对胶体几丁质的Km为0.73mg/ml;酶的最适反应温度是40℃;最适反应pH值为4.6;在温度30℃以下,pH4-8之间较为稳定。硼酸对几丁质酶有一定的促进作用;Mg2+,Mn2+,Al3+,K+,Ca2+,Ba2+等金属离子对酶活力有明显的激活作用,尤其是Mn2+离子对几丁质酶的激活作用非常明显;而Zn2+, Cu2+离子等对该酶有明显的抑制作用;Cu2+离子对该酶活有较大程度的抑制作用。本研究通过设计基因保守区的特异性简并引物,运用SMART RACE RT-PCR技术,首次从粉棒束孢中克隆出完整的几丁质酶基因。该基因cDNA全长1549bp, 5’端非翻译区89bp,3’端非翻译区有188bp,开放阅读框(ORF)1272bp,编码423个氨基酸。信号肽长度为22个氨基酸。信号肽很可能需要两次剪切。成熟的蛋白理论分子量为43.9kD,理论等电点为5.67。氨基酸序列具有几丁质酶18族的两个高度保守的活性区域,一个是酶作用活性位点,另一个是几丁质结合区域。该蛋白可归于几丁质酶18族V类。成熟蛋白的氨基酸序列与裂虫壳AAV98691、白色扁丝霉CAA45468、菌生轮枝孢AAP45631、莱氏野村菌AAP04616和球孢白僵菌AAN41261的同源性分别为91%,89%,80%,76%和75%。更多还原

【Abstract】 In the present thesis, 28 strains of Isaria farinosa were studied for their chitinases activities, and strain RCEF0622 of Isaria farinosa with the highest chitinase activity was chosen to study the optimum of fermentation conditions for chitinase, chitinase purification and enzymological character. The cDNA gene of chitinase from isolate RCEF0685 was also clonied.Chitinases were induced when Isaria farinosa grew on a complete medium covered by cellophane to pre-logarithmic phase at first, and then was transferred to the medium containing colloidal chitin as a sole carbon and nitrogen source.The effects of pH, liquid medium capacity, inocula amount and temperature were studied for its optimal fermentation conditions by single factor level. Basing on the above study, its optimal fermentation conditions were further studied by orthogonal experiments.The results showed that the optimal culture conditions were the following: the colloid chitin 2%, initial pH of culture 6.0, inoculating amount 6%, 100 ml flask containing 15 ml cultural liquid, temperature 19℃, and cultural time 36h. The optimal fermentation process of strain RCEF0622 showed the enzyme activity of chitinase increased to a great extent by 55.61%, and the productive of protein also increased 55.34%. In fact, its specific activity did not increase, and was kept in a same level. The chitinase was determined as a kind of endochitinase with special test method.With the process of enzyme purification, the optimal concentration was 80% saturation determined by two ammonium sulfate precipitation experiments, the lowest specific activity of cultural supernatant, the most specific activity of precipitation and the most yields. The pH 8.0 was determined by the optimal adsorption for DEAE-Sepharose Fast Flow.An extracellular chitinase was purified by ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Sephadex G-100 chromatography. The purified enzyme had a specific activity of chitinase which was 12.65 times that of the culture supernatant with 5.9% primary yield, and showed one band on PAGE. Its molecular weight was estimated to be about 38.5kD by SDS-PAGE. When colloidal chitin was substrate, the michaelis constant (Km) of chitinase from strain RCEF0622 was 0.73mg/ml. The optimal reaction temperature was 40℃, and the optimal pH for reaction was 4.6. The enzyme activity was stable at a pH between 4 and 8 and under temperature 30℃. Boracic acid can also promote for enzyme activity more or less. The enzyme activity was enhanced by Mg2+, Mn2+, Al3+, K+, Ca2+and Ba2+, and Mn2+was obviously for the enzyme activity elevation, especially. However, enzyme activity was strongly inhibited by Zn2+ and Cu2+.This thesis also reports the complete full-length cDNA coding the chitinases produced by the biocontrol agent Isaria farinosa using SMART RACE RT-PCR. Analysis of the cloned complete cDNA, with a whole sequence of 1549bp, showed that it encompassed an open reading frame (ORF) with 1272bp encoding 423 amino acids with a stretch of 22 amino acid residues displaying characteristics of signal peptide. Tool revealed that the mature chitinase (without signal sequence) has a molecular mass of 43.9 kDa with a calculated pI of 5.67. This sequence contained two highly conserved regions of the active domain of the family 18 glycosyl hydrolases including a presumed enzymatic active site and a potential chitin-binding domain. The cloned Isaria chinitase belongs to the class V in 18 family of glycosyl hydrolase. Alignments with the deduced amino acid of mature proteins in 5 species of fungi showed 91%, 89%, 80%, 76% and 75%, respectively, identical with those of Torrubiella confragosa(AAV98691),Aphanocladium album(CAA45468);Verticillium fungicola(AAP45631),Nomuraea rileyi(AAP04616)and Beauveria bassiana(AAN41261), respectively.更多还原