青霉素菌丝中蛋白质酶法水解工艺

Enzymatic hydrolysis of penicillin mycelium protein

采用碱热法溶解青霉素菌丝,研究酶法催化水解菌丝溶解液中蛋白质转化为氨基酸的过程。考察酶种类、溶解液pH值、酶与蛋白质质量比、反应温度和时间等因素对蛋白质水解度的影响,建立最佳水解工艺。结果表明,酶种类、酶与蛋白质之比、p H、温度和时间均对蛋白质水解过程产生影响。以单酶为催化剂,碱性蛋白酶催化水解效果最好,蛋白质水解度达31.43%。以碱性蛋白酶(A)和菠萝蛋白酶(B)组成的复合酶(A:B=2:1)催化水解效果更好,在复合酶与蛋白质的质量比9%、p H=10、反应温度50℃、反应时间3 h条件下,蛋白质水解度达到42.73%,比单酶法提高了11.30个百分点。在最佳复合酶水解工艺下,酶解液中含16种氨基酸,主要有谷氨酸、天冬氨酸、甘氨酸、亮氨酸和丙氨酸等氨基酸。本工作为抗生素菌丝作为发酵培养基循环利用提供了一条新途径。

More than 2000000 tons of antibiotics fermentation residues(AFRs) were produced in China every year, which were hazardous solid waste, and how to safely dispose of them is attracting attention. AFRs are rich in organic substances, such as protein and carbohydrates, which accounts for about 50% of dry penicillin AFRs. So it is significant to develop new methods for recycling and utilization of mycelium protein in AFRs. In this work, penicillin mycelium was dissolved by alkali-thermal method, and penicillin mycelium protein solution was prepared, and then enzymatic hydrolysis performance 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 effects of types of enzymes, p H of the solution, the ratio of enzyme to protein, reaction temperature and time on protein hydrolysis process were systematically studied, and an optimization scheme was proposed. In a single enzyme-catalyzed hydrolysis process, alcalase showed the optimal performance, the hydrolysis degree of protein reached 31.43% at following conditions, the mass ratio of alcalase to protein as 6%, p H=11, the reaction time of 3 h and temperature of 50℃. In the complex enzyme-catalyzed hydrolysis process, the mixture of alcalase and bromelain as catalysts was demonstrated got the best efficiency. The optimum parameters were as follows: the mass ratio of complex enzyme to protein was 9%(A:B=2:1), p H=10, the reaction time of 3 h and temperature of 50℃. Under these optimal conditions, the protein hydrolysis degree reached 42.73%, which was 11.30 percentage points higher than the best single-enzyme method. The high performance liquid chromatography(HPLC) analysis found the enzymatic hydrolysate contained 16 amino acids, such as glutamic acid, aspartic acid, glycine, leucine and alanine, the ratio was 15.47 wt%, 11.94 wt%, 9.41 wt%, 9.23 wt%, 9.22 wt%, respectively. This work provides a high efficient enzyme-catalyzed hydrolysis method of penicillin mycelium protein for preparing amino acids, and to carry out utilization of penicillin mycelium protein in AFRs.