BacillussubtilisZJUTZY固定化转化生产γ-PGA及其水解定量检测方法的改进
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摘要
γ-PGA（γ-聚谷氨酸）是一种通过微生物合成的均聚氨基酸化合物，它由谷氨酸单体以γ-羧基与氨基相缩合而成，是一种水溶性和可生物降解的新型生物高分子材料，近年来由于其被作为增稠剂，保湿剂，药物载体等而一直被广泛应用于工业领域，是一种有极大开发价值和前景的多功能性生物制品。
Bacillus subtilis ZJUTZY为本实验室从纳豆中分离出的γ-聚谷氨酸（γ-PGA）高产菌株，本文对其固定化转化产γ-PGA的生产条件进行优选。实验得出海藻酸钙包埋具有较高的产量而且包埋菌量固定，因此确定海藻酸钙为固定化材料，采用包埋法进行固定。后采用单因素法对转化生产γ-PGA的菌种固定化条件进行优选，得到的较优条件如下：温度为37℃，pH为7，摇床转速为200rpm,接种量为12%，固液比为1：3，氯化钙浓度为4%。
通过增加一检测样品，即醇沉物水溶中的谷氨酸含量，并且通过不断地实验，确定了水解液中和所需的NaOH的添加量，我们改进了水解法测γ-PGA产量的检测方法，并在此基础上，研究了Bacillussubtilis ZJUTZY产γ-PGA的产量变化，得出30小时的γ-PGA的产量最高为11.02g/L。
而后根据pgsB在不同菌株中的序列，及与本实验室菌株同源性较高的Bacillus subtilis ZJU-7的pgsB序列，设计了2对引物。当分别用pgsB-F和pgsB-R为正反向引物，以Bacillus subtilis ZJUTZY基因组DNA为模板时，PCR获得了一段长1221bp的基因片段，对PCR片段进行序列测定pgsB基因，发现该基因片段与多种Bacillus subtilis菌株的pgsB基因具有较高的同源性，相似度达到了99%，故可初步认定PCR得到的DNA片段为pgsB基因的部分序列。
Poly (γ-glutamic acid) (γ-PGA) is an unusual anionic, water-solubleand high viscous polymer in which glutamate is polymerized by amidelinkages between a-amino andγ-carboxylic acid groups. For its excellentcharacters, potential applications ofγ-PGA have been of interest in the pastfew years in a broad range of industrial fields. It has been used asthickeners, humectant and drug carrier.
Bacillus subtilis ZJUTZY, which was aγ-PGA producer, was isolatedfrom natto. And the optimum condition for producingγ-PGA was studiedthrough with immobilized Bacillus subtilis ZJUTZY. The betterimmobilizing material was calcium-alginate and the optimum conditionwas as follows: temperature 30℃, initial pH7, rotation speed 200 rpm,inoculum size 12%，beads to liquid 1:3, and CaCl2 concentration 4%.
We improved the method of mearing the yield ofγ-PGA by two steps.One is adding a meaturement of the concentration of glutamic acid in the deposit and the other is the confirmation of the dosage of NaOH in the stepof neutralization. Then we found that the highest yield ofγ-PGA was11.02 g/L when the culture time reached 30 hours.
Two pairs of degenerate primers was designed to be used to amplifythe partial fragment of pgsB from Bacillus subtilis ZJUTZY according tothe conservative regions of pgsB and the sequence of pgsB from Bacillussubtilis ZJU-7. A DNA band of 817bp was obtained when the latter one wasused. The DNA fragment had 99% identity with the pgsB from manyBacillus subtilis. Based on these results, it was confirmed that partial ofpgsB gene was successfully cloned from the Bacillus subtilis ZJUTZY.
Bacillus subtilis ZJUTZY为本实验室从纳豆中分离出的γ-聚谷氨酸（γ-PGA）高产菌株，本文对其固定化转化产γ-PGA的生产条件进行优选。实验得出海藻酸钙包埋具有较高的产量而且包埋菌量固定，因此确定海藻酸钙为固定化材料，采用包埋法进行固定。后采用单因素法对转化生产γ-PGA的菌种固定化条件进行优选，得到的较优条件如下：温度为37℃，pH为7，摇床转速为200rpm,接种量为12%，固液比为1：3，氯化钙浓度为4%。
通过增加一检测样品，即醇沉物水溶中的谷氨酸含量，并且通过不断地实验，确定了水解液中和所需的NaOH的添加量，我们改进了水解法测γ-PGA产量的检测方法，并在此基础上，研究了Bacillussubtilis ZJUTZY产γ-PGA的产量变化，得出30小时的γ-PGA的产量最高为11.02g/L。
而后根据pgsB在不同菌株中的序列，及与本实验室菌株同源性较高的Bacillus subtilis ZJU-7的pgsB序列，设计了2对引物。当分别用pgsB-F和pgsB-R为正反向引物，以Bacillus subtilis ZJUTZY基因组DNA为模板时，PCR获得了一段长1221bp的基因片段，对PCR片段进行序列测定pgsB基因，发现该基因片段与多种Bacillus subtilis菌株的pgsB基因具有较高的同源性，相似度达到了99%，故可初步认定PCR得到的DNA片段为pgsB基因的部分序列。
Poly (γ-glutamic acid) (γ-PGA) is an unusual anionic, water-solubleand high viscous polymer in which glutamate is polymerized by amidelinkages between a-amino andγ-carboxylic acid groups. For its excellentcharacters, potential applications ofγ-PGA have been of interest in the pastfew years in a broad range of industrial fields. It has been used asthickeners, humectant and drug carrier.
Bacillus subtilis ZJUTZY, which was aγ-PGA producer, was isolatedfrom natto. And the optimum condition for producingγ-PGA was studiedthrough with immobilized Bacillus subtilis ZJUTZY. The betterimmobilizing material was calcium-alginate and the optimum conditionwas as follows: temperature 30℃, initial pH7, rotation speed 200 rpm,inoculum size 12%，beads to liquid 1:3, and CaCl2 concentration 4%.
We improved the method of mearing the yield ofγ-PGA by two steps.One is adding a meaturement of the concentration of glutamic acid in the deposit and the other is the confirmation of the dosage of NaOH in the stepof neutralization. Then we found that the highest yield ofγ-PGA was11.02 g/L when the culture time reached 30 hours.
Two pairs of degenerate primers was designed to be used to amplifythe partial fragment of pgsB from Bacillus subtilis ZJUTZY according tothe conservative regions of pgsB and the sequence of pgsB from Bacillussubtilis ZJU-7. A DNA band of 817bp was obtained when the latter one wasused. The DNA fragment had 99% identity with the pgsB from manyBacillus subtilis. Based on these results, it was confirmed that partial ofpgsB gene was successfully cloned from the Bacillus subtilis ZJUTZY.
引文
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[2] Ashiuchi M, Kamei T, Misono H. Poly-gamma-glutamate synthetase of Bacillussubtilis. Journal of Molecular Catalysis B-Enzymatic, 2003, 23(2-6):101～106.
[3] Hezayen FF, Rehm BHA, Tindall BJ, et al. Transfer of Natrialba asiatica B1T toNatrialba taiwanensis sp nov and description of Natrialba aegyptiaca sp nov., anovel extremely halophilic, aerobic, non-pigmented member of the Archaea fromEgypt that produces extracellular poly(glutamic acid). International Journal ofSystematic and Evolutionary Microbiology, 2001, 51:1133～1142.
[4] Ashiuchi M, Kamei T, Baek DH, et al. Isolation of Bacillus subtilis (chungkookjang),a poly-gamma-glutamate producer with high genetic competence. AppliedMicrobiology and Biotechnology, 2001, 57 (5-6): 764～769.
[5] Ivanovics G., Bruckner V., Chemishe and immunologiche Studien uber denMechanismus der milzbrandinjektin and Immunitat; die chemische Struktur derKapselsubstanz des Milzbrandbazillus and der serologisch dentischenspezifischen Subtanz des Bazillus mesentericus[J]. Z. Immunitatsforsch, 1937. 90:304-318
[6] Fujio Y., Distribution of plasmids in poly glutamate producing bacillus strainsisolated from "natto"-like fermented soybeans,“thua nao”，in Thailand[J]. Gen.AppL, 1986, 32(2): 241-249
[7] Nagai, T., Koguchi K., and Itoh Y., Chemical analysis of poly-gamma-glutamic acidproduced by plasmid-free Bacillus subtilis (natto): Evidence that plasmids are notinvolved in poly-gamma-glutamic acid production[J]. J Gen Appl Microbiol, 1997,43(3): 139-143
[8] Crescenzi V., Dentini, Aqueous solution properties of bacterial poly- -D-glutamate[J].ACS Symp.Ser., 1996, 627: 233-242
[9] Rao M.V., Solution conformation of poly (L-lysyl-L-glutamic acid) and poly (Llysyl-L-glutamine) [J]. Int J Pept Protein Res, 1984, 24(1):48-54
[10] Kim K.S., Graham N., Controlled elease behavior of prodrugs based on thebiodegradable poly (γ-glutamicacid) microspheres[J]. Polymer Journal, 1999, 31(5):813-816
[11] Shih I.L., Van Y.T., The production of poly (γ-glutamic acid) from microorganismsand its various applications[J]. Bioresource Technology, 2001, 79: 207-225
[12]杨革，陈坚，曲音波等，碳源和Mn2+对地衣芽孢杆菌Bacillus licheniformisWBL-3生产聚γ-谷氨酸的影响[J].化工学报，2002, 53(3): 317-320
[13] Ashiuchi M., Misono H., Biochemistry and molecular genetics of poly-γ- glutamatesynthesis[J]. Appl Microbiol Biotechnol, 2002, 59: 9-14
[14] Ashiuchi M., Nakamura H., Yamamoto T., Poly-γ-glutamate depolymerade ofBacillus subtilis: production, simple purification and substrate selectivity[J]. J MolCat B: Enzym, 2003, 23: 249-255
[15] Mclean R.J.C., Beauchemin D, Clapham L, Metal-binding characteristics of thegamma-glutamyl capsular polymer of Bacillus licheniformis ATCC 9945[J]. ApplEnviron Microbial, 1990, 59: 3671-3677
[16] Mitsuiki M., Mizuno A., Tanimoto H., Relationship between the antifreeze activitiesand the chemical structures of oligo-and poly (glutamic acid)s[J]. J Agr Food Chem,1998, 46:891-895
[17] Kunioka M., Choi H. J., Hydrolytic degradation and mechanical properties ofhydrogels prepared from microbial poly (amino acids) [J]. Polym Degrad Stabil,1998, 59: 33-37
[18]黄金，陈宁，γ-聚谷氨酸的性质与生产方法[J]，发酵科技通讯, 2005, 34(3):4-8
[19] Sanda F., Fujiyama T., Endo T., Chemical synthesis of poly-gamma-glutamic acidby polycondensation of gamma-glutamic acid dimmer: synthesis and reaction ofpoly-gamma-glutamic acid methyester[J]. J Polym Science Part A-1, 2001,39(5):732-741
[20]彭银仙，徐虹，陈国广等，新型药物载体聚谷氨酸的合成及其应用[J].中国新药杂志，2002, 11(7): 515-519
[21] Green BD, Battisti L, Koehler TM, et al. Demonstration of a Capsule Plasmid inBacillus-Anthracis. Infection and Immunity, 1985, 49(2):291～297.
[22] Candela T, Mock M, Fouet A. CapE, a 47-amino-acid peptide, is necessary forBacillus anthracis polyglutamate capsule synthesis. Journal of Bacteriology, 2005,187(22):7765～7772.
[23] Ashiuchi M, Soda K, Misono H. A poly-gamma-glutamate synthetic system ofBacillus subtilis IFO 3336: Gene cloning and biochemical analysis of polygamma-glutamate produced by Escherichia coli clone cells. Biochemical andBiophysical Research Communications, 1999, 263(1):6～12.
[24] Ashiuchi M, Nawa C, Kamei T, et al. Physiological and biochemical characteristicsof poly gamma-glutamate synthetase complex of Bacillus subtilis. EuropeanJournal of Biochemistry, 2001, 268(20):5321～5328.
[25] Tarui Y, Iida H, Ono E, et al. Biosynthesis of poly-gamma-glutamic acid in plants:Transient expression of poly-gamma-glutamate synthetase complex in tobaccoleaves. Journal of Bioscience and Bioengineering, 2005, 100(4):443～448.
[26] Candela T, Fouet A. Poly-gamma-glutamate in bacteria. Molecular Microbiology,2006, 60(5):1091～1098.
[27] Ashiuchi M, Tani K, Soda K, et al. Properties of glutamate racemase from Bacillussubtilis IFO3336 producing poly-gamma-glutamate. Journal of Biochemistry, 1998,123(6):1156～1163.
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