Ali Sikander, Zahra Hijab, Ahmad M Usman, Ullah Najeeb, Sarwar Abid, Aziz Tariq, Alharbi Metab, Alasmari Abdullah F, Albekairi Thamer H
Department of Microbiology, Dr. Ikram-ul-Haq Institute of Industrial Biotechnology (IIIB), GC University, 54000, Lahore, Pakistan.
Department of Biotechnology, Dr. Ikram-ul-Haq Institute of Industrial Biotechnology (IIIB), GC University, 54000, Lahore, Pakistan.
Int Microbiol. 2024 Dec 27. doi: 10.1007/s10123-024-00627-2.
The present research work is concerned with the production and optimization of the dopa-oxidase enzyme by using pre-grown mycelia of Aspergillus oryzae. Different strains of A. oryzae were collected and isolated from various soil samples. Out of 32 isolated strains, isolates 19 and 27 were selected as they showed higher dopa-oxidase activity. Biomass harvesting was accomplished in a medium containing chloramphenicol as an antibiotic. The mycelia were filtered, washed with cold water, and stored at 4 ºC. A dopa-oxidase assay was performed, and absorbance was measured at a wavelength of 505 nm. Different physical parameters such as medium pH (6), temperature (30 ºC), and inoculum size (1.5% v/v) were optimized after the results of the assay. Other parameters like nitrogen requirements, biomass level (2 mg/ml), L-tyrosine (3.75 mg/ml), and L-ascorbic acid (8.75 mg/ml) concentrations were evaluated by reaction procedure. Certain micro and macronutrients and stabilizers, including Rochelle salt (20 µM), glycerol (25 µM), orthophosphoric acid (15 µM), and ethanol (20 µM), can also increase dopa-oxidase activity. In the last stage, the time of incubation (48 h) was optimized for maximum dopa-oxidase activity as well as L-dopaquinone production. Hence, from the results of the present study, it was observed that the activity of dopa-oxidase could be increased in the reaction mixture by the addition of various substances. They enhanced the dopa-oxidase activity up to 34.18 and 29.02 U/ml for isolates 19 and 27, respectively. In the future, dopa-oxidase can be used to produce stable L-dopaquinone from L-phenylalanine, which will provide clinical applications.
本研究工作涉及利用米曲霉预先生长的菌丝体生产和优化多巴氧化酶。从各种土壤样品中收集并分离出不同的米曲霉菌株。在32株分离菌株中，分离株19和27因其显示出较高的多巴氧化酶活性而被选中。在含有氯霉素作为抗生素的培养基中完成生物量收获。将菌丝体过滤，用冷水洗涤，并储存在4℃。进行多巴氧化酶测定，并在505nm波长下测量吸光度。根据测定结果优化了不同的物理参数，如培养基pH值（6）、温度（30℃）和接种量（1.5%v/v）。通过反应程序评估了其他参数，如氮需求、生物量水平（2mg/ml）、L-酪氨酸（3.75mg/ml）和L-抗坏血酸（8.75mg/ml）浓度。某些微量和常量营养素以及稳定剂，包括酒石酸钾钠（20μM）、甘油（25μM）、正磷酸（15μM）和乙醇（20μM），也可以提高多巴氧化酶活性。在最后阶段，优化孵育时间（48小时）以实现最大的多巴氧化酶活性以及L-多巴醌产量。因此，从本研究结果可以看出，通过添加各种物质可以提高反应混合物中多巴氧化酶的活性。它们分别将分离株19和27的多巴氧化酶活性提高到34.18和29.02U/ml。未来，多巴氧化酶可用于从L-苯丙氨酸生产稳定的L-多巴醌，这将提供临床应用。
