A new, mild cross‐linking methodology to prepare cross‐linked enzyme aggregates

Mateo, César; Palomo, José M.; Langen, Luuk M. van; Rantwijk, Fred van; Sheldon, Roger A.

doi:10.1002/bit.20033

Cited by 272 publications

(176 citation statements)

References 19 publications

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“…Calcium chloride was added to the enzyme solution to remove ammonium sulfate until the final concentration of calcium ions in the solution reached 0.1 M. The enzyme solution was used without any further purification. CLEA of glucoamylase was prepared by using a modified procedure from the literature [31,32]. A mixture of 400 µL glucoamylase solution, 600 µL sodium acetic buffer (200 mM, pH 5.6) and 70% (w/w) powdered (NH 4 ) 2 SO 4 (created by adding dextrin power or xanthan gum to prepared CLEA-D or CLEA-XG before precipitation and stirring for 20 min at room temperature) was prepared and aged for 15 min at room temperature with occasional gentle stirring.…”

Section: Glucoamylasementioning

confidence: 99%

Preparation of Cross-Linked Glucoamylase Aggregates Immobilization by Using Dextrin and Xanthan Gum as Protecting Agents

Jia

et al. 2016

Catalysts

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Abstract:In this paper glucoamylase from Aspergillus niger was immobilized by using a modified version of cross-linked enzyme aggregates (CLEA). The co-aggregates were cross-linked with glutaraldehyde; meanwhile dextrin and xanthan gum as protecting agents were added, which provides high affinity with the enzyme molecules. The immobilized glucoamylase was stable over a broad range of pH (3.0-8.0) and temperature (55-75˝C); dependence shows more catalytic activity than a free enzyme. The thermostability, kinetic behavior, and first-order inactivation rate constant (k i ) were investigated. The two types of protector made the immobilized glucoamylase more robust than the free form. Both of the immobilized enzymes have excellent recyclability, retaining over 45% of the relative activity after 24 runs. In addition, immobilized enzymes reduced only 40% of the initial activity after three months by the storability measure, indicating high activity.

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Section: Glucoamylasementioning

confidence: 99%

Preparation of Cross-Linked Glucoamylase Aggregates Immobilization by Using Dextrin and Xanthan Gum as Protecting Agents

Jia

et al. 2016

Catalysts

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“…Higher ratios and longer incubation times result in a considerable reduction in catalytic activity, most likely because glutaraldehyde then starts to react with residues in the active site. Indeed, this problem can sometimes be avoided by the use of polymeric cross-linkers that are too large to access the active site [27], but these have not been tested in this work.…”

Section: Production Of Spase Cleasmentioning

confidence: 99%

Sucrose phosphorylase as cross‐linked enzyme aggregate: Improved thermal stability for industrial applications

Cerdobbel

Winter

Desmet

et al. 2010

Biotechnology Journal

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Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross‐linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60°C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity.

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“…In some cases, dextran polyaldehyde has been found to be a milder cross linker than glutaraldehyde. It is believed, that this bifunctional reagent does not diffuse in the interior of the protein molecule because of its big size and crosslinking is limited to the involvement of surface amino groups (Mateo et al, 2004). In the second approach, chitosan has been inserted with the enzyme molecule using carbodiimide coupling of the amino groups on chitosan and the carboxyl groups on the enzyme (Arsenault et al, 2011).…”

Section: Skills In Biocatalysis Are Essential For Establishing a Sounmentioning

confidence: 99%

Skills in biocatalysis are essential for establishing a sound biotechnological base in India

Gupta¹

2015

Proceedings of the Indian National Science Academy

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Techniques developed by enzymologists and protein chemists have been indispensible in many areas of biotechnology. Bio-separation of proteins, biocatalyst engineering and medium engineering are important examples of this. Our understanding of enzyme specificity has helped us in gaining insight into catalytic promiscuity. This, along with the possibility of carrying out biocatalysis in organic solvents has widened the scope of using enzymes for organic synthesis.Reestablishing a strong base in enzymology is crucial towards creating a strong base for the field of biotechnology in India. Keywords IntroductionQuite a few aspects of biotechnology involve production and applications of enzymes. In the old days, most of these aspects were covered under industrial enzymology (Godfrey and Reichelt, 1983). Gradually, a more broad based term 'applied biocatalysis' came into vogue (Straathof and Adlercreutz, 2000) which was an interface between enzymology and biochemical engineering. This phase was characterized by few new developments: (1) As a result of the rDNA technology, our capability to produce mutant properties grew exponentially. There was a scramble to produce proteins at a lower cost. The stakes were no longer limited to low cost industrial grade enzymes. Costlier proteins used in molecular biology, diagnostics and biosensors and pharmaceutical proteins/enzymes could be produced by cloning and their properties tailored to suit the applications. The result was a paradigm shift in the downstream processing methods. Shorter and efficient protocols emerged. Affinity chromatography, rather than being a 'polishing' step at the end, was promoted to the earlier steps of purification. In fact, upstream and downstream processing steps were integrated (Gupta, 2002;Przybycien et al., 2004;Mondal et al., 2006). (2) It was realized that enzymes could be used in non aqueous media (Carrea and Riva 2000; Gupta 2000;Patel 2000; Vulfson et al., 2001). This opened up novel applications; especially in the synthesis of chiral compounds. Table 1 lists some of the milestones in the area of applied biocatalysis.It is remarkable how much enzymology contributed to all this.The present review attempts to emphasize the importance of the skills which are part of the toolbox Published Online on 10 December 2015 1114Munishwar Nath Gupta and Joyeeta Mukherjee et al., 1973; Mosbach 1976 Mosbach , 1980 Nilsson and Mosbach 1981; Mattiasson 1983;Mattiasson 1988) Berezin's group in Moscow was the third one (Berezin 1978; Mozhaev et al., 1987;Martinek and Mozhaev 1993). Current work is adequately discussed at many places ( Enzyme deactivation models Most people wrongly give half lives without analysing the kinetics of deactivation. Sadana's work needs to be known more widely. (Sadana 1991(Sadana , 1993 Enzymes as reverse micelles This is another approach in low water enzymology. (Luisi 1985; Larsson et al., 1987; Adlercreutz et al., 1988) Enzyme use in biosensors Immobilization allows enzymes to interface with transducers. (Yarapolov et al...

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A new, mild cross‐linking methodology to prepare cross‐linked enzyme aggregates

Cited by 272 publications

References 19 publications

Preparation of Cross-Linked Glucoamylase Aggregates Immobilization by Using Dextrin and Xanthan Gum as Protecting Agents

Preparation of Cross-Linked Glucoamylase Aggregates Immobilization by Using Dextrin and Xanthan Gum as Protecting Agents

Sucrose phosphorylase as cross‐linked enzyme aggregate: Improved thermal stability for industrial applications

Skills in biocatalysis are essential for establishing a sound biotechnological base in India

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