【作者】 汪章勋；

【导师】 黄勃； 樊美珍；

【作者基本信息】 安徽农业大学 ， 微生物学， 2005， 硕士

【摘要】 本文较系统地研究了粉拟青霉(Paecilomyces farinosus)不同菌株的生物学特性和类枯草杆菌蛋白酶酶活;筛选出具优良生物学性状的类枯草杆菌蛋白酶高产菌株;确定了高产菌株最佳发酵培养条件;并对其产 Pr1 蛋白酶进行了进一步分离、纯化;同时也克隆出该菌株的类枯草杆菌蛋白酶基因;从而为改造昆虫病原真菌奠定了基础。 通过预备实验和参考各种文献资料,以下述培养条件诱导 Pr1 蛋白酶的产生:先将粉拟青霉菌株接种在 SDY 培养基上,25℃,170rpm 振荡培养 4d。菌丝生长接近最大量时,离心,将洗净的菌丝体(沉淀)接种到添加了几丁质的诱导培养基中,初始pH 值为 8.0,22℃、170rpm 振荡培养 12h。通过对来自不同地域和寄主的 24 株粉拟青霉进行了类枯草杆菌蛋白酶(Pr1)活力的测定,结果表明 Pf102 与 Pf96 菌株相比其他研究的菌株其酶活力高,A405值分别为 0.2028 和 0.1749。 选取产 Pr1 蛋白酶活力相对较高的 15 株粉拟青霉菌株为实验材料,比较了它们的生物学特性,分析了它们在产孢量、生长速度、抗旱能力、抗紫外能力等方面的差异。结果表明,粉拟青霉菌落生长和产生孢子的最适温度为 20℃。在此温度下,24h孢子萌发率在 80%~100%之间;此外不同菌株的抗旱力差异较大,Pf01 抗旱能力最强,而 Pf127 的抗旱能力最弱。经紫外线照射 5min,菌株的萌发率在 44%~81%范围内。Pf96 的生长速度,产孢量,萌发率和抗紫外线能力均为最高或次高。 通过对供试菌株产蛋白酶及生物学特性方面的研究比较,最终确定了 Pf96 菌株作为筛选最佳发酵培养条件,进一步分离、纯化 Pr1 蛋白酶以及克隆其类枯草杆菌蛋白酶基因的研究对象。 不同接种量对 Pf96 菌株产 Pr1 蛋白酶活力影响的试验结果表明:接种量的大小对 Pf96 菌株产 Pr1 蛋白酶的相对活力影响不大,但相对来讲 10%的接种量对产 Pr1蛋白酶更为有利。根据对 Pf96 菌株 Pr1 蛋白酶产酶曲线的研究得知:虽然在诱导后12h 有一个产酶高峰,但在 60h 时又出现另一产酶高峰,根据各种参考文献综合报道,一般虫生真菌在加入诱导物 12h 后,便达到了产酶高峰。在以后的发酵条件研究过程中,采用诱导 12h 后的发酵液作为原始的粗酶液,测定其酶活。 在相同培养条件下,以酶的相对活力为指标,通过单因素诱导物、温度、pH 值、菌龄,培养基不同组分配比的正交试验,结果表明:粉拟青霉菌株 Pf96 的最适培养条件为:添加 1%蝉蜕的基本盐培养基、培养温度 25℃、pH8.0 和接种菌龄 6d。 在蛋白酶纯化过程中,首先向粗酶液中添加硫酸铵粉末分别至不同饱和度,试验结果表明:在 80%硫酸铵饱和度时,虽然回收率达到 41.9%(不是最高),但其纯化倍数最高达 2.6;因而选择 80%的硫酸铵饱和度为试验标准。更多还原

【Abstract】 In the present thesis, the biological characters of 15 strains Paecilomyces farinosuswere studied, and 24 isolates Paecilomyces farinosus were screened basing on the activityof sublitisin-like protease. The strain Pf96 was selected as a research object basing on theabove study. The optimal cultivation method of strain Pf96 was confirmed, and the Pr1protease from this isolates also was partly purified. The complete cDNA gene ofsublitisin-like protease was cloned from strain Pf96. The enzyme activities from 24 isolates of Paecilomyces farinosus with differentgeographic origins and hosts were detected. The results showed that the enzyme activity ofPf102 was the highest in these studied isolates, and Pf96 had higher enzyme activity too. Fifteen isolates of Paecilomyces farinosus differed from one another in conidiation,growth rate, drought resistance, UV-protective ability and germination rate. The resultsshowed that the optimal temperature was 20℃ for conidiation and growth, and thegermination rate of spores was in the range of 80%~100% in 24 hours. In addition thedrought resistance of Pf01 was the strongest among 15 isolates, while Pf127 was theweakest. The range of germination rates from 15 isolates was 44%~81% by UV irradiationfor 5 minutes. The strain Pf96 has the most excellent characters in conidiation, growth,germination rate and UV irradiation resistance among these studied isolates. Ultimately, the strain Pf96 was selected as a research object for studying the optimalcultivation method, purifying sublitisin-like protease, and cloning the gene ofsublitisin-like protease basing on comparing the biological characters and producing Pr1protease of different strains. In the same cultivation conditions, the relative activities of enzymes were used fortargets. Ultimately, the optimal cultivation method of Pf96 was: chitin 1%, temperature25℃, pH8.0, age 6d. The cuticle-degrading proteases from entomopathogenic fungus, Paecilomycesfarinosus, were induced by adding the cuticle of cicada into liquid media. After ammoniumsulfate precipitation with 80% saturation, dialysis, an ion-exchange chromatography withDEAE-cellulose, and gel filtration with Sephadex G-100, a protease was purified partly.Thepartly purified enzyme had a specific sublitisin-like protease activity which was 18.32times that of the culture supernatant with 4.42% primary yield, and showed two bands onPAGE. In order to clone a complete cDNA of sublitisin-like protease from Paecilomycesfarinosus, a pair of primers was designed according to the high conserved nucleotidesequences in GenBank, and RT-PCR, 3’/5’-RACE PCR were used. The cloned completecDNA with 2060bp length was obtained, and the GenBank accession number is DQ022189.Alignments with the deduced amino acid of mature proteins in four species of fungi show69%,71%,68% and 68%,identity with Metarhizium anisopliae var. anisopliae(CAB63913),Gibberella zeae(EAA68914), M.anisopliae var.acridum(CAC95047)and Podosporaanserina(AAC03564), respectively. Meanwhile, the active site residues for serine proteasescan be readily identified in the mature protease.更多还原