Rodrigues David, Pillaca-Pullo Omar, Torres-Obreque Karin, Flores-Santos Juan, Sánchez-Moguel Ignacio, Pimenta Marcela V, Basi Tajindar, Converti Attilio, Lopes André M, Monteiro Gisele, Fonseca Luís P, Pessoa Adalberto Jr
Bioengineering Department of Instituto Superior Técnico, Institute of Bioengineering and Biosciences, Universidade de Lisboa, Lisbon, Portugal.
Department of Pharmaceutical-Biochemical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
Front Bioeng Biotechnol. 2019 Feb 8;7:16. doi: 10.3389/fbioe.2019.00016. eCollection 2019.
L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative ASNase II coded by the gene was biosynthesized by recombinant under the control of the promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 g.L during glycerol batch phase, corresponding to a biomass yield of 0.77 gg and a specific growth rate of 0.21 h. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gL while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gL, periplasmic specific enzyme activity 37.1 U.g , volumetric enzyme activity 3,315 U.L, overall enzyme volumetric productivity 31 U.L.h, while the specific growth rate fell to 0.039 h. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.
L-天冬酰胺酶（ASNase）用于治疗急性淋巴细胞白血病，目前仅由细菌来源生产和商业化。由该基因编码的替代ASNase II在启动子的控制下通过重组体进行生物合成，采用了不同的培养策略。特别是，我们应用了分为四个不同阶段的多级补料分批培养来生产ASNase II并确定发酵参数，即比生长速率、生物量产量和酶活性。在改良的限定培养基中于有利条件下培养重组体，在甘油分批阶段确保了31 g/L的干生物量浓度，对应于0.77 g/g的生物量产量和0.21 h⁻¹的比生长速率。在有限条件下进行12 h甘油补料后，细胞浓度达到65 g/L，而乙醇浓度非常低。在甲醇诱导阶段，生物量浓度达到91 g/L，周质比酶活性为37.1 U/g，体积酶活性为3315 U/L，总酶体积生产力为31 U/L·h，而比生长速率降至0.039 h⁻¹。我们的结果表明，用于生产ASNase II的最佳策略是采用伪指数补料的甘油补料分批阶段加连续甲醇补料诱导。
