A new pH indicator dye-based method for rapid and efficient screening of l -asparaginase producing microorganisms

Mihooliya, Kanti N.; Nandal, J. K.; Swami, Laxmi; Verma, Himanshu; Chopra, Lipsy; Sahoo, Debendra K.

doi:10.1016/j.enzmictec.2017.08.004

Cited by 19 publications

(12 citation statements)

References 12 publications

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“…However, few reports have applied high-throughput screening for L-asparaginase. Instead, some reports have used pH indicator dye-based plate assays to screen for L-asparaginase-producing microorganisms 16 – 18 . In these methods, the pH increases due to the generation of ammonia when strains secrete L-asparaginase, and a differentiable zone form around the strain on the L-asparagine plate.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Zhang

et al. 2018

Sci Rep

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L-asparaginase, which catalyses the hydrolysis of L-asparagine to L-aspartate, has attracted the attention of researchers due to its expanded applications in medicine and the food industry. In this study, a novel thermostable L-asparaginase from Pyrococcus yayanosii CH1 was cloned and over-expressed in Bacillus subtilis 168. To obtain thermostable L-asparaginase mutants with higher activity, a robust high-throughput screening process was developed specifically for thermophilic enzymes. In this process, cell disruption and enzyme activity assays are simultaneously performed in 96-deep well plates. By combining error-prone PCR and screening, six brilliant positive variants and four key amino acid residue mutations were identified. Combined mutation of the four residues showed relatively high specific activity (3108 U/mg) that was 2.1 times greater than that of the wild-type enzyme. Fermentation with the mutant strain in a 5-L fermenter yielded L-asparaginase activity of 2168 U/mL.

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Section: Introductionmentioning

confidence: 99%

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Zhang

et al. 2018

Sci Rep

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“…The intensity of change in color of the dye is related to the production of the enzyme. In the present investigation, eighty-five (85) bacterial isolates were tested positive for asparaginase production on M9 media plates (Table S3) in the presence of 0.009% (w/v) indicator dye phenol red/bromothymol blue/cresol red (Gulati et al 1997;Mahajan et al 2013;Mihooliya et al 2017). The purpose of using different dyes was to find out the best dye for the qualitative visualization of asparaginase activity.…”

Section: Qualitative Screening Of L-asparaginase Producersmentioning

confidence: 99%

“…The change in the pH due to the production of ammonia in the presence of l-asparaginase activity results in the measurable color change on plates in the form of zones. Recently, Mihooliya et al (2017) have screened different pH indicator dyes, i.e., phenol red, bromothymol blue, cresol red, methylene blue, bromocresol purple, bromocresol green, chlorophenol red, and litmus for the screening of l-asparaginase-producing bacteria. The zone of hydrolysis was recorded for each bacterial isolate (Table S3).…”

Section: Qualitative Screening Of L-asparaginase Producersmentioning

confidence: 99%

“…The color intensity was poorly visible below 0.003% dye concentration. Earlier, researchers used 0.009% of dyes for the screening of l-asparaginase (Gulati et al 1997;Mahajan et al 2013;Mihooliya et al 2017).…”

Section: Qualitative Screening Of L-asparaginase Producersmentioning

confidence: 99%

“…Besides, new species in the genus Pseudomonas have been identified which has the potential to produce l-asparaginase (Table S4). Asparaginase production by Pseudomonas pseudoalcaligenes (Badoei-Dalfard 2016), Pseudomonas fluorescens (Sindhu and Manonmani, 2018), Pseudomonas resinovorans strain IGS-131 (Mihooliya et al 2017), Pseudomonas otitidis (Husain et al 2016), and Pseudomonas aeruginosa (El-Bessoumy et al 2004) have been reported in the past.…”

Section: Qualitative Screening Of L-asparaginase Producersmentioning

confidence: 99%

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Optimized chromogenic dyes-based identification and quantitative evaluation of bacterial l-asparaginase with low/no glutaminase activity bioprospected from pristine niches in Indian trans-Himalaya

Kumar

Darnal

et al. 2019

3 Biotech

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Here, we report on the isolation of bacterial isolates from Himalayan niches, which produced extracellular l-asparaginase with low/no glutaminase activity. From the 235 isolates, 85 asparaginase positive bacterial isolates were identified by qualitative screening using optimized chromogenic dyes assay. Optimized concentration of different dyes revealed maximum color visualization in phenol red (0.003%). The diversity analysis of asparaginase positive isolates revealed that Proteobacteria (83%) are the most dominant, followed by Actinobacteria (12%), Firmicutes (3%), and Bacteriodetes (2%). Eleven isolates, which represented seven Pseudomonas species, one species each of the genus Arthrobacter, Janthinobacterium, Lelliottia, and Rahnella, were selected for further studies based on highest zone ratio and novel aspects for l-asparaginase production. Of these, five isolates, namely, Pseudomonas sp. PCH133, Pseudomonas sp. PCH146, Pseudomonas sp. PCH182, Rahnella sp. PCH162, and Arthrobacter sp. PCH138, produced l-asparaginase without glutaminase activity after 55 h of growth with the former isolate showing the highest l-asparaginase activity (1.67 U/ml). Interestingly, this is the first report of l-asparaginase production by members of the genera Janthinobacterium, Rahnella, and Lelliottia.

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Post‐refolding stability considerations for optimization of in‐vitro refolding: L‐asparaginase as a case study

Mihooliya

Nitika

Bhambure

et al. 2022

Biotechnology Journal

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Background L‐Asparaginase is an essential enzyme for the food and biopharmaceutical industry. The stability, however, of L‐asparaginase is widely known to be an issue. Commercial manufacturing of any biopharmaceutical involves hold‐ups during processing, and can result in product loss if stability is an issue, as is the case with L‐asparaginase. This interplay of product intermediate stability and process design is the focus of this investigation. Methods and Results In this study, we propose a strategy to simultaneously increase the refolding yield and stability of refolded L‐asparaginase so as to improve overall process yield. Using one variable at a time (OVAT) experiments, urea (6 M), solubilized inclusion bodies (15 mg/ml), refolding method (step dilution), and pH (8.6) were identified as significant process parameters. A design of experiment (DOE)‐based optimization was then performed for the refolding step. The net outcome was more than a three‐fold increase in enzyme recovery (i.e., 4.90 IU/ml) compared to unoptimized conditions (i.e., 1.26 IU/ml). Further, the L‐asparaginase process intermediate was found to be stable for more than a week at room temperature and 2–8°C, while the unoptimized sample was stable at 2–8°C but did not show any activity at room temperature after 72 h. Conclusions The current study elucidates how process intermediate stability needs to be given due consideration during process optimization, particularly for products such as L‐asparaginase which are labile.

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A new pH indicator dye-based method for rapid and efficient screening of l -asparaginase producing microorganisms

Cited by 19 publications

References 12 publications

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Optimized chromogenic dyes-based identification and quantitative evaluation of bacterial l-asparaginase with low/no glutaminase activity bioprospected from pristine niches in Indian trans-Himalaya

Post‐refolding stability considerations for optimization of in‐vitro refolding: L‐asparaginase as a case study

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