Preparation and characterization of a Combi-CLEAs from pectinases and cellulases: a potential biocatalyst for grape juice clarification

Magro, Lucas Dal; Hertz, Plinho Francisco; Fernández-Lafuente, Roberto; Klein, Manuela Poletto; Rodrigues, Rafael C.

doi:10.1039/c6ra03940e

Cited by 58 publications

(37 citation statements)

References 47 publications

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“…As discussed in the Introduction section, crosslinking is a crucial step in CLEA preparation . It is well‐documented that feeder proteins (lysine‐rich proteins) may facilitate the preparation of CLEAs of enzymes that present low concentration of superficial amino groups (necessary for an effective crosslinking) . For example, CLEAs of Pseudomonas cepacia lipase and penicillin acylase in absence and presence of BSA were prepared by Shah et al The CLEA of lipase prepared in the presence of BSA retained 100% activity, whereas the CLEA prepared without BSA retained only 0.4% activity.…”

Section: Resultsmentioning

confidence: 99%

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1,3‐Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates

Ramos

Miranda

Giordano

et al. 2018

Biotechnology Progress

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The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL-CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA' observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 μmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL-SOY-CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL-SOY-CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12-fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2-fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL-SOY-CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier-free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910-920, 2018.

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Section: Resultsmentioning

confidence: 99%

“…A simplest strategy is the use of an aminated polymer, like polyethylenimine . However, a preferred alternative is to use a feeder protein, rich in amino groups, as this prevents the generation of ionic environments around the enzyme . Moreover, that way the enzyme is diluted and the diffusion problems may be reduced.…”

Section: Introductionmentioning

confidence: 99%

1,3‐Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates

Ramos

Miranda

Giordano

et al. 2018

Biotechnology Progress

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“…Xylanases and pectinases are the two most valuable enzymes of commercial sector holding biotechnological significance and are being exploited since last decade due to their tremendous applicability in a spectrum of industrial processes, viz bleaching in paper and pulp industry, textile industry, recycling of paper, extraction and clarification of various fruit juices, etc. Production of xylanase and pectinase individually from different types of microorganisms has been reported by several researchers but only few reports are available till date mentioning their synthesis in combination, from a single microbial isolate …”

Section: Introductionmentioning

confidence: 99%

A novel and cost‐effective methodology for enhanced production of industrially valuable alkaline xylano‐pectinolytic enzymes cocktail in short solid‐state fermentation cycle

Agrawal

Varghese

Mahajan

2019

Biotechnology Progress

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The aim of this study was to enhance the production of xylano‐pectinolytic enzymes concurrently and also to reduce the fermentation period. In this study, the effect of agro‐residues extract‐based inoculum on yield and fermentation time of xylano‐pectinolytic enzymes was studied. Microbial inoculum and fermentation media were supplemented with xylan and pectin polysaccharides derived from agro‐based residues. Enzymes production parameters were optimized through two‐stage statistical design approach. Under optimized conditions (temperature 37°C, pH 7.2, K2HPO4 0.22%, MgSO4 0.1%, gram flour 5.6%, substrate: moisture ratio 1:2, inoculum size 20%, agro‐based crude xylan in production media 0.45%, and agro‐based crude xylan–pectin in inoculum 0.13%), nearly 28,255 ± 565 and 9,202 ± 193 IU of xylanase and pectinase, respectively, were obtained per gram of substrate in a time interval of 6 days only. The yield of both xylano‐pectinolytic enzymes was enhanced along with a reduction of nearly 24 h in fermentation time in comparison with control, using polysaccharides extracted from agro‐residues. The activity of different types of pectinase enzymes such as exo‐polymethylgalacturonase (exo‐PMG), endo‐PMG, exo‐polygalacturonase (exo‐PG), endo‐PG, pectin lyase, pectate lyase, and pectin esterase was obtained as 1,601, 12.13, 5637, 24.86, 118.62, 124.32, and 12.56 IU/g, respectively, and was nearly twofold higher than obtained for all seven types in control samples. This is the first report mentioning the methodology for enhanced production of xylano‐pectinolytic enzymes in short solid‐state fermentation cycle using agro‐residues extract‐based inoculum and production media.

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“…have been successfully immobilized as CLEAs with high specific activity and activity recovery [31,32,35]. Interestingly, combined cross-linked enzyme aggregates (combi-CLEAs) are a novel prospect for co-immobilization of two or more enzymes, which can catalyze various cascade and non-cascade bioconversion processes [36]. For example, acombi-CLEAs of glucose dehydrogenase (GDH) and ketoreductase were developed and employed repeatedly in the reduction of ethyl 4-chloro-3-oxobutanoate to syntheses the key atorvastatin intermediate ethyl 4-chloro-3-hydroxybutyrate [37].…”

Section: Introductionmentioning

confidence: 99%

Magnetic Combined Cross-Linked Enzyme Aggregates of Ketoreductase and Alcohol Dehydrogenase: An Efficient and Stable Biocatalyst for Asymmetric Synthesis of (R)-3-Quinuclidinol with Regeneration of Coenzymes In Situ

et al. 2018

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Enzymes are biocatalysts. In this study, a novel biocatalyst consisting of magnetic combined cross-linked enzyme aggregates (combi-CLEAs) of 3-quinuclidinone reductase (QNR) and glucose dehydrogenase (GDH) for enantioselective synthesis of (R)-3-quinuclidinolwith regeneration of cofactors in situ was developed. The magnetic combi-CLEAs were fabricated with the use of ammonium sulfate as a precipitant and glutaraldehyde as a cross-linker for direct immobilization of QNR and GDH from E. coli BL(21) cell lysates onto amino-functionalized Fe 3 O 4 nanoparticles. The physicochemical properties of the magnetic combi-CLEAs were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and magnetic measurements. Field emission scanning electron microscope (FE-SEM) images revealed a spherical structure with numerous pores which facilitate the movement of the substrates and coenzymes. Moreover, the magnetic combi-CLEAs exhibited improved operational and thermal stability, enhanced catalytic performance for transformation of 3-quinuclidinone (33 g/L) into (R)-3-quinuclidinol in 100% conversion yield and 100% enantiomeric excess (ee) after 3 h of reaction. The activity of the biocatalysts was preserved about 80% after 70 days storage and retained more than 40% of its initial activity after ten cycles. These results demonstrated that the magnetic combi-CLEAs, as cost-effective and environmentally friendly biocatalysts, were suitable for application in synthesis of (R)-3-quinuclidinol essential for the production of solifenacin and aclidinium with better performance than those currently available.

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Preparation and characterization of a Combi-CLEAs from pectinases and cellulases: a potential biocatalyst for grape juice clarification

Abstract: Combi-CLEAs of pectinases and cellulases were prepared for grape juice clarification.

Cited by 58 publications

References 47 publications

1,3‐Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates

1,3‐Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates

A novel and cost‐effective methodology for enhanced production of industrially valuable alkaline xylano‐pectinolytic enzymes cocktail in short solid‐state fermentation cycle

Magnetic Combined Cross-Linked Enzyme Aggregates of Ketoreductase and Alcohol Dehydrogenase: An Efficient and Stable Biocatalyst for Asymmetric Synthesis of (R)-3-Quinuclidinol with Regeneration of Coenzymes In Situ

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