Knowledge Management System Of Institute of process engineering,CAS
|Thesis Advisor||刘庆芬, 张军立|
我国每年产生约200多万吨抗生素菌渣，抗生素菌渣为危险固体废弃物，其安全处置及利用引起广泛关注。抗生素菌渣中富含蛋白质、葡聚糖等有机物，其中蛋白质的含量约占干菌渣的50%。本论文作者所在团队提出了将抗生素菌渣溶解，并进一步制备氨基酸等营养物质，用于发酵培养，实现抗生素菌渣厂内循环利用的新思路。本文以青霉素菌丝为研究对象，研究碱热法、酸热法和低温氢氧化钠/尿素溶液体系溶解青霉素菌丝过程，建立相应的最佳溶解工艺。在此基础上，以菌丝碱溶液为研究对象，通过等电点沉淀法考察溶液中菌丝蛋白质分布特性，测定其氨基酸组成和含量。研究酶催化水解溶液中菌丝蛋白质制备氨基酸过程，建立最佳酶解工艺。主要研究内容和结论如下： （1）研究碱热法、酸热法和低温氢氧化钠/尿素溶液体系溶解青霉素菌丝过程。研究结果显示，溶剂组成、菌丝与溶液质量比、反应温度和时间等因素对青霉素菌丝溶解过程皆有影响。碱热法溶解最高，青霉素菌丝的溶解率顺序为：碱热法>低温氢氧化钠/尿素溶液体系>酸热法。碱热法溶解菌丝最佳工艺为：氢氧化钠浓度4%、溶液与菌丝质量比5:1、反应温度50℃、溶解反应30 min，菌丝溶解率达到69.5%，菌丝溶解容量为141.3 g/L，溶液中蛋白质和多糖含量分别占菌丝质量的25.3%和9.5%。低温氢氧化钠/尿素溶液体系的最佳溶解工艺为：溶剂体系中含10%氢氧化钠和12%尿素溶液、溶液与菌丝质量比为8:1、溶解温度-8℃、溶解50 min，菌丝溶解率达到69.9%，菌丝溶解容量为88.9 g/L，溶解液中蛋白质和多糖含量分别占菌丝质量的23.5%和15.3%。与碱热法相比，低温氢氧化钠/尿素溶液体系溶液中多糖含量提高了5.8个百分点。 （2）在确定最佳碱热法工艺基础上，通过等电点沉淀法考察了溶液中菌丝蛋白质的分布特性，采用PITC柱前衍生法测定溶解液中菌丝蛋白质的氨基酸组成和含量。研究结果显示，菌丝溶液中蛋白质等电点范围为pH 2-11，在pH 4、7和9时，溶液中皆有蛋白质沉淀出，沉淀率分别为77.7%、30.5%和18.3%，说明菌丝蛋白质的等电点主要分布于酸性、弱酸性和中性范围条件。溶解液中菌丝蛋白质含有16种氨基酸，主要是天冬氨酸、谷氨酸、亮氨酸、丙氨酸和甘氨酸，分别约占检测出氨基酸总量的20.59%、13.20%、10.91%、8.11%和7.15%。 （3）以碱性蛋白酶、中性蛋白酶、酸性蛋白酶和菠萝蛋白酶为催化剂，研究了酶催化水解菌丝溶解液中蛋白质制备氨基酸的过程，确定了菌丝蛋白最佳催化水解工艺。研究结果显示，酶的种类、酶与蛋白质质量比、反应温度和时间等条件对菌丝蛋白质水解过程均有影响，其中碱性蛋白酶催化水解效果最佳。在碱性蛋白酶与蛋白质的质量比6%、溶解液pH 11、50 ℃、反应4 h的工艺条件下，青霉素菌丝蛋白质水解度达到31.43%。为进一步提高蛋白质水解度，以碱性蛋白酶和菠萝蛋白酶组成的复合酶为催化剂，研究复合酶催化水解菌丝蛋白水解过程，确定复合酶催化最佳工艺为：复合酶与蛋白质的质量比为9%、溶解液pH 10、反应温度50 ℃、反应3 h，青霉素菌丝蛋白质水解度达到42.73%，比单酶法提高了11.3个百分点。酶解液中主要含有谷氨酸、天冬氨酸、甘氨酸、亮氨酸和丙氨酸等有16种氨基酸，分别占检测出氨基酸总量的15.47%、11.94%、9.41%、9.23%和9.22%。;More than 2 million tons of antibiotics fermentation residues (AFRs) were produced in china every year, which were hazardous solid waste, and how to safely dispose and utilize them are widely attracting attention. AFRs are rich in organic substances, such as protein and carbohydrates, which account for about 50% in dry penicillin AFRs. The author's team proposed a promising suggestion, in which antibiotic mycelium are dissolved firstly, and then prepared amino acids, and use them to fermentation process. In this paper, the dissolution of penicillin mycelium by Alkali-thermal method, Acid-thermal method and low temperature NaOH/urea solution system were studied, and the distribution of mycelium protein, the amino acid composition and content in mycelium solution were determined. The enzymatic hydrolysis of penicillin mycelium protein in the solution was investigated, and the optimal enzymatic hydrolysis process was established. The main research contents andconclusions are as follows: (1)The dissolution of penicillin mycelium by Alkali-thermal method, Acid-thermal method and low temperature NaOH/urea solution system were studied. respectively. The results showed that the solvent compositions, the mass ratio of the mycelium to the solvent, the reaction temperature and time had significantly effects on dissolution of penicillin mycelium. Alkaline heating method showed the best performance on dissolution rate, the performance order was: Alkaline-thermal method ˃ low temperature NaOH/urea solution system ˃ the acid-thermal method. The optimal dissolution conditions in alkali-thermal method were as follows: sodium hydroxide concentration 4%, mass ratio of solution to mycelium 5:1, temperature 50 ℃ and dissolving time 30 min. The mycelium dissolution rate reached 69.5%. The capacity of mycelium in the solution was 141.3 g/L, in which the protein and polysaccharide accounted for 25.3% and 9.5% to the mycelium mass, respectively. The optimal dissolution parameters of the low temperature NaOH/urea solution system were as follows: the solvent system composition 10% NaOH and 12% urea solution, mass ratio of solution to mycelium 8:1, temperature -8 ℃, and dissolving time 50 min. The mycelium dissolution rate reached 69.3%. The mycelium capacity was 88.9 g/L in the solution, in which the protein and polysaccharide accounted for 23.5% and 15.3% to the mycelium mass, respectively. Compared with the alkaline-thermal method, the polysaccharide content in the low-temperature NaOH/urea solution system increased by 5.8 percentage point. (2) On the basis of penicillin mycelium dissolution by the Alkali-thermal method, the distribution characteristics of mycelium protein in the solution were investigated by isoelectric point precipitation, and the amino acid composition and content in the mycelium solution were determined by PITC pre column derivatization method. The isoelectric points pH of protein in the mycelium solution was at pH 2-11. At pH 4, pH 7 and pH 9, the protein precipitation ratio were 77.7%, 30.5% and 18.3% respectively. That indicated the isoelectric point of mycelium protein at pH 4 and pH 7 were significantly higher than that at pH 9. 16 kinds of amino acids were detected in mycelium solution, and aspartic acid, glutamic acid, leucine, alanine and glycine accounted for 20.59%, 13.20%, 10.91%, 8.11% and 7.15% of the total amino acids,respectively. (3) The enzymatic hydrolysis of penicillin mycelium protein was explored in this solution using enzyme as catalyst, such as Alcalase (A), Bromelain (B) Neutrase (C), and Acid protease (D). The results showed that the enzymes types, the ratio of enzyme to protein, temperature and time had obviously effect on enzymatic hydrolysis of penicillin mycelium protein. In single enzyme catalyzed hydrolysis process, Alcalase showed the best performance, the hydrolysis degree of protein reached 31.43 % at following conditions, the mass ratio of Alcalase to protein as 6%, pH 10, temperature 50 ℃ and reaction time 3 h. The experiment results demonstrated that the complex enzyme constituted of Alcalase and Bromelain as catalyst gave a better performance on enzymatic hydrolysis degree than Alcalase as catalyst. The hydrolysis degree of mycelium protein reached 42.73% at the optimal conditions, the mass ratio of complex enzyme to protein as 9% (A:B=2:1), pH 10, temperature 50 ℃ and reaction time 3 h. The hydrolysis degree of mycelium protein was increased by 11.3% percentage point than the best single-enzyme method. The HPLC analysis measured 16 amino acids in the enzymatic hydrolysate, such as glutamic acid, aspartic acid, glycine, leucine and alanine, in which the ratio was 15.47%, 11.94%, 9.41%, 9.23% , 9.22% , respectively.
|张展敖. 青霉素菌丝溶解及菌丝中蛋白质酶催化水解研究[D]. 中国科学院大学,2020.|
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