Efficient Preparation of Enantiopure D-Phenylalanine through Asymmetric Resolution Using Immobilized Phenylalanine Ammonia-Lyase from Rhodotorula glutinis JN-1 in a Recirculating Packed-Bed Reactor

Abstract

An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase (RgPAL) from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA). The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR) was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h−1 and 0.32 g L−1 h−1, respectively. The optical purity (ee D) of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (ee D>99%) in the scaled-up reactor reached 7.2 g L−1 h−1. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine.

A microparticulate based formulation to protect therapeutic enzymes from proteolytic digestion: phenylalanine ammonia lyase as case study

“…With this type of particles an enzyme loading of 5% was achieved, while with MSP-l, which have pore sizes of 35 nm, a PAL loading of up to 8% was obtained under comparable loading conditions (PAL/MSP mass ratio 2:10). Similarly to what was reported by Zhu et al 22 , while PAL encapsulation increased upon raising the PAL/MSP mass ratio, the activity decreased. This could be due to: (i) the inaccessibility of the substrate (phe) to the PAL located deeper in the pores; (ii) and/or macromolecular crowding, meaning that in pores presenting a high concentration of the enzyme, the mobility of the latter is reduced, which could impact its activity 32 .…”

“…With our protocol an active protein (0.90 ± 0.20 IU/mg) was expressed, but with a low yield (0.65 ± 0.20 mg/L of culture). The expressed AvPAL was incorporated into MSP-s, MSP-l, and MSP-l-NH 2 by adsorption 22 . As reported in Table S2, the drug loading was higher for MSP presenting larger pores (8.5 ± 1.6% w/w) than for MSP with narrower pores (2.9 ± 1.8% w/w).…”

“…Since the PAL diameter is 9.6-14.5 nm, a pore size greater than 15 nm would be desired to accommodate the enzyme 23 . Previously, the encapsulation of PAL in a different type of MSP presenting a pore size ranging from 2 to 12 nm was reported 22 . With this type of particles an enzyme loading of 5% was achieved, while with MSP-l, which have pore sizes of 35 nm, a PAL loading of up to 8% was obtained under comparable loading conditions (PAL/MSP mass ratio 2:10).…”

Concept Design, Development and Preliminary Physical and Chemical Characterization of Tamoxifen-Guided-Mesoporous Silica Nanoparticles

“…Additionally, highly ordered mesopores and the unique crystallized lattices of MCM-41 were observed, as confirmed by the XRD measurement shown in Figure 1 c. The XRD spectrum displays three distinct diffraction peaks, assigned as (100), (110) and (200) planes [ 36 , 40 ]. Furthermore, as demonstrated by FT-IR ( Figure 1 d), the MSNs display a hydroxyl (−OH) signal [ 41 ], which is crucial for further surface grafting with PLH and TAM molecules.…”

“…( c ) MSNs demonstrated three distinct diffraction peaks, assigned as (100), (110) and (200) planes, a characteristic crystalized lattice of MCM-41 [ 36 , 40 ]. ( d ) FT-IR spectra of native MSNs demonstrating the (−OH) signal [ 41 ] which is essential for further surface grafting.…”