Peptide self‐assembly as a strategy for facile immobilization of redox enzymes on carbon electrodes

Redox-enzyme‐mediated electrochemical processes such as hydrogen production,nitrogen fixation,and CO_(2) reduction are at the forefront of the green chemistry revolution.To scale up,the inefficient two‐dimensional(2D)immobilization of redox enzymes on working electrodes must be replaced by an efficient dense 3D system.Fabrication of 3D electrodes was demonstrated by embedding enzymes in polymer matrices.However,several requirements,such as simple immobilization,prolonged stability,and resistance to enzyme leakage,still need to be addressed.The study presented here aims to overcome these gaps by immobilizing enzymes in a supramolecular hydrogel formed by the self‐assembly of the peptide hydrogelator fluorenylmethyloxycarbonyldiphenylalanine.Harnessing the self‐assembly process avoids the need for tedious and potentially harmful chemistry,allowing the rapid loading of enzymes on a 3D electrode under mild conditions.Using the[FeFe]hydrogenase enzyme,high enzyme loads,prolonged resistance against electrophoresis,and highly efficient hydrogen production are demonstrated.Further,this enzyme retention is shown to arise from its interaction with the peptide nanofibrils.Finally,this method is successfully used to retain other redox enzymes,paving the way for a variety of enzyme‐mediated electrochemical applications.

Enzyme Substrates Protective Encapsulation within Polymeric Microspheres

Determination of enzymes activity is an important task of analytical and biomedical oriented fluorimetry. Despite of a long track record of application, there is still some room for improvements in this field. In the case of hydrolases, spontaneous decomposition of substrate leads to substantial errors in determination of enzyme activity. An innovative and effective approach is proposed allowing protection of enzyme substrate within the lipophilic moiety of polyacrylate microspheres. It is shown that the introduction of substrate into microspheres is not only an effective method of prevention of unwanted spontaneous process, but also does not disturb the availability of substrate for enzymatic hydrolysis. The effect of presence of proteins in the sample on fluorimetric responses was studied. In contrary to previous studies related to application of lipophilic polymers in biomedical analysis, it is shown that the presence of bovine serum albumins enhances the sensitivity of fluorimetric determination. It is shown that this surprising effect is related to adsorption of proteins on the microspheres surface and change of surface charge of polymer.

Effects of Encapsulated Enzyme and Yeast Products on in Vitro Rumen Fermentation

Batch cultures of mixed rumen micro-organisms were conducted to evaluate the effects of encapsulated yeast(+EY)and encapsulated enzyme(+EE)using plant proteins(barley and oats grain)on rumen fermentation in vitro,investigate the abilities of encapsulated yeast and encapsulated enzyme to prevent rumen digestion in vitro.Treatments of the study were the control,+EY,+EE products(3.33 mg·mL^(-1) of the incubation medium),unencapsulated yeast(-EY)and enzyme(-EE)products(0.17 and 0.17μL·mL^(-1) of the incubation medium,respectively).+EY group increased dry matter disappearance(DMD,P<0.01)and the total volatile fatty acids(TVFA,P<0.01)at 3 h of the incubation compared with the control,regardless of encapsulation of yeast.Gas production(GP)of+EY group was higher(P=0.05,29.94 mL·mL^(-1) organic matter,OM)than that of the control(25.08 mL·g^(-1) OM)at 3 h of the incubation.Supplementation+EY increased DMD(P=0.04,0.394 vs 0.352,respectively)and acetic proportion(P=0.04,52.6 vs 49.8 mol•100 mL^(-1),respectively)at 6 h of the incubation and increased A:P ratio(P<0.01,3.11 and 2.86,respectively)at 24 h of the incubation,as compared to unencapsulation of yeast.Supplementation of enzyme had higher(P≤0.04)GP,DMD and TVFA at 3 and 6 h of the incubation compared with the control,regardless of encapsulation.Moreover,the addition of+EE produced greater GP at 6(P<0.01,92.35 vs 78.21 mL·g^(-1) OM,respectively),12(218.47 vs 159.18 mL·g^(-1) OM)and 24 h(380.97 vs 297.78 mL·g^(-1) OM,respectively)of the incubation,higher DMD(0.347 vs 0.313,respectively)at 3 h of the incubation as compared to-EE group.The study showed that the encapsulation might protect part of yeast and enzyme from releasing to the rumen throughout the digestion in vitro,resulting in higher or no difference of rumen fermentation parameters compared with unencapsulated groups at any incubation times.In comparison with-EY and-EE,the higher rumen fermentation parameters at the early incubation time were observed,which could be attributed to the higher concentration of yeast or enzyme.However,regardless of the encapsulation,the results indicated that both yeast and enzyme only improved the speed rather than the extent of rumen fermentation in vitro.

Preparation and evaluation of enzyme encapsulated hydrogels(single gels and double network gels) and enzyme immobilized magnetic beads

In the present research,enzyme encapsulated hydrogels（single gels and double network gels）and enzyme immobilized magnetic beads,which allow high-throughput screening,were fabricated and evaluated in terms of the preservation,precision, and repeatability of enzyme activity.The fabricated gels and magnetic beads were analyzed in a 96-well microassay plate.Trypsin was successfully encapsulated in both types of gels and immobilized to the magnetic beads.However,pepsin,either encapsulated in the gels or immobilized to the magnetic beads,could not react with its substrates.The adaptability to various enzymes （e.g.,trypsin,β-glucuronidase,and CYP1A1）in the single gels and magnetic beads was superior to that in double network gels.However,the soak out of the enzymes was observed in the single gels.The double network gels could encapsulate trypsin,whereas the fabrication of the other enzymes（e.g.β-glucuronidase,CYP1A1,and pepsin）failed because of the inactivation of the enzymes by acryl amide and ammonium peroxodisulfate,which are the components of the gel formulation. The enzyme reaction in the magnetic beads exhibited the highest efficiency among the three fabrication methods.Furthermore, the stability of the enzymes immobilized to the magnetic beads was better than that fabricated by the other methods,and the activities of trypsin andβ-glucuronidase did not decline for up to one week.In addition,in the magnetic beads,the activities of trypsin andβ-glucuronidase can be well repeated.Hence,although the adaptability of the double network gels to various enzymes is currently limited,the efficiency of the enzyme encapsulation can be improved by optimizing the formulation of acryl amide gels.