Synthesis and Characterization of Highly Stabilized Polymer–Trypsin Conjugates with Autolysis Resistance

Sasai, Yasushi; Kanno, Hiroshi; Doi, Naoki; Yamauchi, Yukiko; Kuzuya, Masayuki; Kondo, Shinichi

doi:10.3390/catal7010004

Cited by 8 publications

(9 citation statements)

References 36 publications

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“…Since more than half of the residues adjacent to the lysine molecules are hydrophobic amino acids [ 14 ], attachment of the bulky carboxylic groups masks the non-polar hydrophobic residues, thereby protecting them from unfavorable interactions with the aqueous environment and further stabilizing the enzyme structure. Acylation is cost-effective, simple, does not require complex equipment, can be carried out singly or in conjunction with other enzyme engineering protocols, does not require in-depth knowledge of the enzyme protein, and can be applied to native, engineered or mutant enzyme [ 15 , 16 ]. Maleic anhydride (C 4 H 2 O 3 ) and citraconic anhydride also called 2-methyl maleic anhydride (C 5 H 4 O 3 ) owing to its chemical structure are both cyclic anhydrides of aliphatic carboxylic acids which can be used for the modification of enzymes to achieve enzyme stabilization [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius

Nwagu

Aoyagi

Okolo

et al. 2020

Heliyon

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Amylase capable of raw starch digestion presents a cheap and easier means of reducing sugar generation from various starch sources. Unfortunately, its potential for use in numerous industrial processes is hindered by poor stability. In this work, chemical modification by acylation using citraconic anhydride (CA) and maleic anhydride (MA) was used to stabilize the raw starch saccharifying amylase from A. carbonarius . The effect of the anhydrides on the pH and thermal stability of the free amylase was investigated. Enzyme kinetics and thermodynamic studies of the free and modified amylase were also carried out. Blue shifts in fluorescent spectra were observed after modification with both anhydrides. Citraconylation led to increased affinity of the enzyme for raw potato starch, unlike maleylation. The activation energy (kJ mol −1 ) for enzyme inactivation was increased by 94.8% after modification with CA while only 17.9% increase was noted after modification with MA. Acylation led to an increase in Gibb's free energy and enthalpy while a reduction in entropy was observed. At 80 °C the half-life (h) was 5.92, 11.18 and 14.74 for free, MA and CA enzyme samples, respectively. These findings have potential value in all industries interested in starch conversion to sugars.

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Section: Introductionmentioning

confidence: 99%

Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius

Nwagu

Aoyagi

Okolo

et al. 2020

Heliyon

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“…The assay was performed at different bulk pH values to analyze the trypsin activity dependence on the pH. The used pH values were based on the known dependence of the trypsin activity on solution pH …”

Section: Methodsmentioning

confidence: 99%

“…The used pH values were based on the known dependence of the trypsin activity on solution pH. 34 To exclude the impact of specific substrates of "actuator enzymes" (urease or esterase) on the next switching stage (ON or OFF), the glass slides loaded with the SiO 2 NPs carrying both "actuator" and "target" enzymes were transferred to a new buffer solution, without the presence of the substrates of "actuator enzymes". Between the transfer, the glass slides were intensively washed thrice with 3 mM HEPES buffer with the initial pH value for 2 min at medium rotating speed of an orbital shaker.…”

Section: Methodsmentioning

confidence: 99%

Switchable Biocatalytic Reactions Controlled by Interfacial pH Changes Produced by Orthogonal Biocatalytic Processes

Wells

Smutok

Melman

et al. 2021

ACS Appl. Mater. Interfaces

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Enzymes immobilized on a nano-structured surface were used to switch the activity of one enzyme by a local pH change produced by another enzyme. Immobilized amyloglucosidase (AMG) and trypsin were studied as examples of the pH-dependent switchable “target enzymes.” The reactions catalyzed by co-immobilized urease or esterase were increasing or decreasing the local pH, respectively, thus operating as “actuator enzymes.” Both kinds of the enzymes, producing local pH changes and changing biocatalytic activity with the pH variation, were orthogonal in terms of the biocatalytic reactions; however, their operation was coupled with the local pH produced near the surface with the immobilized enzymes. The “target enzymes” (AMG and trypsin) were changed reversibly between the active and inactive states by applying input signals (urea or ester, substrates for the urease or esterase operating as the “actuator enzymes”) and washing them out with a new portion of the background solution. The developed approach can potentially lead to switchable operation of several enzymes, while some of them are inhibited when the others are activated upon receiving external signals processed by the “actuator enzymes.” More complex systems with branched biocatalytic cascades can be controlled by orthogonal biocatalytic reactions activating selected pathways and changing the final output.

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“…Porcine pancreatic alpha-trypsin (PPT), a proteolytic enzyme, is a pancreatic serine protease (EC 3.4.21.4) with specificity for arginine or lysine substrate towards catalytic hydrolysis on esters and amides, under mild reaction conditions [ 1 , 2 , 3 ]. Owing to these facts, this proteolytic enzyme is often utilized in industrial and biomedical applications [ 1 , 4 , 5 ]. However, such enzymes are often immobilized into various substrates to improve stability and reusability without affecting their activity [ 2 , 6 , 7 ].…”

Section: Introductionmentioning

confidence: 99%

Deviation of Trypsin Activity Using Peptide Conformational Imprints

et al. 2021

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In this study, a methodology utilizing peptide conformational imprints (PCIs) as a tool to specifically immobilize porcine pancreatic alpha-trypsin (PPT) at a targeted position is demonstrated. Owing to the fabrication of segment-mediated PCIs on the magnetic particles (PCIMPs), elegant cavities complementary to the PPT structure are constructed. Based on the sequence on targeted PPT, the individual region of the enzyme is trapped with different template-derived PCIMPs to show certain types of inhibition. Upon hydrolysis, N-benzoyl-L-arginine ethyl ester (BAEE) is employed to assess the hydrolytic activity of PCIMPs bound to the trypsin using high-performance liquid chromatography (HPLC) analysis. Further, the kinetic data of four different PCIMPs are compared. As a result, the PCIMPs presented non-competitive inhibition toward trypsin, according to the Lineweaver-Burk plot. Further, the kinetic analysis confirmed that the best parameters of PPT/PCIMPs 233–245+G were Vmax = 1.47 × 10−3 mM s−1, Km = 0.42 mM, kcat = 1.16 s−1, and kcat/Km = 2.79 mM−1 s−1. As PPT is bound tightly to the correct position, its catalytic activities could be sustained. Additionally, our findings stated that the immobilized PPT could maintain stable activity even after four successive cycles.

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Synthesis and Characterization of Highly Stabilized Polymer–Trypsin Conjugates with Autolysis Resistance

Cited by 8 publications

References 36 publications

Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius

Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius

Switchable Biocatalytic Reactions Controlled by Interfacial pH Changes Produced by Orthogonal Biocatalytic Processes

Deviation of Trypsin Activity Using Peptide Conformational Imprints

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