China
Africa
Explore chapters and articles related to this topic
Biofilms in Fermentation for the Production of Value-Added Products
Published in Bakrudeen Ali Ahmed Abdul, Microbial Biofilms, 2020
Ercan and Demirci (2013) constructed PCS-grid biofilm reactors with Kluyveromyces lactis to produce human lysozyme. Lysozyme is an antimicrobial agent heavily used in cheese, wine, cosmetic, and pharmaceutical industries, and human lysozyme is superior to its conventional counterpart from chicken eggs for people allergic to egg products. For this purpose, the researchers screened through four different PCS types with test tube fermentations and selected SFYB for the biofilm reactors. Then, the growth conditions were optimized by RSM for maximum lysozyme production in repeated batch fermentations. Under these conditions of 25˚C, pH 4, and no aeration, highest lysozyme concentrations of 141 U/mL was reached within 72 h of fermentation, which were higher than the levels observed in suspended-cell bioreactors without the presence of biofilm formation (Ercan and Demirci, 2013). In the next step of the study, the researchers used RSM again to optimize components of the synthetic medium used in the biofilm reactors to further improve lysozyme production. The optimum medium was composed of 16.3% lactose, 1.2% casamino acid, and 0.8% yeast nitrogen base, improving lysozyme concentrations to 173 U/mL which were 57% higher than the 110.3 U/mL of lysozyme in suspended-cell bioreactors (Ercan and Demirci, 2014). Then, the researchers investigated fed-batch and continuous fermentation modes in these biofilm reactors with optimum conditions. In fed-batch fermentation with glucose as the initial carbon source, lactose was introduced at constant 0.6 mL/min for 10 h leading to significantly higher lysozyme levels (187 U/mL). By switching to continuous mode, biofilm reactors were able to provide significant higher productivity (7.5 U/mL/h) compared to the maximum productivity in suspended-cell bioreactor (4 U/mL/h), perhaps due to higher cell density at higher dilution rates in biofilm reactors (Ercan and Demirci, 2015b). Finally, the researchers were able to couple the biofilm reactors with an online recovery system using silicic acid as absorbent of the produced lysozyme. The study determined 25˚C, pH 4, and 25% silicic acid: fermentation broth volume ratio for adsorption while 25˚C, pH 6.2, and 5% (wt/v) sodium dodecyl sulfate with 1M NaCl and 20% (v/v) ethanol as eluent were best desorption parameters, leading to 95.6% lysozyme adsorption and 98% desorption. Using the online recovery system, 280.4 U/mL of lysozyme was produced, which was 63% higher than the conditions without recovery (Ercan and Demirci, 2015c).
Heterologous expression of azurin from Pseudomonas aeruginosa in the yeast Pichia pastoris
Published in Preparative Biochemistry & Biotechnology, 2021
Yagmur Unver, Busra Sensoy Gun, Melek Acar, Seyda Yildiz
In this study, intracellular recombinant protein expression, as well as the extracellular expression was achieved through S. cerevisiae α-factor signal sequence. There are two ways for the expression of a foreign protein in P. pastoris, intracellular and extracellular.[53] Although the product of extracellular expression is easy to purify, the yield is usually lower.[54] Extracellular secretion of recombinant protein in high levels into culture media can also be achieved by P. pastoris which secretes endogenous proteins at a minimal level.[27] But, proteolytic degradation of recombinant proteins has known as a permanent problem when yeasts, such as P. pastoris, are used in the expression of these proteins.[55,56] There have been several suggestions like the use of protease-deficient strains or the addition of protease inhibitors or casamino acids to prevent the recombinant protein degradation.[57] Especially, it has been reported that reduction in proteolytic activity in culture media can be provided with casamino acid supplementation and it is also nitrogen and alternative carbon source for cell growth.[27,58] Therefore, avoidance of proteolytic degradation of recombinant azurin was achieved with the addition of 1% (w/v) casamino acid to the secretion media. On the other hand, the yield of the product of intracellular expression can increase because of avoiding the hydrolysis of the metabolic product of the cell.[54]