Implications of cellobiohydrolase glycosylation for use in biomass conversion

The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina), is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline) and phosphoric acid swollen (amorphous) cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A) resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved

Undefined cellulase formulations hinder scientific reproducibility

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations

Surfactant-Free d‑Limonene Encapsulation in Spray-Dried Alginate Microcapsules Cross-Linked by In Situ Internal Gelation

Microencapsulation facilitates incorporating bioactive volatile compounds into products throughout the food, health, and cosmetics industries. To minimize the number of ingredients when microencapsulating volatile oils, we examined surfactant-free encapsulation of d-limonene in cross-linked alginate microcapsules (CLAMs) via in situ cross-linking during spray drying. Surfactant-free CLAMs (SF-CLAMs) were prepared by forming a Pickering d-limonene emulsion stabilized by calcium carbonate nanoparticles (CaCO3-NPs), combining with alginates, and then spray drying. CaCO3-NPs served as both the emulsifier and the reservoir of alginate cross-linking agent. SF-CLAMs, with a volatile retention of 73.1 ± 4.7% and total limonene content of 13.6 ± 8.6% (w/w, d.b.), exhibited core-shell morphology where CaCO3-NPs surrounded large emulsion cores (∼5 μm) encased in densely cross-linked alginate shells. Limonene was fully retained for up to 4 h in SF-CLAMs in water at 37 °C. Moreover, microencapsulation in SF-CLAMs minimized release in simulated gastric fluid (2.2 ± 0.3% in 2 h) while fully releasing in simulated gastric fluid at 37 °C

Chelator Regulation of In Situ

A recently patented one-step in situ cross-linked alginate microencapsulation (CLAM) by spray-drying (i.e., the UC Davis CLAMs technology) can overcome the high cost of scale-up that limits commercial applications. While increasing calcium loading in the CLAMs process can increase the extent of cross-linking and improve retention and protection of the encapsulated cargo, the potential for residual undissolved calcium salt crystals in the final product can be a concern for some applications. Here, we demonstrate an alternate one-step spray-dry CLAMs process using pH-responsive chelation of calcium. The "Chelate CLAMs" process is an improvement over the patented process that controls ion availability based on pH-responsive solubility of the calcium salt. Hyaluronic acid was encapsulated in CLAMs to minimize swelling and release in aqueous formulations. CLAMs with 61% (d.b.) hyaluronic acid (HA-CLAMs) demonstrated restricted plumping, limited water absorption capacity, and reduced leaching, retaining up to 49% hyaluronic acid after 2 h in water. Alternatively, "Chelate HA-CLAMs" formed by the improved process exhibited nearly full retention of hyaluronic acid over 2 h in water and remained visibly insoluble after 1 year of storage in water at 4 °C. Successful hyaluronic acid retention in CLAMs is likely due in part to its ability to cross-link with calcium

Comparative Technoeconomic Process Analysis of Industrial-Scale Microencapsulation of Bioactives in Cross-Linked Alginate

The food, chemical, and biotechnology industries offer many potential applications for calcium alginate microencapsulation, but this technique is largely confined to the laboratory bench due to scalability challenges. Scaling up the traditional external gelation method requires several costly unit operations. Alternatively, a consolidated process accomplishes alginate cross-linking in situ during spray-drying to form cross-linked alginate microcapsules (‘the CLAMs process’). This work examined the process economics of these two microencapsulation processes through technoeconomic analysis. Parallel batch process models were constructed in SuperPro Designer, initially for encapsulating emulsified fish oil. At all production scales examined, the capital investment and annual operating cost were lower for the CLAMs process. Modifying the external gelation process marginally improved the process economics, but costs remained elevated. The CLAMs process’ economic advantage stemmed from reducing the number of unit procedures, which lowered the equipment purchase cost and the dependent components of capital investment and annual operating cost. Upon modifying the models for microencapsulating hydrophilic cargo (e.g. enzymes, vitamins, microbial concentrates), the CLAMs process remained favorable at all cargo material costs and cargo loadings examined. This work demonstrates the utility of technoeconomic analysis for evaluating microencapsulation processes and may justify applying the CLAMs process at the industrial scale. </div

Chelator Regulation of In Situ Calcium Availability to Enable Spray-Dry Microencapsulation in Cross-Linked Alginates.

A recently patented one-step in situ cross-linked alginate microencapsulation (CLAM) by spray-drying (i.e., the UC Davis CLAMs technology) can overcome the high cost of scale-up that limits commercial applications. While increasing calcium loading in the CLAMs process can increase the extent of cross-linking and improve retention and protection of the encapsulated cargo, the potential for residual undissolved calcium salt crystals in the final product can be a concern for some applications. Here, we demonstrate an alternate one-step spray-dry CLAMs process using pH-responsive chelation of calcium. The "Chelate CLAMs" process is an improvement over the patented process that controls ion availability based on pH-responsive solubility of the calcium salt. Hyaluronic acid was encapsulated in CLAMs to minimize swelling and release in aqueous formulations. CLAMs with 61% (d.b.) hyaluronic acid (HA-CLAMs) demonstrated restricted plumping, limited water absorption capacity, and reduced leaching, retaining up to 49% hyaluronic acid after 2 h in water. Alternatively, "Chelate HA-CLAMs" formed by the improved process exhibited nearly full retention of hyaluronic acid over 2 h in water and remained visibly insoluble after 1 year of storage in water at 4 °C. Successful hyaluronic acid retention in CLAMs is likely due in part to its ability to cross-link with calcium

Industrially-Scalable Microencapsulation of Plant Beneficial Bacteria in Dry Cross-Linked Alginate Matrix.

Microencapsulation of plant-beneficial bacteria, such as pink pigmented facultative methylotrophs (PPFM), may greatly extend the shelf life of these Gram-negative microorganisms and facilitate their application to crops for sustainable agriculture. A species of PPFM designated Methylobacterium radiotolerans was microencapsulated in cross-linked alginate microcapsules (CLAMs) prepared by an innovative and industrially scalable process that achieves polymer cross-linking during spray-drying. PPFM survived the spray-drying microencapsulation process with no significant loss in viable population, and the initial population of PPFM in CLAMs exceeded 1010 CFU/g powder. The PPFM population in CLAMs gradually declined by 4 to 5 log CFU/g over one year of storage. The extent of alginate cross-linking, modulated by adjusting the calcium phosphate content in the spray-dryer feed, did not influence cell viability after spray-drying, viability over storage, or dry particle size. However, particle size measurements and light microscopy of aqueous CLAMs suggest that enhanced crosslinking may limit the release of encapsulated bacteria. This work demonstrates an industrially scalable method for producing alginate-based inoculants that may be suitable for on-seed or foliar spray applications