A novel process using immobilized engineered strain HC01 cells with co-expressed D-hydantoinase and D-N-carbamoylase as biocatalyst for the production of D-Valine.

The demand for D-Valine increases because of its wide range of use. A whole-cell biocatalyst for the production of D-Valine from 5'-isopropyl hydantoin by co-expression of the D-hydantoinase (hyd) gene from Pseudomonas putida YZ-26 and D-N-carbamoylase (cab) gene from Sinorhizobium sp. SS-ori in Escherichia coli BL 21 (DE3) was developed. The expression condition of the engineered strain HC01 co-expressing D-hydantoinase (HYD) and D-N-carbamoylase (CAB) was optimized. HYD and CAB reached the highest activities (4.65 and 0.75 U/ml-broth) after inducing for 8-12 h. Subsequently, the cells of HC01 were immobilized in the form of Ca2+-alginate beads, and the optimal conditions for immobilizing were obtained as 2.5% gel concentration and 0.029 g/mL cell concentration in the presence of 3% CaCl2. The thermostability of immobilized cells was 5 ℃ higher than that of free cells in the same condition. And the divalent metal ions such as Mn2+, Mg2+, Cu2+, Co2+, and Ni2+ did not significantly affect the enzymatic activity of HYD and CAB in immobilized cells. Bioconversion rate reached to 91% after a 42-h reaction when the substrate concentration was 50 mmol/L with the initial pH of 9.0 under the nitrogen protection. This method provides D-Valine with optical purity of 97% and an overall yield of 72%. Furthermore, the immobilized cells can be reused for more than 7 cycles and maintain their capacity of over 70%. Hence the immobilized cells of engineered strain HC01 could potentially be used to prepare D-Valine.

Old pesticide, new use: Smart and safe enantiomer of isocarbophos in locust control.

Locust plagues are still worldwide problems. Selecting active enantiomers from current chiral insecticides is necessary for controlling locusts and mitigating the pesticide pollution in agricultural lands. Herein, two enantiomers of isocarbophos (ICP) were separated and the enantioselectivity in insecticidal activity against the pest Locusta migratoria manilensis (L. migratoria) and mechanisms were investigated. The significant difference of LD50 between (+)-ICP (0.609 mg/kg bw) and (-)-ICP (79.412 mg/kg bw) demonstrated that (+)-ICP was a more effective enantiomer. The enantioselectivity in insecticidal activity of ICP enantiomers could be attributed to the selective affinity to acetylcholinesterase (AChE). Results of in vivo and in vitro assays suggested that AChE was more sensitive to (+)-ICP. In addition, molecular docking showed that the -CDOKER energies of (+)-ICP and (-)-ICP were 25.6652 and 24.4169, respectively, which suggested a stronger affinity between (+)-ICP and AChE. Significant selectivity also occurred in detoxifying enzymes activities (carboxylesterases (CarEs) and glutathione S-transferases (GSTs)) and related gene expressions. Suppression of detoxifying enzymes activities with (+)-ICP treatment suggested that (-)-ICP may induce the detoxifying enzyme-mediated ICP resistance. A more comprehensive understanding of the enantioselectivity of ICP is necessary for improving regulation and risk assessment of ICP.

[Analysis of 2, 3-pyridinedicarboximide and its products conversed by engineering strain with high performance liquid chromatography].

A high performance liquid chromatography (HPLC) method was established for the detection of 2, 3-pyridinedicarboximide (PDI) and its enzyme reaction products, 3-carbamoyl-α-picolinic acid (α-3CP), using an engineering strain containing the D-hydantoinase gene expression box.The strain pET3a-hyd/BL21(DE3) was collected after induction and added to a PDI saturated aqueous solution.After reacting at 37℃ for 30 min with constant stirring, the supernatant was separated by centrifugation at 13000 r/min and detected by HPLC.The chromatographic conditions were as follows:HypersilTM GOLD C18 column (250 mm×4.6 mm, 5 µm), H2O-acetonitrile (90:10, v/v) containing 0.1%(v/v) trifluoroacetic acid as the mobile phase with a flow rate of 1 mL/min and a detection wavelength of 254 nm.The specific activity of pET3a-hyd/BL21(DE3) was found to be 0.61 U/(mL·10OD600 nm).This study provides a theoretical basis for the preparation of complicated half-amides using biological methods.

Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

Human enterokinase (synonym: enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia coli codon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104 U/mg.

[Reactivity and function of cysteine residues in imidase from Pseudomonas putida YZ-26].

OBJECTIVE: We investigated the reactivity and function of two cysteine residues in imidase (CIH) by site-directed mutagenesis. METHODS: Three variants of imidase (CIH) were constructed with Cys7 and Cys108 or only one of them substituted with Gly. The two thiol groups of Cys7 and Cys108 of imidase were specifically modified separately or collectively by dithio-bis-nitrobenzoic acid (DTNB) in the native state. It was also confirmed by SDS-PAGE analysis. To further verify the above results, the oligomeric structure and sulfhedryl groups of native and mutant CIH were also examined by measuring zinc binding ability and molecular size under different concentrations of H2O2. RESULTS: Compared with CIH, CIH108 retained 72% activity, while CIH7, 108 and CIH7 had no activity using DL-hydantoin as substrate. The spectral detection result shown that the two thiol groups were both in a free state. It is indicated that CIH and CIH108 are tetramer, CIH7,108 is multimer, and CIH7 is a mixture of monomer and multimer. The zinc binding ability of CIH108 was still relative high, while CIH7 and CIH7,108 decreased obviously. The increased concentration of H2O2 could increase the intrachain disulfide bond of CIH and the interchain disulfide bond of CIH108. CONCLUSION: All data imply that the Cys7 is required for binding zinc ion and maintaining the stable structure of enzymatic molecule.

Potential roles of recombinant acetylcholine receptor α subunit 1-211 in immunoadsorbent and DNA immunization.

The open reading frame of the α-subunit (amino acids 1-211) of human muscle nicotinic acetylcholine receptor (hAChR(α211)) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChR(α211) (recombinant hAChR(α211)) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K-hAChR(α211) with a yield of 25 mg/L. rhAChR(α211) was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DNA fragment of hAChR(α211) was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA-hAChR(α211). Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding antibody, AChRAb, was detected in mice sera by ELISA. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. It provides rapid and efficient methods to remove specific acetylcholine receptor antibody from the patient's sera and establish an animal model of myasthenia gravis by recombinant hAChR(α211) immunization.

Quantitative analysis and functional evaluation of zinc ion in the D-hydantoinase from Pseudomonas putida YZ-26.

D-hydantoinase (HDT) is a metal-dependent enzyme that is widely used in industrial bioconversion to D-amino acids as valuable intermediates in the fields of food, pharmaceutical industry and agriculture. In this report, we prepared apo-HDT (metal-removed HDT) and Zn(2+)-HDT (Zn(2+)-added HDT) in vitro from a recombinant HDT (re-HDT) expressed in E. coli. The Zn(2+)-HDT and re-HDT contain 2.17 and 0.95 mol Zn(2+) per mol subunit, respectively, and they have comparable enzymatic activities. In contrast, the apo-HDT only retains 0.04 mol Zn(2+) per mol subunit with less than 10% activity, compared with the re-HDT. When the apo-HDT was reconstituted with ZnCl(2), the enzymatic activity recovery was about 75%. Moreover, the fluorescence intensity, circular dichroism spectra and thermo-stability of the apo-HDT and Zn(2+)-HDT are quite different from those of the re-HDT. These data suggest that the re-HDT may have two Zn(2+)-binding sites, one is an intrinsic or tight-binding site (zinc-alpha) essential for its activity and the other is a vacant or loose-binding site (zinc-beta) possibly non-essential for the activity.

The flexibility of the non-conservative region at the C terminus of D-hydantoinase from Pseudomonas putida YZ-26 is extremely limited.

We previously reported that a deletion mutant (P478) with a residue Arg deleted at the C terminus of D-hydantoinase (P479) from Pseudomonas putida YZ-26 was dissociated into the monomer from its dimeric state. Based on the above result, a series of mutants of the enzyme with the C-terminal residues either deleted or substituted were prepared. The size-exclusion chromatography and bioactivity assay show that a C-terminal-substituted enzyme (R479D) and several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained. In contrast, two other mutants (R474 and R479A) are expressed in the form of random aggregates without any activity. Our experiments demonstrate that only the last four amino acids (-PVQR) at the C terminus of the enzyme can be deleted without seriously affecting its activity, although the enzyme is dissociated from a dimer into a monomer. These mutants also reveal some unique properties such as the enzymatic activity in vivo or in vitro, the effect of divalent metal ions, and the thermostability etc. in comparison to wild-type enzyme (P479). In addition, the three-dimensional structural modeling shows that the intact structure of the enzyme is essential, and the flexibility of the non-conservative region at the C terminus of the enzyme is quite limited.

Zinc binding site in PICK1 is dominantly located at the CPC motif of its PDZ domain.

PICK1 (protein interacting with Ckinase 1) containing a PDZ domain, a BAR domain, and two short acidic regions is as an adaptor protein that plays an important role in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor trafficking, cell morphology and migration, as well as in some diseases such as cancer, schizophrenia and pain. To better understand the physiological function of PICK1, we expressed the recombinant PICK1 and its truncated mutants in E.coli, and measured their zinc binding properties by fluorescence and competition assay. It is shown that PICK1 has one Zn2+-binding site. The Zn2+-binding properties of PICK1 are not appreciably affected after the removal of BARC domain (involving BAR domain and C-terminal acidic region). Deleting the N-terminal acidic region of NPDZ domain (involving PDZ domain and N-terminal acidic region) in PICK1 impairs its Zn2+-binding capacity. The mutation of the CPC (Cys-Pro-Cys) motif in the PDZ domain of PICK1 abolishes the ability of Zn2+-binding. In addition, Zn2+ can enhance the lipid-binding ability of PDZ domain as observed in both protein-lipid overlay assay and fluorescence analysis. The results presented in this report suggested that Zn2+ plays a regulatory role in the trafficking of PICK1 from the cytoplasm to cell membrane.

Subunit dissociation and stability alteration of D hydantoinase deleted at the terminal amino acid residue.

Two variants of D hydantoinase (HYD), created by deletion of one amino acid residue of at either the N- or C-terminus, were expressed in Escherichia coli and purified by two-step chromatography. Compared with HYD, HYDc1 with the C-terminal Arg deletion retained 43% activity, while HYDn1 with the N-terminal Ser deletion had no activity using DL Hydantoin as substrate. Based on HYD dimer with a molecular weight of 103 kDa, HYDc1 is a monomer of 52 kDa and HYDn1 is a mixture of dimer and monomer. Moreover, HYDc1 displayed higher pH stability and lower thermal stability compared to HYD. In addition, the secondary and tertiary structures of HYDc1 were not significantly changed in contrast to the ones of HYDn1. All data imply that the C-terminal Arg of the HYD is crucial for homodimeric architecture of the enzyme, but non-essential for catalysis, while the N-terminal Ser is required for both conformation and catalysis of the enzyme.

[Molecular form and stability of D-hydantoinase deleted at C-terminal residue Arg].

This report is about only deleting one C-terminal residue of D-hydantoinase to result in obvious changes on its molecular form and stability. A recombinant D-hydantoinase (P479) and its mutant enzyme deleted at C-terminal residue Arg (P478) were prepared by methods of gene cloning, expression and purification. Results show that the subunit molecular weight of P479 and P478 is the same (54kDa) as determined by SDS-PAGE, whilst the molecular form of native P479 and P478 is a dimer and a monomer respectively in the completely operative conditions. Compared with P479, the enzymatic activity of P478 for substrate hydantoin maintained about 40% and pH stability was obviously increased, at the alkaline side in particular, as well as the anti-SDS ability was also raised. However, the thermal stability for P478 was clearly lowed as compared to P479. It implies from above data that the C-terminal residue Arg of the D-hydantoinase is a crucial one for subunit dissociation, but non-essential for catalysis.