1. 毕业设计（论文）的内容和要求

（1）掌握文献查阅的一般方法，学会在中国期刊网、web of science科学引文索引、springer link电子期刊、elsevier sdos电子期刊等检索资源上查阅关于尿酸氧化酶、黄嘌呤脱氢酶及枯草芽孢杆菌中基因表达等的相关文献，并对枯草芽孢杆菌基因操作有全面了解。

（2）文献阅读及综述：阅读与课题相关的中英文文献，了解国内外的研究动态，撰写文献综述。

（3）明确实验任务，拟定实验方案：根据所查阅文献的内容，确定研究内容及方案，拟定科学可行的研究计划。

2. 参考文献

[1] Li W, Xu S, Zhang B, et al. Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis[J]. PloS one, 2017, 12(5): e0177877. [2] Ma P, Patching S G, Ivanova E, et al. Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity[J]. Microbiology, 2016, 162(5): 823-836. [3] Khade S, Srivastava S K. Effect of surfactants and inducers on increased uricase production under submerged fermentations by Bacillus cereus[J]. Preparative Biochemistry and Biotechnology, 2017, 47(1): 81-85. [4] Kotb E. Improvement of uricase production from Bacillus subtilis RNZ-79 by solid state fermentation of shrimp shell wastes[J]. Biologia, 2016, 71(3): 229-238. [5] Feng T, Yang X, Wang D, et al. A practical system for high-throughput screening of mutants of bacillus fastidiosus uricase[J]. Applied biochemistry and biotechnology, 2017, 181(2): 667-681. [6] Chen M H, Chen S J, Hsu H Y, et al. Method of reducing levels of uric acid: U.S. Patent 9,441,210[P]. 2016-9-13. [7] Chityala S, Dasu V V, Ahmad J, et al. High yield expression of novel glutaminase free L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N[J]. Bioprocess and biosystems engineering, 2015, 38(11): 2271-2284. [8] Wei W, Ma J, Guo S, et al. A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine: functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization[J]. Process Biochemistry, 2014, 49(11): 1893-1902. [9] Wu L, Wu S, Qiu J, et al. Green synthesis of isomaltulose from cane molasses by Bacillus subtilis WB800-pHA01-palI in a biologic membrane reactor[J]. Food Chemistry, 2017, 229: 761-768. [10] Chen J, Gai Y, Fu G, et al. Enhanced extracellular production of α-amylase in Bacillus subtilis by optimization of regulatory elements and over-expression of PrsA lipoprotein[J]. Biotechnology letters, 2015, 37(4): 899-906. [11] Chen J, Fu G, Gai Y, et al. Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression[J]. Microbial cell factories, 2015, 14(1): 92. [12] Ho H T, Nguyen H T. Optimization of Bacillus Subtilis Natto Immobilization Process on Alginate-Chitosan Complex and Its Application for Nattokinase Fermentation[J]. International Journal of Pharmaceutical Science Invention, 2016, 30. [13] Nie G, Liu N, Zhang E, et al. Preparation of a novel mixed milk with nattokinase produced by Bacillus subtilis (natto)[J]. Journal of Food Processing and Preservation, 2017. [14] Suwanmanon K, Hsieh P C. Isolating Bacillus subtilis and optimizing its fermentative medium for GABA and nattokinase production[J]. CyTA-Journal of Food, 2014, 12(3): 282-290. [15] Wei X, Zhou Y, Chen J, et al. Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization[J]. Journal of industrial microbiology biotechnology, 2015, 42(2): 287-295. [16] Dabbagh F, Negahdaripour M, Berenjian A, et al. Nattokinase: production and application[J]. Applied microbiology and biotechnology, 2014, 98(22): 9199-9206. [17] Cai D, Wei X, Qiu Y, et al. High‐level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase[J]. Journal of applied microbiology, 2016, 121(3): 704-712. [18] Jhan J K, Chang W F, Wang P M, et al. Production of fermented red beans with multiple bioactivities using co-cultures of Bacillus subtilis and Lactobacillus delbrueckii subsp. bulgaricus[J]. LWT-food science and technology, 2015, 63(2): 1281-1287. [19] Kitagawa M, Shiraishi T, Yamamoto S, et al. Novel antimicrobial activities of a peptide derived from a Japanese soybean fermented food, Natto, against Streptococcus pneumoniae and Bacillus subtilis group strains[J]. AMB Express, 2017, 7(1): 127. [20] Weng M, Deng X, Bao W, et al. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation[J]. Biochemical and biophysical research communications, 2015, 465(3): 580-586. [21] #214;zt#252;rk S, #199;al#305;k P, #214;zdamar T H. Fed-batch biomolecule production by Bacillus subtilis: A state of the art review[J]. Trends in biotechnology, 2016, 34(4): 329-345. [22] Shiu Y L, Wong S L, Guei W C, et al. Increase in the plant protein ratio in the diet of white shrimp, Litopenaeus vannamei (Boone), using Bacillus subtilis E20‐fermented soybean meal as a replacement[J]. Aquaculture Research, 2015, 46(2): 382-394. [23] Cai D, Zhu C, Chen S. Microbial production of nattokinase: current progress, challenge and prospect[J]. World Journal of Microbiology and Biotechnology, 2017, 33(5): 84. [24] Guan C, Cui W, Cheng J, et al. Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis[J]. Microbial cell factories, 2016, 15(1): 66. [25] Kapoor R, Harde H, Jain S, et al. Downstream processing, formulation development and antithrombotic evaluation of microbial nattokinase[J]. Journal of biomedical nanotechnology, 2015, 11(7): 1213-1224. [26] Borriss R, Danchin A, Harwood C R, et al. Bacillus subtilis, the model Gram‐positive bacterium: 20 years of annotation refinement[J]. Microbial biotechnology, 2018, 11(1): 3-17. [27] Yogesh D, Halami P M. Fibrinolytic enzymes of Bacillus spp.: an overview[J]. International Food Research Journal, 2017, 24(1). [28] Bi H, Zhao H, Lu F, et al. Improvement of the Nutritional Quality and Fibrinolytic Enzyme Activity of Soybean Meal by Fermentation of Bacillus subtilis[J]. Journal of food processing and preservation, 2015, 39(6): 1235-1242. [29] Ayala F R, Bauman C, Cogliati S, et al. Microbial flora, probiotics, Bacillus subtilis and the search for a long and healthy human longevity[J]. Microbial Cell, 2017, 4(4): 133. [30] Ma Y, Shen W, Chen X, et al. Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization[J]. Journal of biological engineering, 2016, 10(1): 13. [31] Cai D, Wang H, He P, et al. A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis[J]. Microbial cell factories, 2017, 16(1): 70. [32] Cheng A C, Lin H L, Shiu Y L, et al. Isolation and characterization of antimicrobial peptides derived from Bacillus subtilis E20-fermented soybean meal and its use for preventing Vibrio infection in shrimp aquaculture[J]. Fish Shellfish Immunology, 2017. [33] Mohkam M, Nezafat N, Berenjian A, et al. Role of Bacillus Genus in the Production of Value-Added Compounds[M]//Bacilli and Agrobiotechnology. Springer International Publishing, 2016: 1-33.

3. 毕业设计（论文）进程安排

2018.1-2018.2 熟悉实验原理和实验操作，查阅文献，对课题进行初步探索。

2018.3-2018.4 查找尿酸氧化酶与黄嘌呤脱氢酶编码基因序列，通过基因工程的技术手段，构建重组质粒，将其转化到枯草芽孢杆菌中表达。

对培养温度、诱导时机、诱导剂浓度、诱导表达时间等因素进行分析，确定最佳产酶条件。