本文選題：L-天冬酰胺酶 + Bacillus　； 參考：《南京農業(yè)大學》2016年碩士論文

【摘要】：L-天冬酰胺酶(L-Asparaginase,EC3.5.1.1)能專一催化水解L-天冬酰胺脫氨基生成L-天冬氨酸和氨。L-天冬酰胺酶不僅廣泛應用于急性淋巴細胞白血病(ALL)、惡性淋巴瘤(ML)、霍奇金淋巴瘤(HL)等疾病的治療,而且用于控制高溫油炸、烘焙食品中丙烯酰胺的形成而不影響產(chǎn)品的外觀、味道。目前L-天冬酰胺酶普遍存在穩(wěn)定性差、酶活低、pH適用范圍窄等問題,限制了其在食品加工中的應用。因此,尋找和發(fā)現(xiàn)新型的L-天冬酰胺酶具有深遠理論意義和潛在的應用價值。本文針對以上問題,從土壤中篩選分離到一株高效產(chǎn)L-天冬酰胺酶菌株H-1,對其進行了形態(tài)特征、生理生化特征和分子生物學鑒定,并對其編碼L-天冬酰胺酶基因ansA、ansZ進行了克隆和異源表達,以及對表達產(chǎn)物提純和重組酶的酶學性質作了初步探討。同時,對L-天冬酰胺酶的定向進化及其在油炸薯條中的應用進行了研究。主要研究結果分述如下。1.L-天冬酰胺酶產(chǎn)生菌的篩選與鑒定。采用酚紅/BTB初篩平板篩選到100余株L-天冬酰胺酶產(chǎn)生菌,并通過搖瓶復篩,從江蘇省某天冬酰胺生產(chǎn)企業(yè)排放污水口土壤中篩選一株具有較高酶活的菌株H-1,酶活達0.086±0.0023 IU/mL。對菌株H-1進行菌落形態(tài)觀察、生理生化鑒定及分子生物學鑒定,確定菌株H-1為巨大芽孢桿菌(Bacillus megaterium)。2.B.megaterium L-天冬酰胺酶的克隆與表達。根據(jù)Genbank中B.megaterium WSH-002的全基因組序列成功克隆出B.megateriumH-1 L-天冬酰胺酶基因ansA(1050 bp)、ansZ(1113 bp)序列,分別編碼349與370個氨基酸蛋白。酶基因分別與表達載體pET-30a,pET-32a連接,成功構建了四個重組表達載體,實現(xiàn)L-天冬酰胺酶基因ansA、ansZ在Escherichia coli中異源表達,其中催化活力最高的pET-30a-BM-ansZ重組菌,經(jīng)表達條件優(yōu)化后酶活達4.26±0.22IU/mL,較野生菌的酶活提高約50倍,將該重組酶命名為BmAase。3.BmAase的分離純化及其酶學性質研究。表達產(chǎn)物通過Ni親和柱進行一步純化,BmAase比活達到146.48 IU/mg(谷氨酰胺活性可忽略不計);SDS-PAGE分析與MALDI-TOF-MS檢測出其分子量為39.628 kDa。BmAase的最適催化反應條件為40�！�,pH7.0;BmAase具有一個相對較寬的pH活性范圍pH5.0-8.0,熱穩(wěn)定性好,60℃/70℃處理12 h仍分別保留70%/55%以上相對活力;BmAase動力學參數(shù)米氏常數(shù)Km為0.8 mM,最大反應速度Vmax為1.58IU/μg。4.B.megaterium L-天冬酰胺酶的定向進化。摸索易錯PCR反應體系,按Mg2+、Mn2+濃度分別為2mM、0.06mM進行易錯PCR反應。利用建立的基于96微孔板高通量篩選模型,經(jīng)過2輪易錯PCR,從8000多個突變株中篩選出酶活最高的突變株2MH6,其比活力為188.69 IU/mg,較BmAase提高約30%。以易錯PCR篩選得到的突變體作為親本進行DNA Shuffling,從5000多個突變株中篩選到突變株S-CA3,其比活力達到249.01 IU/mg,較BmAase提高70%,且該酶在pH5.0-9.0保持活力穩(wěn)定,拓寬了堿性pH反應范圍。5.L-天冬酰胺酶在油炸薯條中的應用。油炸前用L-天冬酰胺酶處理土豆條,通過HPLC-MS檢測油炸薯條中丙烯酰胺含量發(fā)現(xiàn),經(jīng)L-天冬酰胺酶處理后油炸薯條中丙烯酰胺含量降低至7.6%(0.056 ± 0.0056 mg/kg),而未處理油炸薯條中丙烯酰胺含量高達0.738 ± 0.0163 mg/kg,說明B.megateriumL-天冬酰胺酶能有效控制薯條加工過程中丙烯酰胺的形成。
[Abstract]:L- asparaginase (L-Asparaginase, EC3.5.1.1) can catalyze the hydrolysis of L- asparagine deamination to L- aspartic acid and ammonia.L- asparaginase not only widely used in the treatment of acute lymphoblastic leukemia (ALL), malignant lymphoma (ML), Hodgkin's lymphoma (HL) and other diseases, but also for controlling high temperature frying and propene in baked food. The formation of amides does not affect the appearance and taste of the products. At present, L- asparaginase has many problems, such as poor stability, low enzyme activity and narrow application of pH, which restrict its application in food processing. Therefore, the search and discovery of new L- asparaginase have far-reaching theoretical meaning and potential application value. This article is aimed at the above problems, A highly efficient L- asparaginase strain H-1 was screened and isolated from the soil, and its morphological characteristics, physiological and biochemical characteristics and molecular biological identification were carried out, and the L- asparaginase gene ansA, ansZ were cloned and heterologous expression, and the enzymatic properties of the purified and recombinant enzyme were preliminarily discussed. The directional evolution of L- asparaginase and its application in fried fries were studied. The main research results were as follows: screening and identification of.1.L- asparaginase producing bacteria. 100 strains of L- asparaginase producing bacteria were screened by phenol red /BTB screen plate, and the production enterprise of asparagine in Jiangsu province was produced by shaking flask rescreening. A strain H-1 with high enzyme activity was screened in the soil of the polluted water, and the enzyme activity reached 0.086 + 0.0023 IU/mL. to observe the colony morphology of strain H-1, physiological and biochemical identification and molecular biological identification. The strain H-1 was identified as the cloning and expression of the.2.B.megaterium L- asparaginase of Bacillus megigantobacilli (Bacillus megaterium). According to B in Genbank B. The whole genome sequence of.Megaterium WSH-002 successfully cloned the B.megateriumH-1 L- asparaginase gene ansA (1050 BP), ansZ (1113 BP) sequence, encoding 349 and 370 amino acid proteins respectively. The enzyme gene was connected with the expression vector pET-30a and pET-32a respectively, and four recombinant expression vectors were successfully constructed to realize L- asparaginase gene ansA. It was expressed in Escherichia coli, in which the recombinant strain of pET-30a-BM-ansZ, which has the highest catalytic activity, was optimized by 4.26 + 0.22IU/mL after the expression conditions, which was about 50 times higher than that of the wild bacteria. The recombinant enzyme was named BmAase.3.BmAase purification and the characterization of its enzymology. The expression product was purified by a Ni affinity column. The BmAase activity reached 146.48 IU/mg (the activity of glutamine was negligible); SDS-PAGE analysis and MALDI-TOF-MS showed that the optimum catalytic reaction conditions for its molecular weight of 39.628 kDa.BmAase were 40. C, pH7.0, BmAase had a relatively wide pH activity range pH5.0-8.0, good thermal stability, 60 C /70 C treatment 12 h still retained 70%/55%, respectively. The BmAase kinetic parameter is 0.8 mM and the maximum reaction rate is Km, and the maximum reaction speed Vmax is the directional evolution of 1.58IU/ micron g.4.B.megaterium L- asparaginase. The error PCR reaction system is groped, the concentration of Mn2+ is 2mM, the 0.06mM carries out the error prone reaction. Using the established high throughput screening model based on the 96 microporous plate, through 2 round easy PCR, the mutant strain 2MH6 was screened out of the highest enzyme activity from more than 8000 mutant strains, its specific activity was 188.69 IU/mg, and BmAase improved about 30%. with the error PCR screening mutant as parent to carry out DNA Shuffling, and screened the mutant S-CA3 from more than 5000 mutant strains, and the specific activity reached 249.01 IU/mg, and was 70% higher than that of BmAase, and the enzyme was 70%. PH5.0-9.0 maintained stable vitality, widened the application of.5.L- asparaginase in the alkaline pH reaction range in fried potato chips. Before frying, L- asparaginase was used to treat potato strips and the content of acrylamide in fried fries was detected by HPLC-MS. The content of acrylamide in fried potato strips after L- asparaginase treatment was reduced to 7.6% (0.056 + 0.005). 6 mg/kg), while the content of acrylamide in the untreated fries was 0.738 + 0.0163 mg/kg, indicating that the B.megateriumL- asparaginase could effectively control the formation of acrylamide during the processing of French fries.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78;Q55;TS215

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