Novel glutaminase free l-asparaginase from Nocardiopsis alba NIOT-VKMA08: production, optimization, functional and molecular characterization

Abstract

Studies were carried out for the optimization and production of novel extracellular glutaminase-free L-asparaginase from Nocardiopsis alba NIOT-VKMA08. Among the tested carbon and nitrogen sources, maximum L-asparaginase production was observed with a combination of L-asparagine and maltose (1.5%) and twofold increase in yield (18.47 IU mL(-1)) was observed with newly optimized NIOT-asparaginase medium. Activity of the purified enzyme was moderately inhibited by various divalent cations and thiol group blocking reagents, with K(m) and V(max) of 0.127 mM and 5.50 U µg(-1). Optimum pH and temperature of purified L-asparaginase for the hydrolysis of L-asparagine was 8.0 and 37 °C, respectively. The enzyme inhibited polyacrylamide formation in 10% solution and it was very specific for its natural substrate L-asparagine. Partial glutaminase activity was not detected, which could reduce the possibility of side effects during cancer therapy. L-Asparaginase biosynthesis gene (ansA) was cloned and transformed in E. coli JM109. The ansA gene sequence reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in altered amino acid sequences of the translated protein.

Purification and characterization of l-asparaginase from Pseudomonas aeruginosa strain SN004: Production optimization by statistical methods

“…(Moorthy et al, 2010), Pseudomonas aeruginosa (Abdel-Fattah and Olama, 2002), Enterobacter aerogenes (Mukherjee et al, 2002), Zymomonas mobilis (Pinheiro et al, 2001), Bacillus licheniformis (Sudhir et al, 2014), Nocardiopsis alba NIOT-VKMA08 (Meena et al, 2015), Bacillus subtilis B11−06 (Jia et al, 2013), and Staphylococcus sp. OJ82 (Sangwon et al, 2014) have been found to produce L-asparaginase.…”

Optimization of L-asparaginase activity of Actinobacteria isolated from Guaviare river sediments in Colombia

“…Different carbon sources have different effects on L-asparaginase production from Streptomyces. S. longsporusflavus and S. albidoflavus produced the maximum amount of Lasparaginase when grown on soluble starch [13,23] whereas S. phaeochromogenes and S. tendae produced the maximum activity on glycerol and sucrose, respectively [24]. However, few studies used lactose for Actinobacterial Lasparaginase production.…”

Avaliação da atividade e resistência à clivagem proteolítica de L-asparaginases recombinantes obtidas por reação em cadeia da polimerase propensa a erro

“…Os sintomas das reações alérgicas à L-asparaginase incluem febre, dor, urticária, exantema, anafilaxia e desconforto respiratório, sendo que a maioria dos pacientes (84%) desenvolve reações alérgicas grau 2, enquanto que 15% destes desenvolvem grau 3, e apenas 0,5% grau 4 (CHEN et al, 2010 (CHAN et al, 2014;MEENA et al, 2015;MEHTA et al 2014;SUDHIR et al, 2014;VERMA et al, 2014;OFFMAN et al, 2011;RAMYA et al, 2011;REINERT et al 2006).…”

“…Dentre os estudos citados, Meena et al (2015), Sudhir et al (2014), Ramya et al (2011) (MEENA et al, 2015;SUDHIR et al, 2014;RAMYA et al, 2011;REINERT et al 2006). Já os pesquisadores Chain et.…”

“…Devido ao alto potencial de cura, mas também aos graves eventos adversos, muitos pesquisadores têm buscado por L-asparaginases alternativas de outros organismos, ou produzidas por mutações sítio-dirigidas, que sejam mais ativas e estáveis, menos imunogênicas, e com menor atividade glutaminase (CHAN et al, 2014;MEENA et al, 2015;MEHTA et al 2014;SUDHIR et al, 2014;VERMA et al, 2014;VIDYA, USHASREE e PANDEY, 2014;OFFMAN et al, 2011;RAMYA et al, 2011;REINERT et al 2006).…”