巨大芽孢杆菌L-天冬酰胺酶基因的克隆表达及定向进化

发布时间：2018-05-17 09:52

本文选题：L-天冬酰胺酶 + Bacillus　；参考：《南京农业大学》2016年硕士论文

【摘要】：L-天冬酰胺酶(L-Asparaginase,EC3.5.1.1)能专一催化水解L-天冬酰胺脱氨基生成L-天冬氨酸和氨。L-天冬酰胺酶不仅广泛应用于急性淋巴细胞白血病(ALL)、恶性淋巴瘤(ML)、霍奇金淋巴瘤(HL)等疾病的治疗,而且用于控制高温油炸、烘焙食品中丙烯酰胺的形成而不影响产品的外观、味道。目前L-天冬酰胺酶普遍存在稳定性差、酶活低、pH适用范围窄等问题,限制了其在食品加工中的应用。因此,寻找和发现新型的L-天冬酰胺酶具有深远理论意义和潜在的应用价值。本文针对以上问题,从土壤中筛选分离到一株高效产L-天冬酰胺酶菌株H-1,对其进行了形态特征、生理生化特征和分子生物学鉴定,并对其编码L-天冬酰胺酶基因ansA、ansZ进行了克隆和异源表达,以及对表达产物提纯和重组酶的酶学性质作了初步探讨。同时,对L-天冬酰胺酶的定向进化及其在油炸薯条中的应用进行了研究。主要研究结果分述如下。1.L-天冬酰胺酶产生菌的筛选与鉴定。采用酚红/BTB初筛平板筛选到100余株L-天冬酰胺酶产生菌,并通过摇瓶复筛,从江苏省某天冬酰胺生产企业排放污水口土壤中筛选一株具有较高酶活的菌株H-1,酶活达0.086±0.0023 IU/mL。对菌株H-1进行菌落形态观察、生理生化鉴定及分子生物学鉴定,确定菌株H-1为巨大芽孢杆菌(Bacillus megaterium)。2.B.megaterium L-天冬酰胺酶的克隆与表达。根据Genbank中B.megaterium WSH-002的全基因组序列成功克隆出B.megateriumH-1 L-天冬酰胺酶基因ansA(1050 bp)、ansZ(1113 bp)序列,分别编码349与370个氨基酸蛋白。酶基因分别与表达载体pET-30a,pET-32a连接,成功构建了四个重组表达载体,实现L-天冬酰胺酶基因ansA、ansZ在Escherichia coli中异源表达,其中催化活力最高的pET-30a-BM-ansZ重组菌,经表达条件优化后酶活达4.26±0.22IU/mL,较野生菌的酶活提高约50倍,将该重组酶命名为BmAase。3.BmAase的分离纯化及其酶学性质研究。表达产物通过Ni亲和柱进行一步纯化,BmAase比活达到146.48 IU/mg(谷氨酰胺活性可忽略不计);SDS-PAGE分析与MALDI-TOF-MS检测出其分子量为39.628 kDa。BmAase的最适催化反应条件为40。℃,pH7.0;BmAase具有一个相对较宽的pH活性范围pH5.0-8.0,热稳定性好,60℃/70℃处理12 h仍分别保留70%/55%以上相对活力;BmAase动力学参数米氏常数Km为0.8 mM,最大反应速度Vmax为1.58IU/μg。4.B.megaterium L-天冬酰胺酶的定向进化。摸索易错PCR反应体系,按Mg2+、Mn2+浓度分别为2mM、0.06mM进行易错PCR反应。利用建立的基于96微孔板高通量筛选模型,经过2轮易错PCR,从8000多个突变株中筛选出酶活最高的突变株2MH6,其比活力为188.69 IU/mg,较BmAase提高约30%。以易错PCR筛选得到的突变体作为亲本进行DNA Shuffling,从5000多个突变株中筛选到突变株S-CA3,其比活力达到249.01 IU/mg,较BmAase提高70%,且该酶在pH5.0-9.0保持活力稳定,拓宽了碱性pH反应范围。5.L-天冬酰胺酶在油炸薯条中的应用。油炸前用L-天冬酰胺酶处理土豆条,通过HPLC-MS检测油炸薯条中丙烯酰胺含量发现,经L-天冬酰胺酶处理后油炸薯条中丙烯酰胺含量降低至7.6%(0.056 ± 0.0056 mg/kg),而未处理油炸薯条中丙烯酰胺含量高达0.738 ± 0.0163 mg/kg,说明B.megateriumL-天冬酰胺酶能有效控制薯条加工过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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;Q55;TS215

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