Co-expression of the recombined alcohol dehydrogenase and glucose dehydrogenase and cross-linked enzyme aggregates stabilization

Hu, Xiaozhi; Liu, Liqin; Chen, Daijie; Wang, Yongzhong; Zhang, Junliang; Shao, Lei

doi:10.1016/j.biortech.2016.10.076

Cited by 20 publications

(19 citation statements)

References 24 publications

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“…HgCl 2 is a thiol‐specific strong inhibitor that reacts with amino and imidazolium groups of histidine and peptide bonds and causes a decrease in enzyme activity. SDS is an excessively denaturating reagent that disrupts the tertiary and quaternary structures of proteins, and its addition to the reaction medium negatively affected enzyme activity …”

Section: Resultsmentioning

confidence: 99%

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Cross‐linked α‐galactosidase aggregates: optimization, characterization and application in the hydrolysis of raffinose‐type oligosaccharides in soymilk

Bayraktar

Önal

2019

J Sci Food Agric

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BACKGROUND Cross‐linked enzyme aggregates (CLEAs) of α‐galactosidase, partially purified from maize (Zea mays) flour, were prepared. The impact of various parameters on enzyme activity was examined to optimize the immobilization procedure. Biochemical characterization of the free and immobilized enzyme was carried out. Stability (thermal, pH, storage and operational stability) and reusability tests were performed. The potential use of the free enzyme and the CLEAs in hydrolysis processes of raffinose‐type oligosaccharides present in soymilk was investigated. RESULTS α‐galactosidase CLEAs were prepared with 47% activity recovery under optimum conditions [1:5 (v/v) enzyme solution:saturated ammonium sulfate solution ratio; 7.5 mg protein and 0.1% (v/v) glutaraldehyde, 6 h, 4 °C, 150 rpm]. α‐galactosidase CLEAs exhibited increased stability in comparison to the free enzyme. The CLEAs and the free enzyme showed a maximum activity at 40°C and their optimal pH values were5.5 and 6.0, respectively. Kinetic constants (KM, Vmax and kcat) were calculated for the free enzyme and the CLEAs in the presence of p‐nitrophenyl‐α‐d‐galactopyranoside, stachyose, melibiose and raffinose. The effect of various chemicals and sugars on enzyme activity showed that both enzyme forms were significantly inhibited by HgCl2 and galactose. The CLEAs hydrolyzed 85% of raffinose and 96% of stachyose. CONCLUSION The α‐galactosidase CLEAs, with their satisfactory enzymatic characteristics, have much potential for use in the food and feed industry. © 2019 Society of Chemical Industry

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Section: Resultsmentioning

confidence: 99%

“…SDS is an excessively denaturating reagent that disrupts the tertiary and quaternary structures of proteins, and its addition to the reaction medium negatively affected enzyme activity. 33 The effect of several sugars on the activity of the free enzyme and CLEAs is shown in Fig. 6(b).…”

Section: Effect Of Chemicals and Sugarsmentioning

confidence: 99%

Cross‐linked α‐galactosidase aggregates: optimization, characterization and application in the hydrolysis of raffinose‐type oligosaccharides in soymilk

Bayraktar

Önal

2019

J Sci Food Agric

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“…CLEAs were prepared according to the protocol described by Hu et al. . First, the ADH was precipitated adding 70 % (w/v) ammonium sulfate to the enzyme solution in 1 mL.…”

Section: Methodsmentioning

confidence: 99%

Enzyme Immobilization on Synthesized Nanoporous Silica Particles and their Application in a Bi‐enzymatic Reaction

et al. 2020

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The application of enzymes presents a great advantage regarding highly selective reactions; however, it involves also challenges due to their sensitivity. Immobilization offers one strategy to overcome those challenges enabling enzyme stabilization, as well as retention. In the present study, covalent attachment on hydrophilic amino-functionalized carriers is found to be the most promising immobilization method for the investigated reaction system. To achieve this, a novel method for preparation of silica particles with subsequent aminofunctionalization is developed to prepare spherical carriers for enzyme immobilization, whereby high porosities are obtained based on polymerization. With these particles, immobilization of an alcohol dehydrogenase and a formate dehydrogenase is realized with residual activities of 70 and 80 % after 12 consecutive batches, respectively. The two immobilized enzymes are used in the reduction of cinnamyl aldehyde with in situ cofactor regeneration, obtaining a conversion of 100 % and up to 10-fold higher turnover numbers compared to the free enzyme. afterwards for immobilization without stirring for 22 h at room temperature. Afterwards the resins were filtered and washed buffer.

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“…The combi-CLEA concept has also been used to advantage in oxidation reactions requiring cofactor regeneration. A pertinent example is the ketoreductase (KRED)/glucose dehydrogenase (GDH) combi-CLEA that was used in the enantioselective reduction of a prochiral ketone with cofactor regeneration with glucose/GDH [149,150]. The combi-CLEA exhibited increased thermal and pH stability and high substrate tolerance compared with the free enzymes, coupled with high volumetric productivity, good operational stability and reusability.…”

Section: Multi-and Combi-cleasmentioning

confidence: 99%

CLEAs, Combi-CLEAs and ‘Smart’ Magnetic CLEAs: Biocatalysis in a Bio-Based Economy

2019

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Biocatalysis has emerged in the last decade as a pre-eminent technology for enabling the envisaged transition to a more sustainable bio-based economy. For industrial viability it is essential that enzymes can be readily recovered and recycled by immobilization as solid, recyclable catalysts. One method to achieve this is via carrier-free immobilization as cross-linked enzyme aggregates (CLEAs). This methodology proved to be very effective with a broad selection of enzymes, in particular carbohydrate-converting enzymes. Methods for optimizing CLEA preparations by, for example, adding proteic feeders to promote cross-linking, and strategies for making the pores accessible for macromolecular substrates are critically reviewed and compared. Co-immobilization of two or more enzymes in combi-CLEAs enables the cost-effective use of multiple enzymes in biocatalytic cascade processes and the use of "smart" magnetic CLEAs to separate the immobilized enzyme from other solids has raised the CLEA technology to a new level of industrial and environmental relevance. Magnetic-CLEAs of polysaccharide-converting enzymes, for example, are eminently suitable for use in the conversion of first and second generation biomass.Catalysts 2019, 9, 261 2 of 31 and deprotection steps. This affords synthetic routes that, compared with conventional organic syntheses, are more step economic, more energy efficient, generate less waste and provide products in exquisite stereochemical purities that are difficult to compete with.Consequently, in the last two decades biocatalysis has emerged as an important technology for meeting the growing demand for green and sustainable processing [2][3][4]. This embraces the whole gamut of chemicals manufacture, from the enantioselective synthesis of chiral drugs [5][6][7][8][9][10][11][12] to the conversion of renewable biomass to liquid fuels and commodity chemicals [13][14][15]. This raises the perennial question: if biocatalysis is so superior why has it not been extensively used in the past? The answer is simple: thanks to advances in molecular biology and biotechnology, biocatalysis has undergone a spectacular leap forward in the last two decades:-Many more enzymes have been identified through (meta)genome mining, that is the in silico analysis of publicly accessible genome sequence data bases that have been generated as a result of next generation genome sequencing [16]. -Advances in gene synthesis have enabled the synthesis of identified genes, ready for cloning into a host production organism, in a few weeks at relatively low cost. This has significantly reduced the cost of development and subsequent production of enzymes at industrial scale. -Advances in protein engineering, using directed evolution techniques [17,18], have enabled optimization of enzyme performance under the challenging conditions encountered in industrial-scale processes, namely, high (stereo)selectivities, activities and space-time yields with non-natural substrates at high substrate concentrations, in the presence of organic solve...

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Co-expression of the recombined alcohol dehydrogenase and glucose dehydrogenase and cross-linked enzyme aggregates stabilization

Cited by 20 publications

References 24 publications

Cross‐linked α‐galactosidase aggregates: optimization, characterization and application in the hydrolysis of raffinose‐type oligosaccharides in soymilk

Cross‐linked α‐galactosidase aggregates: optimization, characterization and application in the hydrolysis of raffinose‐type oligosaccharides in soymilk

Enzyme Immobilization on Synthesized Nanoporous Silica Particles and their Application in a Bi‐enzymatic Reaction

CLEAs, Combi-CLEAs and ‘Smart’ Magnetic CLEAs: Biocatalysis in a Bio-Based Economy

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