Encapsulation of an Agrobacterium radiobacter extract containing d-hydantoinase and d-carbamoylase activities into alginate–chitosan polyelectrolyte complexes: Preparation of the biocatalyst

Alginate–chitosan polyelectrolyte complexes (PECs) have been used for the first time as a suitable matrix for coimmobilisation of enzymes to reproduce a multistep enzymatic route for production of d-amino acids. Encapsulation of a crude cell extract from Agrobacterium radiobacter containing d-hydantoinase and d-carbamoylase activities into the PECs with negligible leakage from the formed capsules was accomplished. All results in this study indicate that the preparation of the biocatalyst (preparation method and chitosan characteristics) play a key role in the biocatalyst's properties. The most suitable biocatalysts were prepared using a chitosan with a medium molecular weight (600 kDa) and a degree of deacetylation of 0.9. For all of the preparation conditions under study, an encapsulation yield of around 60% was achieved and the enzymatic activity yields ranged from 30 to 80% for d-hydantoinase activity and from 40 to 128% for d-carbamoylase activity relative to the activities of the soluble extract. All of the biocatalysts were able to hydrolyze l,d-hydroxyphenylhydantoin into p-hydroxyphenylglycine with yields ranging from 30 to 80%.     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.     

Purification of a -hydantoinase using a laboratory-scale Streamline phenyl column as the initial step

A -hydantoinase from Thermus sp. was overexpressed in Escherichia coli and purified to homogeneity for subsequent crystallization. The purification was performed with hydrophobic interaction chromatography as the capture step followed by anion-exchange chromatography and gel permeation chromatography as intermediate purification and polishing steps, respectively. The hydrophobic interaction step was done in fluidized bed mode in a laboratory-scale Streamline column made from conventional laboratory equipment. The whole purification protocol could be finished within one day. The purified enzyme crystallizes. The crystals are suitable for X-ray protein structure analysis and diffract to at least 2.3 Å resolution. Complete data sets have been measured up to 2.6 Å resolution. The X-ray structure is currently being solved.     

Structure-based mutational analysis of the active site residues of -hydantoinase

We previously proposed the hydrophobic and bulky residues of the three loops, designated stereochemistry gate loops (SGLs), to constitute a hydrophobic substrate binding pocket of -hydantoinase from Bacillus stearothermophilus SD1. Simulation of substrate binding in the active site of -hydantoinase and sequence alignment of various -hydantoinases revealed the critical hydrophobic residues closely located around the exocyclic substituent of substrate. To evaluate the roles of these residues in substrate binding pocket, site-directed mutagenesis was performed specifically for Leu 65, Tyr 155, and Phe 159. When Tyr 155 was mutated to Phe and Glu, both mutants Y155F and Y155E were totally inactive for nonsubstituted hydantoin and -5-hydroxyphenyl hydantoin (HPH), which indicates that Tyr 155 is involved in substrate binding via a hydrogen bond with the hydantoinic ring. Furthermore, replacement of the hydrophobic residues Leu 65 and Phe 159 with Glu, a charged amino acid, resulted in a significant decrease in activity for nonsubstituted hydantoin, but not for HPH. The K_cat values of both mutants for nonsubstituted hydantoin also severely decreased, but a slight change in the K_cat values was observed towards HPH. These results suggest that the hydrophobic residues in SGLs play an essential role in substrate binding, and differentially interact according to the property of the exocyclic substituent.     

Immobilization of d-hydantoinase in polyaniline

Polyaniline (PANI) is a water-insoluble polymer that has been used as support for enzyme immobilization due to its desirable characteristics, such as ease of preparation, high synthesis yield, high stability to temperature and pH, and resistance to microbial attack. In this work an investigation was carried out to determine the best conditions to immobilize d-hydantoinase (E.C. 3.5.2.2) in this support. As result, a simple and fast methodology for d-hydantoinase immobilization in PANI is described. 100% of proteins were immobilized on the support in concentrations up to 2 mg solid/ml. Higher concentrations led to a lower protein percentage immobilized. After five reaction cycles about a half of d-hydantoinase initial activity was conserved.     

The immobilization of d-hydantoinase and characterization under classic condition and microwave irradiation

d-Hydantoinase was covalently immobilized onto polystyrene anion exchange resin via glutaraldehyde. Immobilization conditions were optimized: the carrier as D-92 type polystyrene anion exchange resin, temperature as 25 °C, immobilization time as 12 h, and initial concentration of protein as 6 mg/ml. Under the optimized reaction conditions the activity of the free and immobilized d-hydantoinase was determined. The free and immobilized d-hydantoinase samples were characterized with their kinetic parameters, thermal, and storage stability. The K_m and V_max values were 14.985 mM and 0.6 mM/min for the free, and 27.030 mM and 1.187 mM/min for the immobilized, respectively. Operational stability of the immobilized d-hydantoinase was also detected in a circulating packed-bed reactor. The half-time of the immobilized d-hydantoinase was 11 days. Nearly 90% of activity of the immobilized d-hydantoinase was reserved for 100 days stored at 4 °C. The free and immobilized d-hydantoinases were also characterized under microwave irradiation. Results shown that the reactions catalyzed by both free and immobilized d-hydantoinase were accelerated under microwave irradiation. The half-time of the immobilized d-hydantoinase reduced to 16 min under microwave irradiation.