A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications

Cassimjee, Karim Engelmark; Kadow, Maria; Wikmark, Ylva; Humble, Maria Svedendahl; Rothstein, M. L.; Rothstein, David M.; Bäckvall, Jan‐E.

doi:10.1039/c4cc02605e

Cited by 69 publications

(63 citation statements)

References 34 publications

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“…(56% e.e., entry 7), the best results were obtained with EziG3, a carrier with a semi‐hydrophilic surface (Table , entry 10 Figure S8). The carrier EziG, based on glass beads, was used to overcome the compressibility of the alginate beads which may cause problems when used in flow. The carrier links the enzyme via Fe 2+ ions at the surface which possess according to the supplier enhanced binding strength toward the His‐tag.…”

Section: Resultsmentioning

confidence: 99%

Library of Norcoclaurine Synthases and Their Immobilization for Biocatalytic Transformations

Lechner

Soriano

Poschner

et al. 2017

Biotechnology Journal

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Norcoclaurine synthases (NCS), catalyzing a Pictet-Spengler reaction in plants as one of the first enzymes in the biosynthetic benzylisoquinoline pathway, are investigated for biocatalytic transformations. The library of NCS available is extended by two novel NCSs from Argemone mexicana (AmNCS1, AmNCS2) and one new NCS from Corydalis saxicola (CsNCS); furthermore, it is shown that the NCS from Papaver bracteatum (PbNCS) is a highly productive catalyst leading to the isoquinoline product with up to >99% e.e. Under certain conditions lyophilized whole Escherichia coli cells containing the various overexpressed NCS turned out to be suitable catalysts. The reaction using dopamine as substrate bears several challenges such as the spontaneous nonstereoselective background reaction and side reactions. The PbNCS enzyme is successfully immobilized on various carriers whereby EziG3 proved to be the best suited for biotransformations. Dopamine showed limited stability in solution resulting in the coating of the catalyst over time, which could be solved by the addition of ascorbic acid (e.g., 1 mg ml À1 ) as antioxidant.

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Section: Resultsmentioning

confidence: 99%

Library of Norcoclaurine Synthases and Their Immobilization for Biocatalytic Transformations

Lechner

Soriano

Poschner

et al. 2017

Biotechnology Journal

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“…5MP functionalized solid supports were prepared by treating commercially available long chain aminoalkyl controlled porosity glass (LCAA-CPG) 37 with freshly prepared 1′ at pH 7.5 for 4 h. Quantitation of free amines on the support before and after the reaction using 4-chloro-7-nitrobenzofurazan 38 revealed that 5MP functionalization proceeded essentially to completion. Mixing ilvN with 5MP-CPG resulted in quantitative protein immobilization in less than 10 min (Figure S49).…”

Section: Resultsmentioning

confidence: 99%

Thiol Specific and Tracelessly Removable Bioconjugation via Michael Addition to 5-Methylene Pyrrolones

et al. 2017

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5-Methylene pyrrolones (5MPs) are highly thiol-specific and tracelessly removable bioconjugation tools. 5MPs are readily prepared from primary amines in one step. 5MPs exhibit significantly improved stability under physiologically relevant conditions and cysteine specificity compared to commonly used analogues, maleimides. Michael addition of thiol to 5MPs occurs rapidly, cleanly and does not generate a stereocenter. The conjugates efficiently release thiols via retro-Michael reaction in alkaline buffer (pH 9.5) or via thiol exchange at pH 7.5. This unique property makes 5MPs valuable for the controlled release of conjugated cargo and temporary thiol protection. The utilization of 5MPs for protein immobilization and pull-down of active complexes is illustrated using E. coli. acetohydroxyacid synthase isozyme I.

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“…Immobilization on EziG and co‐lyophilization with surfactants : Immobilization of SC on hydrophobic EziG 2 material was made by using sterile, filtered (0.45 and 0.2 μm) cell culture supernatant to obtain simultaneous protein purification and immobilization . The EziG 2 material was added in fourfold excess relative to the amount of protein in solution.…”

Section: Methodsmentioning

confidence: 99%

“…Immobilization of SC on hydrophobic EziG 2m aterial was made by using sterile, filtered (0.45 and 0.2 mm) cell culture supernatant to obtain simultaneous protein purification and immobilization. [64] The EziG 2m aterial was added in fourfold excess relative to the amount of protein in solution. For SC wild type, approximately 300 mg EziG 2p er liter cell culture supernatant was applied.…”

Section: Immobilization On Ezig and Co-lyophilization With Surfactantsmentioning

confidence: 99%

Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

2017

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Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36 %), enantioselectivity (E values up to 400), substrate scope, and stability in THF.

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A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications

Abstract: PostprintThis is the accepted version of a paper published in Chemical Communications. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination.

Cited by 69 publications

References 34 publications

Library of Norcoclaurine Synthases and Their Immobilization for Biocatalytic Transformations

Library of Norcoclaurine Synthases and Their Immobilization for Biocatalytic Transformations

Thiol Specific and Tracelessly Removable Bioconjugation via Michael Addition to 5-Methylene Pyrrolones

Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

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