Moving adsorption belt system for continuous bioproduct recovery utilizing composite fibrous adsorbents.

A continuous protein recovery and purification system based on the true moving bed concept is presented. A novel adsorbent material, in the form of an elastic and robust woven fabric, served as a moving belt following the general designs observed in known belt conveyors. The composite fibrous material that forms the said woven fabric showed high protein binding capacity, reaching a static binding capacity equal to 107.3 mg/g, as determined via isotherm experiments. Moreover, testing the same cation exchange fibrous material in a packed bed format resulted in excellent dynamic binding capacity values (54.5 mg/g) even when operating at high flow rates (480 cm/h). In a subsequent step, a benchtop prototype was designed, constructed, and tested. Results indicated that the moving belt system could recover a model protein (hen egg white lysozyme) with a productivity up to 0.5 mg/cm2/h. Likewise, a monoclonal antibody was directly recovered from unclarified CHO_K1 cell line culture with high purity, as judged by SDS-PAGE, high purification factor (5.8), and in a single step, confirming the suitability and selectivity of the purification procedure.

Production of a novel milk-clotting enzyme from solid-substrate Mucor spp. culture.

Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk-clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk-clotting activity under a solid-state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal-sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost-effective production, milk-clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese-making industry. PRACTICAL APPLICATION: The milk-clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers.

Megaporous monolithic adsorbents for bioproduct recovery as prepared on the basis of nonwoven fabrics.

Megaporous adsorbents were prepared based on nonwoven polyethylene terephthalate (PET) fabrics and functionalized by covalent modification with polyvinylamine (PVAm) or monotriazinyl-ß-cyclodextrin-substituted polyvinylamine (PVAm-MCT-ß-CD). Mechanical properties of the resulting fabrics were maintained, as judged by tensile strength tests and scanning electron microscopy. Exceptional porosity (≥82%) and preserved hydrodynamic characteristics (Pe ≥ 63) indicated excellent structural stability when packed. The performance of the constructed adsorbents was evaluated with high molecular weight (proteins) and low molecular weight (dyes) model compounds. The static binding capacity (SBC) for bovine serum albumin (BSA) was 79.7 ± 1.3 and 92.9 ± 8.2 mg/g for PVAm-modified and PVAm-MCT-ß-CD-modified fabrics, respectively. The mentioned materials also adsorbed Orange II, an acidic dye (92.4 ± 2.6 and 101.9 ± 2.6 mg/g, respectively), indicating that the hydrophobicity was a prevailing binding mechanism operating at a pH close to isoelectric point. SBC for lysozyme and toluidine blue O (TBO, a basic dye) onto PVAm-MCT-ß-CD functionalized PET was 52.7 ± 5.1 and 73.3 ± 0.6 mg/g, respectively. TBOs have also shown some affinity for PVAm functionalized PET, but this was most likely to be mediated by hydrophobicity. On the other hand, operating at a superficial velocity of 90 cm/h, dynamic binding capacity for BSA was 11.4 ± 3.5 and 2.5 ± 0.6 mg/g indicating the importance of possible aggregation mechanisms during protein binding at equilibrium. Thus, PET-based adsorbents require further functional improvement for chromatography applications. However, the easy-to-construct, scalable nonwoven adsorbents deserve further attention as a potential alternative to packed-bed-chromatography adsorbents.

Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation.

This study reports the optimization of milk-clotting protease production from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) in both one-variable-at-a-time and response surface methodology (RSM). The production and optimization of milk-clotting protease obtained from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) using different agro-industrial wastes as solid substrates were studied. The agro-industrial wastes used included wheat bran, rice bran, pea bran, and grass pea bran. The chemical composition of the best solid substrate was tested using standard methods. Others cultivation parameters were studied, and the results showed that the optimum fermentation medium composed of wheat bran, casein (1% w/w), and glucose (0.5% w/w) and the conditions for maximum milk-clotting protease production were at the moisture content of 55.0%, inoculum of 0.5*106 spores/mL, incubation temperature of 30 °C, pH of 6.0, and fermentation time of 5 days. The highest milk-clotting activity was obtained from the crude enzyme extracted using 0.1 M NaCl and partial purification of the crude enzyme using chilled acetone, and 80% (NH4)2SO4 increased the ratio of MCA/PA from 0.56 to 1.30 and 0.65, respectively. Moreover, the highest MCA (137.58 U/mL) was obtained at a casein concentration of 0.5%, pH 4.0, and 25 °C, using RSM. Thus, results from the present study showed that the optimization of milk-clotting protease production from A. oryzae DRDFS 13 under SSF by both one-variable-at-a-time and RSM significantly increased the milk-clotting activity. This is the first report from a fungus in the Ethiopian setting and a modest contribution to highlight the potential of harnessing microbial protease enzymes for industrial applications.

Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability.

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.