Cellulase cross-linked enzyme aggregates (CLEA) activities can be modulated and enhanced by precipitant selection

Abstract

BACKGROUND Crosslinked enzyme aggregates (CLEA) technology is a rapid and versatile method to produce immobilized enzymes via precipitation and cross‐linking. A direct relationship between CLEA final activity and process parameters is however yet to be clarified. To address the issue, we have used a factorial design to test the formation and optimization of CLEA made from technical grade cellulase (EC 3.2.1.4). Three types of precipitant (ammonium sulfate, polyethylene glycol, tert‐butyl alcohol) were used at varied levels of cross‐linker concentration, cross‐linking time, and temperature. RESULTS It was found that the CLEAs produced with tert‐butyl alcohol were inactive, whereas the polyethylene glycol‐ and ammonium sulfate‐CLEA, recovered 29 and 17% of the free enzyme activity, respectively. Testing for re‐usability, we observed that the polyethylene glycol‐CLEA maintained 40% of the initial activity after four cycles, in contrast the ammonium sulfate‐CLEA only maintained 10% of its activity after one cycle. CONCLUSION Our study highlights the importance of evaluating the effect of the precipitant on final CLEA activity rather than re‐solubilized enzyme activity. It was demonstrated that polyethylene glycol, despite not being able to precipitate the enzymes as readily as ammonium sulfate, resulted in better performing CLEA. © 2016 Society of Chemical Industry

Cross-Linked Enzyme Aggregates of Feruloyl Esterase Preparations from Thermothelomyces thermophila and Talaromyces wortmannii

“…The residual activity of the obtained CLEAs was in agreement with the available data from the literature: for example, in the work of Cruz-Izquierdo et al [28], the maximum retention of activity from Candida antarctica lipase (48%) was observed in the case where ammonium sulfate was used as the precipitant, while the maximum residual activity of halohydrin dehalogenase from Agrobacterium radiobacter in the same conditions was 70.5% [29]. On the other hand, in most samples precipitated with acetone and tert-butyl alcohol, a single aggregate could be observed, which could not be broken into smaller aggregates with prolonged agitation, or ultrasound treatment, in accordance with the findings of Perzon et al [24]. These CLEAs exhibited lower residual activities, probably due to severe mass transfer limitations.…”

“…As regards the type of the precipitant, the selected precipitants were ammonium sulfate, a very common precipitant for CLEA preparations, due to its low cost and mild effect on the protein structure; acetone, which has been used extensively in the past for the precipitation and subsequent purification of esterases [20,21]; tert-butyl alcohol, previously shown to be a suitable precipitant for FAE CLEAs [4]; and PEG-4000, a hydrophilic polymer with a stabilizing effect on the protein structure [22] that has been used before for CLEA preparation [23]. The selection of a proper precipitant might even enhance the residual activity of the obtained CLEAs, by stabilizing their structure, or by inducing a more favorable conformation of the active site towards the substrate, as shown in previous studies [4,24,25]. Especially acetone is reported to enhance the activity of FAEs, even in soluble form [26], while the stabilizing effects of acetone on enzyme activity have been known since 1967 [27].…”

Techniques for Preparation of Cross-Linked Enzyme Aggregates and Their Applications in Bioconversions

“…The activity of cellulase-CLEAs prepared with (NH 4 ) 2 SO 4 was lower than that of cellulase-CLEAs produced with PEG. The CLEAs prepared with t-butanol were inactive [41]. Similar results were reported in the case of lipase-CLEAs.…”

“…Several cellulase-CLEAs produced using organic solvents showed better activities and reusability [39,40]. Recently, a systematic study of the effects of different precipitants and various cross-linking parameters was reported [41]. The effects of different precipitants, namely (NH 4 ) 2 SO 4 , poly(ethylene glycol) (PEG), and t-butanol, on the resolubilized enzyme activity and final CLEA activity were examined.…”

Cross-linked enzyme-polymer conjugates with excellent stability and detergent-enhanced activity for efficient organophosphate degradation

“…g The transmission electron microscopy of CLEPC showed the aggregation of immobilized enzymes with the mean diameter of 0.66 ± 0.12 μm and detergents. However, this activity improvement was lower than that of PoOPH M9 CLEPC incubated with detergents (2-and 2.5-fold), indicating that the enzyme aggregation and cross-linkage in PoOPH M9 CLEPC are very important in the activity enhancement (Akbar et al 2007;Perzon et al 2017;Wang et al 2011). In summary, the significant enhancement in the activity of PoOPH M9 CLEPC was caused by the synergic effect of Pluronic F127, enzyme immobilization and detergents.…”