RESUMO
L-asparaginase synthetase, an ATP-dependent enzyme, necessitates ATP for its catalytic activity. However, the integration of L-asparaginase synthetase into industrial processes is curtailed by the prohibitive cost of ATP. To address this limitation, this study explores the construction of an efficient ATP regeneration system using the glucose metabolism of Escherichia coli, synergistically coupled with L-asparaginase synthetase catalysis. The optimal conditions for L-asparagine yield were determined in shake flasks. A total of 2.7 g/L was the highest yield achieved under specific parameters, including 0.1 mol/L of substrate, 0.2 mol/L glucose, 0.01 mol/L MgCl2 at pH 7.5, a temperature of 37 °C, and agitation at 300 r/min over 12 h. The process was then scaled to a 3-L fermenter, optimizing the addition rates of the substrate and magnesium chloride, and employing a constant glucose feed of 10 g/L/h. The scale-up process led to a significant enhancement in the production of L-asparagine. The yield of L-asparagine was increased to 38.49 g/L after 20 h of conversion, and the molar conversion rate reached 29.16%. This strategy has proven to be effective in improving the efficiency of L-asparagine production. When compared to in vitro ATP regeneration methods, this in vivo approach showcased superior efficiency and reduced costs. These findings furnish pivotal insights that may propel the enzymatic synthesis of L-asparagine toward viable industrial application.
RESUMO
L-Asparagine, a crucial amino acid widely used in both food and medicine, presents pollution-related and side reaction challenges when prepared using chemical synthesis method. Although biotransformation methods offer significant advantages such as high efficiency and mild reaction conditions, they also entail increased costs due to the need for ATP supplementation. This study aimed to address the challenges associated with biopreparation of L-asparagine. Firstly, the functionality and characteristics of recombinant L-asparagine synthetase enzymes derived from Escherichia coli and Lactobacillus salivarius were evaluated to determine their practical applicability. Subsequently, recombinant expression of polyphosphate kinase from Erysipelotrichaceae bacterium was conducted. A reaction system for L-asparagine synthesis was established using a dual enzyme-coupled conversion approach. Under the optimal reaction conditions, a maximum yield of 11.67 g/L of L-asparagine was achieved, with an 88.43% conversion rate, representing a 5.03-fold increase compared to the initial conversion conditions. Notably, the initial addition of ATP was reduced to only 5.66% of the theoretical demand, indicating the effectiveness of our ATP regeneration system. These findings highlight the potential of our approach in enhancing the efficiency of L-asparagine preparation, offering promising prospects for the food and medical industries.
RESUMO
BACKGROUND: Both Wood's lamp and reflective confidential laser scanning microcopy are helpful for the diagnosis and treatment of vitiligo. However, there is few research that contains large samples and consistent observations. AIMS: To analyze the characteristics of Wood's lamp images and reflectance confocal microscopy (RCM) images of vitiligo lesions and to evaluate their significance in vitiligo staging. METHODS: We analyzed the characteristics of RCM images, Wood's lamp images, the vitiligo disease activity (VIDA) score, and clinical features to guide vitiligo staging and treatment. RESULTS: The expert consensus based on the clinical features, VIDA score, Wood's lamp findings, and isomorphic response was consistent with the Wood's lamp findings (χ2 = 3.63, p > 0.05) and RCM findings (χ2 = 3.60, p > 0.05) in diagnosing vitiligo and assessing the disease stage. There was a correlation between the three lesion grades based on the Wood's lamp findings and the stage of vitiligo (p < 0.01). Lesions that appeared porcelain white under the Wood's lamp were in the slowly progressive stage; lesions that appeared gray-white or trichromatic under the Wood's lamp were in the rapidly progressive stage; lesions with clear borders under the Wood's lamp needed further analysis by RCM for the stage to be determined; lesions with blurred borders under the Wood's lamp were in the rapidly progressive stage; lesions that were visible under the naked eye and under the Wood's lamp were in the rapidly progressive stage. CONCLUSION: The study demonstrates a reliable correlation between the findings of RCM (a sophisticated expensive tool) and Wood's lamp examination (a simple, readily available, inexpensive tool) in the assessment of the disease activity of vitiligo lesions.
