Nitrile hydratase CLEAs: The immobilization and stabilization of an industrially important enzyme

Pelt, Sander van; Quignard, Sandrine; Kubáč, David; Sorokin, Dimitry Y.; Rantwijk, Fred van; Sheldon, Roger A.

doi:10.1039/b714258g

Cited by 65 publications

(35 citation statements)

References 25 publications

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“…[8,11] NHase CLEAs were stored at + 4 8C in Tris-HCl buffer (0.01 M, pH 8), while the MeHnL CLEAs were stored at À20 8C in lyophilised form. Hexanenitrile (98%, Aldrich), hexanamide (98%, Aldrich), sodium borohydride (> 96%, Fluka), diisopropyl ether (DIPE, > 99%, Fluka), acrolein (> 95%, Fluka), propionaldehyde (99 + %, Janssen), isobutyraldehyde (99 + %, Acros), butyraldehyde (> 99.5%, Aldrich), valeraldehyde (> 97%, Fluka), a-hydroxybutyronitrile (Fluka, purum), a-hydroxybutyric acid (Fluka, > 97%), and a-hydroxycaproic acid (Aldrich, 98%) were used in the experiments as received without additional purification.…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, NHase CLEAs suffer from a high loss of activity during freeze drying and are most stable when stored in an aqueous environment. [8] Incubation of the NHase CLEAs in different DIPE/ buffer mixtures resulted in an average activity loss of 40% in one hour. In addition to the low stability, it is cumbersome to suspend the NHase CLEAs in these mixtures, since the particles coagulate easily and tend to stick to the walls of the reaction vessel.…”

Section: Stability Of Nhase Cleas Under Hnl Hydrocyanation Conditionsmentioning

confidence: 99%

“…Recently, a new NHase-containing organism from soda lakes was isolated by our group [7] and subsequently the cell-free NHase from this organism was stabilised by the formation of CLEAs. [8] When a bienzymatic cascade of HnL and NHase is used for the production of a-hydroxycarboxylic amides, the low selectivity of the NHase is no longer a drawback since either an (R)-or (S)-selective HnL will impart its enantioselectivity on the reaction (Scheme 1).…”

Section: Introductionmentioning

confidence: 99%

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Synthesis of Aliphatic (S)‐α‐Hydroxycarboxylic Amides using a One‐Pot Bienzymatic Cascade of Immobilised Oxynitrilase and Nitrile Hydratase

2009

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A one-pot bienzymatic cascade combining a hydroxynitrile lyase (Manihot esculenta, E.C. 4.1.2.10) and a nitrile hydratase (Nitriliruptor alkaliphilus, E.C. 4.2.1.84) for the synthesis of enantiopure aliphatic a-hydroxycarboxylic amides from aldehydes is described. Both enzymes were immobilised as cross-linked enzyme aggregates (CLEAs). Stability tests show that the nitrile hydratase CLEAs are sensitive to water-immiscible organic solvents as well as to aldehydes and hydrogen cyanide (HCN), but are remarkably stable and show useful activity in acidic aqueous environments of pH 4-5. The cascade reactions are consequently carried out by using a portionwise feed of HCN and moderate concentrations of aldehyde in acidic aqueous buffer to suppress the uncatalysed hydrocyanation background reaction. After optimisation, this method was used to synthesise five different kinds of aliphatic a-hydroxycarboxylic amides from the corresponding aldehydes with good yields and with enantiomeric purities comparable to those obtained for the a-hydroxynitriles in the microaqueous hydrocyanation using hydroxynitrile lyase and an excess of HCN.

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Section: Methodsmentioning

confidence: 99%

Section: Stability Of Nhase Cleas Under Hnl Hydrocyanation Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Synthesis of Aliphatic (S)‐α‐Hydroxycarboxylic Amides using a One‐Pot Bienzymatic Cascade of Immobilised Oxynitrilase and Nitrile Hydratase

2009

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“…However, as a rule, the immobilization of most proteins on such supports results in low activity/weight ratios. Recently, to overcome this important limitation, procedures for the production of insoluble but catalytically functional enzyme aggregates have been proposed (Cao et al, 2003;Sheldon et al, 2005;Sheldon, 2007a,b;van Pelt et al, 2008). The general approach involves the precipitation of soluble enzyme with an appropriate aggregation additive and its subsequent cross-linking with a bifunctional agent leading to the formation of so-called cross-linked enzyme aggregates (CLEAs) that is, biocatalysts that exhibit high activity/ volume ratio (Cao et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Utilization of cross‐linked laccase aggregates in a perfusion basket reactor for the continuous elimination of endocrine‐disrupting chemicals

Cabana

Jones

Agathos

2008

Biotech & Bioengineering

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A perfusion basket reactor (BR) was developed for the continuous utilization of insolubilized laccase as cross-linked enzyme aggregates (CLEAs). The BR consisted of an unbaffled basket made of a metallic filtration module filled with CLEAs and continuously agitated by a 3-blade marine propeller. The agitation conditions influenced both the apparent laccase activity in the reactor and the stability of the biocatalyst. Optimal laccase activity was obtained at a rotational speed of 12.5 rps and the highest stability was reached at speeds of 1.7 rps or lower. The activity and stability of the biocatalyst were affected drastically upon the appearance of vortices in the reaction medium. This reactor was used for the continuous elimination of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA), and triclosan (TCS). Optimization of EDC elimination by laccase CLEAs as a function of temperature and pH was achieved by response surface methodology using a central composite factorial design. The optimal conditions of pH and temperature were, respectively, 4.8 and 40.3 degrees C for the elimination of p353NP (a branched isomer of NP), 4.7 and 48.0 degrees C for BPA, and 4.9 and 41.2 degrees C for TCS. Finally, the BR was used for the continuous elimination of these EDCs from a 5 mg L(-1) aqueous solution using 1 mg of CLEAs at pH 5 and room temperature. Our results showed that at least 85% of these EDCs could be eliminated with a hydraulic retention time of 325 min. The performances of the BR were quite stable over a 7-day period of continuous treatment. Furthermore, this system could eliminate the same EDCs from a 100 mg L(-1) solution. Finally, a mathematical model combining the Michaelis-Menten kinetics of the laccase CLEAs and the continuous stirred tank reactor behavior of the BR was developed to predict the elimination of these xenobiotics.

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“…[245][246][247] In the case of the Fl-Hals, this cross-linking may help to stabilise interdomain interactions, or effectively "protect" some of the biocatalyst from high substrate concentration by shielding it within the heterogeneous catalyst. 244,248 The preparation of such biocatalysts from crude cell lysates, rather than needing to purify enzymes, 244 is also advantageous.…”

Section: -237mentioning

confidence: 99%

Development of Halogenase Enzymes for Use in Synthesis

Latham

Brandenburger

Shepherd³

et al. 2017

Chem. Rev.

286

244

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Nature has evolved halogenase enzymes to regioselectively halogenate a diverse range of biosynthetic precursors, with the halogens introduced often having a profound effect on the biological activity of the resulting natural products. Synthetic endeavours to create non-natural bioactive small molecules for pharmaceutical and agrochemical applications have also arrived at a similar conclusion -halogens can dramatically improve the properties of organic molecules for selective modulation of biological targets in vivo. Consequently a high proportion of pharmaceuticals and agrochemicals on the market today possess halogens. Halogenated organic compounds are also common intermediates in synthesis and are particularly valuable in metal catalyzed cross-coupling reactions.Despite the potential utility of organohalogens, traditional non-enzymatic halogenation chemistry utilizes deleterious reagents and often lacks regiocontrol. Reliable, facile and cleaner methods for the regioselective halogenation of organic compounds are therefore essential in the development of economical and environmentally-friendly industrial processes. A potential avenue towards such methods is the use of halogenase enzymes, responsible for the biosynthesis of halogenated natural products, as biocatalysts. This review will discuss the advances towards this goal thus far, in addition to untapped potential sources of such biocatalysts and how further development of the enzymes may be focused in order to achieve the goal of industrial scale biohalogenation.

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Nitrile hydratase CLEAs: The immobilization and stabilization of an industrially important enzyme

Cited by 65 publications

References 25 publications

Synthesis of Aliphatic (S)‐α‐Hydroxycarboxylic Amides using a One‐Pot Bienzymatic Cascade of Immobilised Oxynitrilase and Nitrile Hydratase

Synthesis of Aliphatic (S)‐α‐Hydroxycarboxylic Amides using a One‐Pot Bienzymatic Cascade of Immobilised Oxynitrilase and Nitrile Hydratase

Utilization of cross‐linked laccase aggregates in a perfusion basket reactor for the continuous elimination of endocrine‐disrupting chemicals

Development of Halogenase Enzymes for Use in Synthesis

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