Development of Processes for Recombinant L-Asparaginase II Production by Escherichia coli Bl21 (De3): From Shaker to Bioreactors

Since 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparaginase, this work aimed to produce this enzyme from Escherichia coli in a shaker and in a 3 L bioreactor. Three culture media were tested: defined, semi-defined and complex medium. L-asparaginase activity was quantified using the β-hydroxamate aspartic acid method. The defined medium provided the highest L-asparaginase activity. In induction studies, two inducers, lactose and its analog IPTG, were compared. Lactose was chosen as an inducer for the experiments conducted in the bioreactor due to its natural source, lower cost and lower toxicity. Batch and fed-batch cultures were carried out to reach high cell density and then start the induction. Batch cultivation provided a final cell concentration of 11 g L−1 and fed-batch cultivation produced 69.90 g L−1 of cells, which produced a volumetric activity of 43,954.79 U L−1 after lactose induction. L-asparaginase was produced in a shaker and scaled up to a bioreactor, increasing 23-fold the cell concentration and thus, the enzyme productivity.

Lactose Inducible Fermentation in Escherichia coli for Improved Production of Recombinant Urate Oxidase: Optimization by Statistical Experimental Designs

The enzyme urate oxidase (UOX) is used as a drug for preventing and treatment of chemotherapy-induced hyperuricemia. This study deals with the statistical optimization of lactose inducible fermentation for production of soluble recombinant Aspergillus flavus UOX. 10 variables were investigated by Plackett–Burman design (PBD), and the most significant factors were further optimized by central composite design (CCD). PBD results indicated that glycerol, yeast extract, tryptone, and lactose affected UOX activity significantly. The CCD results showed that the maximum enzyme activity (19.34 U/ml) could be achieved under the optimum conditions of glycerol 0.87 g/L, yeast extract 9.11 g/L, tryptone 10.29 g/L, K2HPO4 1.81 g/L, and lactose 12.79 g/L. When the same induction strategy was tested at shake flask, 19.34 U/mL of UOX activity was obtained, which was 12.5 folds higher than IPTG induction protocol. Furthermore, the lower total cost (0.7 vs. 13.5 €) was additionally feature that confirmed the suitability of the lactose induction method. Collectively, our results showed that design of experiment methodology can be applied as a suitable tool for improved production of UOX using lactose as the inducer.

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

Density of Streptococcus mutans biofilm protein induced by glucose, lactose, soy protein and iron

Background: Caries constitute an infectious disease that result from the interaction of bacteria with the host and the oral environment. Streptococcus mutans (S. mutans) represents the main bacterium that causes caries. The ability of S. mutans to form biofilms in the oral cavity is influenced by daily nutrient intake. This study of bacterial biofilm proteins can be used in the manufacture of kits for the detection of infectious diseases such as caries in the oral cavity. A biomarker is required for the manufacture of the detection kit. Consequently, research must first be conducted to determine the molecular weight and density of S. mutans biofilm proteins induced by several different daily nutrients, namely; 5% glucose, 5% lactose, soy protein and 5% iron. Purpose: This study aimed to analyse the density of S. mutans biofilm protein induced by 5% glucose, 5% lactose, soy protein, and 5% iron. Methods: The density of the S. mutans biofilm protein bands induced were measured using EZ Imager Gel DocTM software. Results: A band of biofilm protein (61.7 kDa) was obtained from S. mutans induced by 5% glucose, four bands of biofilm protein (180 kDa; 153,9 kDa; 43,9 kDa; 37,5 kDa) from 5% lactose induction and seven bands of biofilm protein (157,9 kDa; 86,6 kDa; 66,5 kDa; 50,1 kDa; 37,9 kDa; 32,3 kDa; 29,4 kDa) from soy protein induction. In contrast, S. mutans induced by 5% iron did not show any protein bands. The proteins that result from each inducer are of differing densities. Conclusion: The protein bands from each inducer are of different densities which can be used in the further test to make a biomarker for dental caries detection kits.

Intracellular β-Glucosidases CEL1a and CEL1b Are Essential for Cellulase Induction on Lactose in Trichoderma reesei

ABSTRACT Lactose (1,4- O -β- d -galacto-pyranosyl- d -glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular β-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei . We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular β-glucosidases abolished cbh1 gene expression on lactose. d -Galactose restored the efficient cellulase gene induction in the Δ cel1a strain independently of its reductive metabolism, but not in the Δ cel1a Δ cel1b strain. A further comparison of the transcriptional responses of the Δ cel1a Δ cel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the Δ cel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis β-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular β-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes.

Optimization of the Fusion Expression of Chicken β-Defensin Gal-3 in E Coli

The expression level of chicken β-defensin Gal-3 was influenced by lactose concentration, induction time and initial cell density, and IPTG induction was used as a control. The optimal parameters of various factors were confirmed. the optimal parameters with lactose induction was 37°C of culture temperature, 2 g/L of joining lactose final concentration when the recombinant strain growth density A600 achieved 0.8 and induction maintained 3 hours, the production of fusion protein reached the highest level. This study provides a good experimental basis for the large production of Gal-3.