Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases

Lončar, Nikola; Slavić, Marinela Šokarda; Vujčić, Zoran; Božić, Nataša

doi:10.1038/srep15772

Cited by 6 publications

(6 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…On the other hand even RSDA that contains SBD are not always fully recovered from raw starch granule when raw starch was used as affinity support [59]. Recently, by using mixed mode Nuvia™ cPrime™ resin with the idea to exploit the hydrophobic patches on RSDA surface, we were able to simultaneously concentrate, remove pigment and to purify RSDA with yields of 96% directly from the fermentation broth in a single step [56]. This method may be useful in the purification of recombinant proteins without tags since there are many cases where the N-or C-terminus of a protein is not exposed to solvent and thus the addition of tags is unfeasible.…”

Section: Recent Advances In Rsda Upstream and Downstream Processingmentioning

confidence: 95%

See 1 more Smart Citation

Raw starch degrading α-amylases: an unsolved riddle

Božić

Lončar

Slavić

et al. 2017

Amylase

Self Cite

View full text Add to dashboard Cite

Starch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

show abstract

Section: Recent Advances In Rsda Upstream and Downstream Processingmentioning

confidence: 95%

“…Total of 0.7 g/L of enzyme was obtained this way [55]. Furthermore, by simple change of expression host to E. coli C43 (DE3), total amylase reached 1.2 g/L [56].…”

Section: Recent Advances In Rsda Upstream and Downstream Processingmentioning

confidence: 99%

Raw starch degrading α-amylases: an unsolved riddle

Božić

Lončar

Slavić

et al. 2017

Amylase

Self Cite

View full text Add to dashboard Cite

show abstract

“…The untagged wild-type BliAmy protein (GenBank accession number JN042159) was overexpressed and purified as previously described [23,29]. Purity (>95%) was checked by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE).…”

Section: Protein Preparation For Crystallographymentioning

confidence: 99%

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

Božić

Rozeboom

Lončar³

et al. 2020

International Journal of Biological Macromolecules

Self Cite

View full text Add to dashboard Cite

Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

show abstract

“…7,8 To address these hurdles, alternative methods including aqueous twophase systems and mixed-mode chromatography resins have been investigated for recombinant proteins. 9,10 However, the scalability of liquid-liquid extraction and the additional complexity and time required for developing unit operations with multimodal adsorbents compared with single-mode resins remain as challenges. Therefore, there is a need for additional purification tools beyond those commonly used across the industry.…”

Section: Introductionmentioning

confidence: 99%

“…These impurities often associate with the molecule and co‐purify, presenting challenges in achieving the desired final drug substance product quality 7,8 . To address these hurdles, alternative methods including aqueous two‐phase systems and mixed‐mode chromatography resins have been investigated for recombinant proteins 9,10 . However, the scalability of liquid–liquid extraction and the additional complexity and time required for developing unit operations with multimodal adsorbents compared with single‐mode resins remain as challenges.…”

Section: Introductionmentioning

confidence: 99%

Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process

Slocum

Santora

et al. 2021

Biotechnol Progress

View full text Add to dashboard Cite

An increasing number of non-mAb recombinant proteins are being developed today.These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new.As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log 10 reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.

show abstract

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases

Cited by 6 publications

References 27 publications

Raw starch degrading α-amylases: an unsolved riddle

Raw starch degrading α-amylases: an unsolved riddle

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process

Contact Info

Product

Resources

About