Biochemical Engineering Research and Process Development Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
Biochemical Engineering Research and Process Development Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
Enzyme Microb Technol. 2017 Dec;107:72-81. doi: 10.1016/j.enzmictec.2017.08.004. Epub 2017 Aug 18.
l-asparaginase is a pharmaceutically and industrially important enzyme as it has potential to treat different cancers and inhibit acrylamide formation in fried and baked food products. In the present study, an attempt was made to screen for new and novel l-asparaginase producers using a widely applied phenol red and bromothymol blue (BTB) dye-based plate assay. Screening of four different soil samples for l-asparaginase producers resulted in the isolation of three new potential l-asparaginase producing bacteria. These three strains identified (by 16S rRNA sequencing) as a Pseudomonas resinovorans strain IGS-131, a Bacillus safensis strain IGS-81, and a Glutamicibacter arilaitensis strain ICS-13 with enzyme activities of 10.91 IU/ml, 6.65 IU/ml, and 1.47 IU/ml, respectively. These three strains of bacteria have not been reported as l-asparaginase producers previously. Also, we developed a new pH indicator dye-based plate assay for the screening of l-asparaginase producers after testing eight different pH indicator dyes. This cresol red dye-based method gave a better differentiable zone of hydrolysis and consistent results as compared to previously reported phenol red and BTB-based plate assay. It was also found to be efficient in comparison to all other dyes studied. It produced a bright yellow color at acidic pH (5.5) and turned into a dark red or maroon color when pH was increased (above 7.5). This finding is expected to make screening of all kinds of l-asparaginases more comfortable, rapid, and efficient.
天冬酰胺酶是一种具有重要药用和工业价值的酶，因为它有潜力治疗不同的癌症，并抑制油炸和烘焙食品中丙烯酰胺的形成。在本研究中，我们试图使用广泛应用的酚红和溴甲酚蓝（BTB）染料平板检测法筛选新的和新型的天冬酰胺酶产生菌。对四种不同土壤样本进行天冬酰胺酶产生菌的筛选，结果分离到三种新的潜在天冬酰胺酶产生菌。这三种菌株（通过 16S rRNA 测序鉴定）分别为腐生假单胞菌（Pseudomonas resinovorans）菌株 IGS-131、巴氏芽孢杆菌（Bacillus safensis）菌株 IGS-81 和谷氨酸棒杆菌（Glutamicibacter arilaitensis）菌株 ICS-13，其酶活性分别为 10.91 IU/ml、6.65 IU/ml 和 1.47 IU/ml。这三种细菌以前都没有报道过是天冬酰胺酶产生菌。此外，我们在测试了八种不同的 pH 指示剂染料后，开发了一种新的基于 pH 指示剂染料的平板检测法来筛选天冬酰胺酶产生菌。与之前报道的酚红和 BTB 平板检测法相比，这种甲酚红染料法产生的水解区分带更好，结果更一致。与所有研究的其他染料相比，该方法也更有效。在酸性 pH（5.5）下，它呈现出鲜艳的黄色，当 pH 升高（高于 7.5）时，它变成暗红色或栗色。这一发现有望使各种天冬酰胺酶的筛选更加舒适、快速和高效。
