Hybrid Magnetic Cross-Linked Enzyme Aggregates of Phenylalanine Ammonia Lyase from Rhodotorula glutinis

Abstract

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

Phenylalanine ammonia-lyase, a key component used for phenylpropanoids production by metabolic engineering

“…35,36,[49][50][51][170][171][172][173] Besides the forward reaction of spontaneous, nonoxidative deamination of L-Phe to t-CA and ammonia, PALs can also catalyze the reverse reaction, namely formation of L-Phe under conditions of high concentration of trans-cinnamic acids and ammonia at an elevated pH. 49,50,170-172 L-Phe is a key precursor of articial sweetener aspartame.…”

“…The great demand on aspartame stimulated considerable researches on the enzymatic production of L-Phe catalyzed by PALs. [170][171][172] In order to improve PAL activity, a combine promoter was used to regulate the expression of PAL. 174 The biotransformation approach catalyzed by PALs has the advantage of asymmetric synthesis of an optically pure product, L-Phe.…”

“…174 The biotransformation approach catalyzed by PALs has the advantage of asymmetric synthesis of an optically pure product, L-Phe. 49,[170][171][172] Compared to the free enzyme, the aggregates exhibited the higher thermal stability, denaturant stability and storage stability, retaining 30% initial activity even aer 11 cycles of reuse. [170][171][172] In order to improve PAL activity, a combine promoter was used to regulate the expression of PAL.…”

Preparation of spherical cross-linked lipase aggregates with improved activity, stability and reusability characteristic in water-in-ionic liquid microemulsion

“…CLEAs proved significantly more stable to denaturation by heat, organic solvents and proteolysis than the corresponding soluble enzyme . Despite the process of preparing CLEAs being simple and cost effective, CLEAs prepared in aqueous solution by conventional methods form amorphous large clusters that result in significant mass‐transfer limitations, and hamper CLEAs dispersing again in solution, leading to low catalytic efficiency …”

Solid state fermentation with recovery of Amyloglucosidase from extract by direct immobilization in cross linked enzyme aggregate for starch hydrolysis

“…Whereas, methodology of CLEA does not require purified enzymes, as it involve cross-linking of precipitated enzymes which need not be in it's pure form (Aytar and Bakir, 2008;Pan et al, 2011;Talekar et al, 2012a;Talekar et al, 2012b). The general methodology of CLEA has been widely used in various earlier studies (Agyei and He, 2015;Cui et al, 2014;García-García et al, 2012;Hormigo et al, 2012;Maria et al, 2011;Pan et al, 2011;Talekar et al, 2012a;Vaidya et al, 2012;Wang et al, 2011) and is also used in this study.…”