Search Results (9)

Unspecific peroxygenases (UPOs) are promising biocatalysts for oxyfunctionalizations in future sustainable economies and can be efficiently immobilized on the cell surface of their heterologous production yeast. This immobilization has versatile uses, ranging from the mL to m³ scale; but the production of the yeast surface displayed UPOs, and their handling has yet to be optimized to advance sustainable industrial processes in light of the UN’s sustainable development goals. Here, we present optimized production protocols for surface-displayed UPOs for shaken and stirred systems in different scales and describe suitable storage conditions and a sterilization method. We utilized one-factor-at-a-time and design of experiments approaches. We were able to streamline published protocols for shaken flask cultivations to achieve a 60% increase in volumetric activity, using reduced amounts of media. We also show at least a doubling of final activity for bioreactor cultivations by utilizing a different medium than the industry standard. Finally, we present a novel, robust protocol for parallelized methanol-induced enzyme production in Komagataella phaffii in a BioLector XT^® reactor. Enzyme activity did not decrease and even increased by our recommended sterilization method and during storage over 87 days. This study aims to advance the yeast surface display immobilization method by providing methods for efficient production, storage and utilization of this promising biocatalyst. Full article

Cellobionic acid (CBA) can be obtained through the oxidation of cellobiose, the monomer of cellulose. CBA serves as a plant-based alternative to its stereoisomer lactobionic acid, which is used in the pharmaceutical, cosmetic, and food industries. Gluconobacter oxydans is a well-established whole-cell biocatalyst with membrane-bound dehydrogenases (mDH) for regio-specific oxidations. As G. oxydans wildtype cells show low cellobiose oxidation activities, the glucose mDH from Pseudomonas taetrolens was overexpressed in G. oxydans BP9, a multi mDH deletion strain. Whole-cell biotransformation studies were performed with resting cells of the engineered G. oxydans in stirred tank bioreactors. Initial biomass specific cellobionate formation rates increased with increasing cellobiose concentrations up to 190 g L⁻¹, and were constant until the solubility limit. The maximal volumetric CBA formation rates and the oxygen uptake rates increased linearly with the concentration of engineered G. oxydans. This enables the estimation of the maximum biocatalyst concentration limited by the maximum oxygen transfer rate of any bioreactor. Thus, 5.2 g L⁻¹ G. oxydans was sufficient to produce 502 g L⁻¹ CBA with >99% yield in a simple aerobic batch process. The highly concentrated CBA will reduce downstream processing costs considerably after cell separation. Full article

Phenylacetylcarbinol (PAC) is a precursor for the synthesis of several pharmaceuticals, including ephedrine, pseudoephedrine, and norephedrine. PAC is commonly produced through biotransformation using microbial pyruvate decarboxylase (PDC) in the form of frozen–thawed whole cells. However, the lack of microorganisms capable of high PDC activity is the main factor in the production of PAC. In addition, researchers are also looking for ways to utilize agro-industrial residues as an inexpensive carbon source through an integrated biorefinery approach in which sugars can be utilized for bioethanol production and frozen–thawed whole cells for PAC synthesis. In the present study, Candida tropicalis, Saccharomyces cerevisiae, and the co-culture of both strains were compared for their biomass and ethanol concentrations, as well as for their volumetric and specific PDC activities when cultivated in a sugarcane bagasse (SCB) hydrolysate medium (SCBHM). The co-culture that resulted in a higher level of PAC (8.65 ± 0.08 mM) with 26.4 ± 0.9 g L⁻¹ ethanol production was chosen for further experiments. Biomass production was scaled up to 100 L and the kinetic parameters were studied. The biomass harvested from the bioreactor was utilized as frozen–thawed whole cells for the selection of an initial pyruvate (Pyr)-to-benzaldehyde (Bz) concentration ([Pyr]/[Bz]) ratio suitable for the PAC biotransformation in a single-phase emulsion system. The initial [Pyr]/[Bz] at 100/120 mM resulted in higher PAC levels with 10.5 ± 0.2 mM when compared to 200/240 mM (8.60 ± 0.01 mM). A subsequent two-phase emulsion system with Pyr in the aqueous phase, Bz in the organic phase, and frozen–thawed whole cells of the co-culture as the biocatalyst produced a 1.46-fold higher PAC level when compared to a single-phase emulsion system. In addition, the cost analysis strategy indicated preliminary costs of USD 0.82 and 1.01/kg PAC for the single-phase and two-phase emulsion systems, respectively. The results of the present study suggested that the co-culture of C. tropicalis and S. cerevisiae can effectively produce bioethanol and PAC from SCB and would decrease the overall production cost on an industrial scale utilizing the two-phase emulsion system with the proposed multiple-pass strategy. Full article

Because of their high volumetric catalytic activity, in addition to their high chemical and thermal resistances, enzymes in the form of protein crystals are an excellent choice for application as immobilized biocatalysts. However, mechanical stability is a requirement for the processability of immobilisates, in addition to the protein crystals retaining their enzymatic activity, and this is closely related to the crystal structure. In this study, the influence of protein engineering on the mechanical stability of cross-linked enzyme crystals (CLECs) was investigated using a genetically modified model protein in which additionally cysteines were introduced on the protein surface for targeted cross-linking. The results showed that the mechanical stability of crystals of the mutant proteins in the native form was decreased compared to native wild-type crystals. However, specific cross-linking of the introduced amino acid residues in the mutant proteins resulted in their increased mechanical stability compared to wild-type CLECs. In order to determine the correlation between the crystal structure and the resulting mechanical properties of CLECs to enable targeted cross-linking, a previously developed model was revised and then used for the two model proteins. This model can explain the mechanically investigated relationships, such as the anisotropic crystal behavior and the influence of a linker or mutation on the micromechanical properties and, hence, can be helpful for the tailor-made production of CLECs. Full article

Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P_GAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (P_AOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel). Full article

Recombinant Rhizopus oryzae lipase (mature sequence, rROL) was modified by adding to its N-terminal 28 additional amino acids from the C-terminal of the prosequence (proROL) to obtain a biocatalyst more suitable for the biodiesel industry. Both enzymes were expressed in Pichia pastoris and compared in terms of production bioprocess parameters, biochemical properties, and stability. Growth kinetics, production, and yields were better for proROL harboring strain than rROL one in batch cultures. When different fed-batch strategies were applied, lipase production and volumetric productivity of proROL-strain were always higher (5.4 and 4.4-fold, respectively) in the best case. rROL and proROL enzymatic activity was dependent on ionic strength and peaked in 200 mM Tris-HCl buffer. The optimum temperature and pH for rROL were influenced by ionic strength, but those for proROL were not. The presence of these amino acids altered lipase substrate specificity and increased proROL stability when different temperature, pH, and methanol/ethanol concentrations were employed. The 28 amino acids were found to be preferably removed by proteases, leading to the transformation of proROL into rROL. Nevertheless, the truncated prosequence enhanced Rhizopus oryzae lipase heterologous production and stability, making it more appropriate as industrial biocatalyst. Full article

A new strategy to increase the enzyme-loading capacity of porous supports was investigated. Lipase from Pseudomonas fluorescens (PFL) was immobilized on octyl-agarose (OA) beads and treated with polyethyleneimine (PEI). Then, PFL was immobilized on the previous PFL layer. Next, the biocatalyst was coated with PEI and a third layer of PFL was added. Sodium dodecyl sulfate polyacrylamide electrophoresis showed that the amount of PFL proportionally increased with each enzyme layer; however, the effects on biocatalyst activity were not as clear. Hydrolyzing 50 mM of triacetin at 25 °C, the activity of the three-layer biocatalyst was even lower than that of the bi-layer one; on the contrary its activity was higher when the activity was measured at 4 °C in the presence of 30% acetonitrile (that reduced the activity and thus the relevance of the substrate diffusion limitations). That is, the advantage of the multilayer formation depends on the specific activity of the enzyme and on the diffusion limitations of the substrate. When octyl agarose (OA)-PFL-PEI-PFL preparation was treated with glutaraldehyde, the activity was reduced, although the enzyme stability increased and the immobilization of the last PFL layer offered results similar to the one obtained using the three-layer preparation without glutaraldehyde modification (90%). Full article

The preparation of highly porous magnetic crosslinked aggregates (pm-CLEA) of porcine pancreas lipase (PPL) is reported. Some strategies to improve the volumetric activity of the immobilized biocatalyst were evaluated, such as treatment of PPL with enzyme surface-modifying agents (polyethyleneimine or dodecyl aldehyde), co-aggregation with protein co-feeders (bovine serum albumin and/or soy protein), use of silica magnetic nanoparticles functionalized with amino groups (SMNPs) as separation aid, and starch as pore-making agent. The combination of enzyme surface modification with dodecyl aldehyde, co-aggregation with SMNPs and soy protein, in the presence of 0.8% starch (followed by hydrolysis of the starch with α-amylase), yielded CLEAs expressing high activity (immobilization yield around 100% and recovered activity around 80%), high effectiveness factor (approximately 65% of the equivalent free enzyme activity) and high stability at 40 °C and pH 8.0, i.e., PPL CLEAs co-aggregated with SMNPs/bovine serum albumin or SMNPs/soy protein retained 80% and 50% activity after 10 h incubation, respectively, while free PPL was fully inactivated after 2 h. Besides, highly porous magnetic CLEAs co-aggregated with soy protein and magnetic nanoparticles (pm-SP-CLEAs) showed good performance and reusability in the hydrolysis of tributyrin for five 4h-batches. Full article

The preparation of Cross-Linked Enzyme Aggregates (CLEAs) is a simple and cost-effective technique capable of generating insoluble biocatalysts with high volumetric activity and improved stability. The standard CLEA preparation consists of the aggregation of the enzyme and its further crosslinking, usually with glutaraldehyde. However, some enzymes have too low a content of surface lysine groups to permit effective crosslinking with glutaraldehyde, requiring co-aggregation with feeders rich in amino groups to aid the formation of CLEAs. The co-aggregation with magnetic particles makes their handling easier. In this work, CLEAs of a commercial amyloglucosidase (AMG) produced by Aspergillus niger were prepared by co-aggregation in the presence of polyethyleneimine (PEI) or starch with aminated magnetic nanoparticles (MNPs) or bovine serum albumin (BSA). First, CLEAs were prepared only with MNPs at different glutaraldehyde concentrations, yielding a recovered activity of around 20%. The addition of starch during the precipitation and crosslinking steps nearly doubled the recovered activity. Similar recovered activity (around 40%) was achieved when changing starch by PEI. Moreover, under the same conditions, AMG co-aggregated with BSA was also synthesized, yielding CLEAs with very similar recovered activity. Both CLEAs (co-aggregated with MNPs or BSA) were four times more stable than the soluble enzyme. These CLEAs were evaluated in the hydrolysis of starch at typical industrial conditions, achieving more than 95% starch-to-glucose conversion, measured as Dextrose Equivalent (DE). Moreover, both CLEAS could be reused for five cycles, maintaining a DE of around 90%. Although both CLEAs had good properties, magnetic CLEAs could be more attractive for industrial purposes because of their easy separation by an external magnetic field, avoiding the formation of clusters during the filtration or centrifugation recovery methods usually used. Full article