BackgroundExtracellular aspartic proteinase (MCAP) produced by Mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. Despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. This work focuses on MCAP cloning and optimization of heterologous expression in Pichia pastoris, and characterization of the enzyme.ResultsThe cloning of cDNA sequence encoding MCAP from M. circinelloides was performed using a fragment of approximately 1 kbp as a probe. The fragment was amplified using non-specific primers designed from the NDIEYYG and KNNYVVFN consensus motifs from aspartic proteinases of different fungi. Gene specific primers were designed to amplify a full-length cDNA using SMART™ RACE PCR. MCAP was expressed in P. pastoris under the control of the constitutive GAP promoter. It was shown that P. pastoris secreted non-glycosylated and glycosylated MCAPs with molecular weights of 33 and 37 kDa, respectively.ConclusionA novel MCAP was expressed in P. pastoris and efficiently secreted into the culture medium. The expression of the heterologous proteins was significantly increased due to advantages in codon usage as compared to other expression systems. The results suggest that P. pastoris could be exploited as a safe production platform for the milk clotting enzyme.
The major platform for high level recombinant protein production is based on genetically modified microorganisms like Escherichia coli (E. coli) due to its short dividing time, ability to use inexpensive substrates and additionally, its genetics is comparatively simple, well characterized and can be manipulated easily. Here, we investigated the possibilities of finding the best media for high cell density fermentation, by analyzing different media samples, focusing on improving fermentation techniques and recombinant protein production. Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria–Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively). However, in fed-batch fermentation processes when using MMM after 25 h cultivation, it was possible to yield an optical density (OD600) of 139 corresponding to 172 g/L of wet biomass was produced in a 30 L TV Techfors-S Infors HT fermenter, with a computer controlled nutrient supply (glucose as a carbon source) delivery system, indicating nearly 1.5 times that obtained from TB. Upon purification, a total of 1.65 mg/g of protein per gram cell biomass was obtained and the purified AviPure showed affinity for immunoglobulin. High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition. It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0155-y) contains supplementary material, which is available to authorized users.
Methylotrophic yeasts have widely been used as model organisms for understanding cellular functions and biochemical activities in lower eukaryotes. The gene encoding an aspartic protease (MCAP) from Mucor circinelloides DSM 2183 was cloned and expressed into Pichia pastoris using both the native M. circinelloides signal peptide (mcSP) and α-factor secretion signal from Saccharomyces cerevisiae (α-MF). When expressed in P. pastoris using α-MF and mcSP, MCAP was secreted into the culture medium at a concentration 200 mg L−1 (410 MCU mL−1) and 110 mg L−1 (249 MCU mL−1), respectively. The SDS-PAGE analysis of each culture shows that the protein was secreted in the media in two forms with molecular weights of approximately 33 and 37 kDa. Upon digestion using endoglycosidase H (Endo H), only one band at 33 kDa was observed, indicating that the protein might be glycosylated. One putative N-glycosylation site was found and a site-directed mutagenesis at position Asn331-Gln of the sequence produce only one form of the protein of 33 kDa, similar to that obtained when digested with Endo H. The optimum temperature and pH activity of the expressed MCAP was found to be at 60 °C and 3.6, respectively.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0691-3) contains supplementary material, which is available to authorized users.
The influence of internal mass transfer on productivity as well as the performance of packed bed bioreactor was determined by varying a number of parameters; chitosan coating, flow rate, glucose concentration and particle size. Saccharomyces cerevisiae cells were immobilized in chitosan and non-chitosan coated alginate beads to demonstrate the effect on particle side mass transfer on substrate consumption time, lag phase and ethanol production. The results indicate that chitosan coating, beads size, glucose concentration and flow rate have a significant effect on lag phase duration. The duration of lag phase for different size of beads (0.8, 2 and 4 mm) decreases by increasing flow rate and by decreasing the size of beads. Moreover, longer lag phase were found at higher glucose medium concentration and also with chitosan coated beads. It was observed that by increasing flow rates; lag phase and glucose consumption time decreased. The reason is due to the reduction of external (fluid side) mass transfer as a result of increase in flow rate as glucose is easily transported to the surface of the beads. Varying the size of beads is an additional factor: as it reduces the internal (particle side) mass transfer by reducing the size of beads. The reason behind this is the distance for reactants to reach active site of catalyst (cells) and the thickness of fluid created layer around alginate beads is reduced. The optimum combination of parameters consisting of smaller beads size (0.8 mm), higher flow rate of 90 ml/min and glucose concentration of 10 g/l were found to be the maximum condition for ethanol production.
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