Enzyme behavior at surfaces. Site-specific variants of subtilisin BPN' with enhanced surface stability.

Brode, P. F.; Erwin, Christopher R.; Rauch, Deborah S.; Lucas, Donald S.; Rubingh, Donn N.

doi:10.1016/s0021-9258(17)31549-1

Cited by 21 publications

(21 citation statements)

References 21 publications

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“…Mutagenesis. Site-directed mutagenesis was done as previously described (Brode et al, 1994). For some of the mutants, a modified pSS-5 with a frame-shift stop codon mutation at amino acid 217 was used to produce the uracil template.…”

Section: Enzymesmentioning

confidence: 99%

“…Fermentation and Purification. Fermentation was done using a Biostat ED fermenter (B. Braun Biotech, Inc., Allentown, PA), and purification to g95% activity was done as previously described (Brode et al, 1994).…”

Section: Enzymesmentioning

confidence: 99%

“…Three columns of wells contained the wild-type BPN′ in known amounts for standard curve generation, and three columns contained a variant with a known conversion factor. Conversion factors for these standards had been determined previously (Brode et al, 1994) via batch and column active site titration methods. The new variants were placed in the remaining columns of the plates.…”

Section: Enzymesmentioning

confidence: 99%

“…Although these studies were done on very different substrates, some common notable features were found which support the importance of the surface concentration of enzyme in controlling the rate of catalyzed hydrolysis of surface-bound substrates. In a related study, the stability of subtilisin BPN′ on hydrophilic and hydrophobic surfaces was shown to be increased by designing variants that adsorb at a lower level on these surfaces (Brode et al, 1994). Recent work on a closely related alkaline protease describes the engineering of surface charge to change adsorption properties (Egmond et al, 1994;Aehle et al, 1993).…”

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confidence: 99%

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Subtilisin BPN‘ Variants: Increased Hydrolytic Activity on Surface-Bound Substrates via Decreased Surface Activity

et al. 1996

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Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties. Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface. While most variants adsorb at a level very similar to that of native BPN', several variants were identified which adsorb either more or less. For surface-bound substrates we report a linear dependence between the concentration of adsorbed protease enzyme and substrate hydrolysis, similar to the linear dependence between enzyme solution concentration and hydrolysis of soluble substrates. On the basis of this knowledge we hypothesized that variants designed to adsorb at a higher level on a surface-bound peptide substrate would hydrolyze that surface-bound substrate faster. Contrary to our original expectations, the variants that adsorb more on the covalently bound peptide surface hydrolyze this substrate slower. In addition, variants of BPN' which adsorb at a lower level than native BPN' hydrolyze the surface-bound substrate faster. Enzyme adsorption and the subsequent peptide hydrolysis are altered by substituting amino acids that modify the surface charge or hydrophobicity of the native enzyme. This effect is most dramatic when the changes were made at surface-exposed sites around the binding pocket/active site of the enzyme. One mechanism that is consistent with the data is based on the relationship between the level of adsorption and the enzyme's affinity for the surface. In this mechanism weakly adsorbed enzymes are postulated to move more rapidly from site to site on the surface, thereby increasing substrate hydrolysis.

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Section: Enzymesmentioning

confidence: 99%

Section: Enzymesmentioning

confidence: 99%

Section: Enzymesmentioning

confidence: 99%

mentioning

confidence: 99%

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Subtilisin BPN‘ Variants: Increased Hydrolytic Activity on Surface-Bound Substrates via Decreased Surface Activity

et al. 1996

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“…The observed catalytic efficiency of many enzymes are dependent on other extraneous contributions, such as surfaceinduced deactivation. 28 This effect can be significant when working with enzymes at very low concentrations, as is necessary to collect the linear initial rate for highly efficient enzymes such as arPTE. Experimentally, these adsorption effects can be prevented with the addition of bovine serum albumin (BSA) to block nonspecific protein binding.…”

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confidence: 99%

Sequential Electrostatic Assembly of a Polymer Surfactant Corona Increases Activity of the Phosphotriesterase arPTE

et al. 2019

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We present a new methodology for the generation of discrete molecularly dispersed enzyme− polymer−surfactant bioconjugates. Significantly, we demonstrate that >3-fold increase in the catalytic efficiency of the diffusion-limited phosphotriesterase arPTE can be achieved through sequential electrostatic addition of cationic and anionic polymer surfactants, respectively. Here, the polymer surfactants assemble on the surface of the enzyme via ion exchange to yield a compact corona. The observed rate enhancement is consistent with a mechanism whereby the polymer−surfactant corona gives rise to a decrease in the dielectric constant in the vicinity of the active site of the enzyme, accelerating the rate-determining product diffusion step. The facile methodology has significant potential for increasing the efficiency of enzymes and could therefore have a substantially positive impact for industrial enzymology.

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Tailoring Particle‐Enzyme Nanoconjugates for Biocatalysis at the Organic‐Organic Interface

et al. 2020

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Nonaqueous Pickering emulsions (PEs) are a powerful platform for catalysis design, offering both a large interface contact and a preferable environment for water‐sensitive synthesis. However, up to now, little progress has been made to incorporate insoluble enzymes into the nonaqueous system for biotransformation. Herein, we present biocatalytically active nonaqueous PEs, stabilized by particle‐enzyme nanoconjugates, for the fast transesterification and esterification, and eventually for biodiesel synthesis. Our nanoconjugates are the hybrid biocatalysts tailor‐made by loading hydrophilic Candida antarctica lipase B onto hydrophobic silica nanoparticles, resulting in not only catalytically active but highly amphiphilic particles for stabilization of a methanol‐decane emulsion. The enzyme activity in these PEs is significantly enhanced, ca. 375‐fold higher than in the nonaqueous biphasic control. Moreover, the PEs can be multiply reused without significant loss of enzyme performance. With this proof‐of‐concept, this system can be expanded for many advanced syntheses using different enzymes in the future.

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Enzyme behavior at surfaces. Site-specific variants of subtilisin BPN' with enhanced surface stability.

Cited by 21 publications

References 21 publications

Subtilisin BPN‘ Variants: Increased Hydrolytic Activity on Surface-Bound Substrates via Decreased Surface Activity

Subtilisin BPN‘ Variants: Increased Hydrolytic Activity on Surface-Bound Substrates via Decreased Surface Activity

Sequential Electrostatic Assembly of a Polymer Surfactant Corona Increases Activity of the Phosphotriesterase arPTE

Tailoring Particle‐Enzyme Nanoconjugates for Biocatalysis at the Organic‐Organic Interface

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