Hypoxia-Inducible Factor-1α-Activated Protein Switch Based on Allosteric Self-Splicing Reduces Nonspecific Cytotoxicity of Pharmaceutical Drugs.

Protein-based therapeutic agents currently used for targeted tumor therapy exhibit limited penetrability, nonspecific toxicity, and a short circulation half-life. Although targeting cell surface receptors improves cancer selectivity, the receptors are also slightly expressed in normal cells; consequently, the nonspecific toxicity of recombinant protein-based therapeutic agents has not been eliminated. In this study, an allosteric-regulated protein switch was designed that achieved cytoplasmic reorganization of engineered immunotoxins in tumor cells via interactions between allosteric self-splicing elements and cancer markers. It can target the accumulated HIF-1α in hypoxic cancer cells and undergo allosteric activation, and the splicing products were present in hypoxic cancer cells but were absent in normoxic cells, selectively killing tumor cells and reducing nonspecific toxicity to normal cells. The engineered pro-protein provides a platform for targeted therapy of tumors while offering a novel universal strategy for combining the activation of therapeutic functions with specific cancer markers. The allosteric self-splicing element is a powerful tool that significantly reduces the nonspecific cytotoxicity of therapeutic proteins.

Application of plasmid stabilization systems for heterologous protein expression in Escherichia coli.

BACKGROUND: Plasmids are the most commonly used vectors for heterologous protein expression in Escherichia coli. However, the plasmid copy number decreases with the segregational instability, which inevitably leads to a decrease in the yield of heterologous protein. METHODS AND RESULTS: In this study, plasmid stabilization systems were used to enhance the expression level of heterologous proteins in E. coli. With the investigation of protein expression level, biomass and plasmid retention rate in different plasmid stabilization systems, the hok/sok system had the greatest potential on plasmid stabilization. In order to further investigate the molecular mechanism of hok/sok system, the structure of the binding region of hok mRNA and sok antisense RNA was modified based on the minimum free energy of mRNA, which resulted in the reduction of the binding efficiency of hok mRNA and sok asRNA, and then the toxicity of the Hok protein led to the decreased viability of the host cells. Finally, the hok/sok plasmid stabilization system was testified in 5 L fermenter, and the plasmid retention rate and protein expression level were significantly increased without the addition of antibiotics. CONCLUSIONS: This study lays a solid foundation for a deeper understanding of the mechanism of the hok/sok plasmid stabilization system and improving the productivity of heterologous protein in E. coli.

Single-cell atlas of the immune microenvironment reveals macrophage reprogramming and the potential dual macrophage-targeted strategy in multiple myeloma.

The tumour microenvironment (TME) plays a critical role in disease progression in multiple myeloma (MM). This study aimed to present an atlas of MM-TME in disease progression and explore TME-directed therapeutic strategies. We performed single-cell RNA sequencing (scRNAseq) in samples from different disease stages. We validated the findings by bulk RNAseq, flow cytometry (FCM) and in vitro and in vivo functional experiments. We delineated a compromised TME during disease progression, characterized by enrichment of exhausted NK cells and CD8+ T cells and reprogramming of macrophages (MPs). The reprogrammed tumour-associated MPs (TAMs) displayed a mixed phenotype showing both M1 and M2 features, with two TAM clusters exclusively present in the MM stage showing higher M2 scores. We validated the mixed M1/M2 phenotype in TAMs in a clinical cohort and verified phagocytic dysfunction in reprogrammed TAMs. Cellular interaction analysis identified two enriched ligand-receptor pairs between MPs and malignant plasma cells (PCs), including the SIRPA-CD47 pathway suppressing phagocytosis and the CD74-MIF (macrophage inhibitory factor) reshaping the phenotype of MPs. The expression of CD47 and MIF correlated with disease progression and adverse outcomes. We designed a dual-MP-targeted strategy by combining an anti-CD47 antibody and MIF inhibitor to activate phagocytosis and repolarize MP to a functional phenotype and proved its potent antitumour effect in vitro and in vivo. We drafted alterations in MM-TME during disease progression and unravelled TAM's reprogramming. The dual MP-targeted approach blocking both CD47 and MIF showed potent antitumour effects.

Novel Oxygen-Dependent Degradable Immunotoxin Regulated by the Ubiquitin-Proteasome System Reduces Nonspecific Cytotoxicity.

The use of bacterial toxins as antitumor agents has received considerable attention. Immunotoxins based on antigen recognition of single-chain antibodies have been widely explored for cancer therapy. Despite their impressive killing effect on tumor cells, immunotoxins still display unspecific toxicity with undesired side effects. High levels of hypoxia-inducible factor 1α (HIF-1α) are well-known indicators of hypoxia in cancer cells. In this study, different linkers were employed to fuse the immunotoxin DAB389-4D5 scFv (DS) with the oxygen-dependent degradation domain (ODDD) of HIF-1α, a domain selectively facilitating the accumulation of HIF-1α under hypoxia, to construct the oxygen-dependent degradable immunotoxin DS-ODDD (DSO). The engineered fusion protein DSO-2 containing a linker (G4S)3 possesses the best killing effect on cancer cells under hypoxia and displayed considerably reduced nonspecific toxicity to normal cells under normoxic conditions. Flow cytometry, immunofluorescence, and immunoblot analyses demonstrated that DSO-2 was degraded via the ubiquitin-proteasome pathway regulated by the oxygen-sensitive mechanism. Western blot analysis indicated that the degradation of DSO-2 significantly decreased the activation of apoptosis-related molecules in normal cells. The engineered immunotoxin with oxygen-sensing properties developed herein is a potential therapeutic agent for cancer treatment.

A Machine Learning Model to Predict Survival and Therapeutic Responses in Multiple Myeloma.

Multiple myeloma (MM) is a highly heterogeneous hematologic tumor. Ubiquitin proteasome pathways (UPP) play a vital role in its initiation and development. We used cox regression analysis and least absolute shrinkage and selector operation (LASSO) to select ubiquitin proteasome pathway associated genes (UPPGs) correlated with the overall survival (OS) of MM patients in a Gene Expression Omnibus (GEO) dataset, and we formed this into ubiquitin proteasome pathway risk score (UPPRS). The association between clinical outcomes and responses triggered by proteasome inhibitors (PIs) and UPPRS were evaluated. MMRF CoMMpass was used for validation. We applied machine learning algorithms to MM clinical and UPPRS in the whole cohort to make a prognostic nomogram. Single-cell data and vitro experiments were performed to unravel the mechanism and functions of UPPRS. UPPRS consisting of 9 genes showed a strong ability to predict OS in MM patients. Additionally, UPPRS can be used to sort out the patients who would gain more benefits from PIs. A machine learning model incorporating UPPRS and International Staging System (ISS) improved survival prediction in both datasets compared to the revisions of ISS. At the single-cell level, high-risk UPPRS myeloma cells exhibited increased cell adhesion. Targeted UPPGs effectively inhibited myeloma cells in vitro. The UPP genes risk score is a helpful tool for risk stratification in MM patients, particularly those treated with PIs.

Rational engineering in Escherichia coli for high-titer production of baicalein based on genome-scale target identification.

Baicalein is a bioactive flavonoid isolated from the traditional Chinese medicinal plant, Scutellaria baicalensis Georgi. Microbial synthesis of flavonoids has been intensively developed owing to the eco-friendly nature of the process. However, the titer of the flavonoids obtained is still at a low level, and effective methods to enhance these titers are lacking. In this study, the synthetic performance of baicalein-producing engineered Escherichia coli was rationally evaluated to enhance the expression of key enzymes. Transcriptional analyses of baicalein-overproducing strain and a control strain enabled the identification of 13 beneficial genes, including eight genes that are seemingly irrelevant to baicalein metabolism. With the combination of the enzyme assembly and modularization strategy, the engineered DN-8 strain produced 367.8 mg/L baicalein in fed-batch fermentation, the maximum titer reported to date.

SAC-TRAIL, a novel anticancer fusion protein: expression, purification, and functional characterization.

Recombinant protein pharmaceutical agents have been widely used for cancer treatment. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has broad-spectrum antitumor activity, its clinical applications are limited because most tumor cells eventually develop resistance to TRAIL-induced apoptosis through various pathways. Prostate apoptosis response-4 (Par-4) selectively induces apoptosis in cancer cells after binding to the cell surface receptor, GRP78. In this study, TRAIL was fused with the core domain of Par-4 (SAC) to produce a novel recombinant fusion protein. To obtain solubly expressed fusion protein, a small ubiquitin-related modifier (SUMO) was added to the N-terminus of the target protein. Cytotoxicity assays showed that the purified fusion protein exhibited more significant antitumor activity on cancer cells than that by native TRAIL. The connection order and linker sequence of the fusion proteins were optimized. In vitro cytotoxicity assay showed that the SAC-TRAIL fusion protein, which contained a flexible linker (G4S)3, optimally inhibited the proliferation of cancer cells. Immunofluorescence assays demonstrated that SAC-TRAIL could efficiently and specifically bind to cancer cells. Additionally, circular dichroism assays showed that the secondary structure of the recombinant protein with a flexible linker (G4S)3 has both a lower α-helix and higher random coiling, which facilitates the specific binding of SAC-TRAIL to the receptor. Collectively, these results suggest that the novel recombinant fusion protein SAC-(G4S)3-TRAIL is a potential therapeutic agent for cancer. KEY POINTS: • Improved tumor growth suppression and apoptosis induction potency of SAC-TRAIL. • Enhanced targeting selectivity of SAC-TRAIL in cancer cells. • Lower α-helix and higher random coiling in SAC-TRAIL with flexible linker (G4S)3.

Screening and characterization of a nitrilase with significant nitrile hydratase activity.

PURPOSE: We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase. METHODS: A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as "amide-formation hotspots") as molecular probes to identify putative amide-forming nitrilases. RESULTS: Based on the previously reported "amide-formation hotspots," we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various benzylic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation-mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase. CONCLUSIONS: NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of benzylic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.

Rapid Mining of Novel α-Glucosidase and Lipase Inhibitors from Streptomyces sp. HO1518 Using UPLC-QTOF-MS/MS.

A rapid and sensitive method using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) was applied for the analysis of the metabolic profile of acarviostatin-containing aminooligosaccharides derived from Streptomyces sp. HO1518. A total of ninety-eight aminooligosaccharides, including eighty potential new compounds, were detected mainly based on the characteristic fragment ions originating from quinovosidic bond cleavages in their molecules. Following an LC-MS-guided separation technique, seven new aminooligosaccharides (10-16) along with four known related compounds (17-20) were obtained directly from the crude extract of strain HO1518. Compounds 10-13 represent the first examples of aminooligosaccharides with a rare acarviostatin II02-type structure. In addition, all isolates displayed considerable inhibitory effects on three digestive enzymes, which revealed that the number of the pseudo-trisaccharide core(s), the feasible length of the oligosaccharides, and acyl side chain exerted a crucial influence on their bioactivities. These results demonstrated that the UPLC-QTOF-MS/MS-based metabolomics approach could be applied for the rapid identification of aminooligosaccharides and other similar structures in complex samples. Furthermore, this study highlights the potential of acylated aminooligosaccharides with conspicuous α-glucosidase and lipase inhibition for the future development of multi-target anti-diabetic drugs.

Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction.

Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.

The expression, purification, and functional evaluation of the novel tumor suppressor fusion protein IL-24-CN.

Interleukin-24 (IL-24) can specifically induce apoptosis in a broad range of cancer cells without harming normal cells. The interaction of contortrostatin (CN) with integrins on angiogenic vascular endothelial and tumor cells is modulated by the RGD motifs that can significantly inhibit metastasis and angiogenesis. To achieve superior therapeutic efficacy by combining anti-metastasis with tumor-selective apoptosis activity, CN was fused at the C-terminus of IL-24 with a flexible linker (G4S)2, and the recombinant IL-24-CN was expressed in Escherichia coli as a Thioredoxin (Trx)/IL-24-CN fusion protein. The target protein was purified using nickel affinity chromatography. Furthermore, we simplified the purification process by purifying Trx-IL-24-CN and cleaving the Trx tag in one step. The final yield of IL-24-CN was 27.6 mg/L based on flask fermentation. In vitro activity assay demonstrated that the recombinant IL-24-CN could more effectively suppress tumor growth and induce apoptosis of melanoma cells. Scratch and transwell assays suggested that IL-24-CN strongly reduced the migration and invasion behavior of melanoma cells. Immunofluorescence analysis and cell adhesion assay showed that CN could evidently improve the tumor inhibition capability of IL-24 by enhancing the affinity of recombinant protein toward cancer cells. In summary, a highly efficient strategy was developed for producing the bioactive IL-24-CN from prokaryotic cells, supporting IL-24-CN in melanoma therapy.Key points• Efficient heterologous production of recombinant IL-24-CN in E. coli using Trx fusion strategy.• Improved tumor growth suppression and apoptosis induction potency of IL-24-CN.• Enhanced cell adhesion ability of IL-24-CN in cancer cells.

The yeast peroxisome: A dynamic storage depot and subcellular factory for squalene overproduction.

Engineering microbes to produce terpenes from renewable feedstock is a promising alternative to traditional production approaches. Generally, terpenes are not readily secreted by microbial cells, and their distribution within cells is usually obscure and often a restricting factor for the overproduction of terpenes due to the storage limitation. Here, we determined that squalene overproduced in the cytoplasm of Saccharomyces cerevisiae was distributed in a form similar to oil droplets. Interestingly, these suspected oil droplets were confirmed to be inflated peroxisomes that were swollen along with the production of squalene, indicating that peroxisomes in S. cerevisiae are dynamic depots for the storage of squalene. In view of this, harnessing peroxisomes as subcellular compartments for squalene synthesis was performed, achieving a 138-fold improvement in squalene titer (1312.82 mg/L) relative to the parent strain, suggesting that the peroxisome of S. cerevisiae is an efficient subcellular factory for the synthesis of terpenes. By dual modulation of cytoplasmic and peroxisomal engineering, the squalene titer was further improved to 1698.02 mg/L. After optimizing a two-stage fed-batch fermentation method, the squalene titer reached 11.00 g/L, the highest ever reported. This provides new insight into the synthesis and storage of squalene in peroxisomes and reveals the potential of harnessing peroxisomes to overproduce terpenes in S. cerevisiae through dual cytoplasmic-peroxisomal engineering.

Acylated Aminooligosaccharides from the Yellow Sea Streptomyces sp. HO1518 as Both α-Glucosidase and Lipase Inhibitors.

Three new acylated aminooligosaccharide (1-3), along with five known congeners (4-8), were isolated from the marine-derived Streptomyces sp. HO1518. Their structures were fully elucidated by extensive spectroscopic analysis, mainly based on 1D-selective and 2D TOCSY, HSQC-TOCSY, and HRESIMS spectrometry measurements, and by chemical transformations. All of the compounds were evaluated for their α-glucosidase and pancreatic lipase inhibitory activities. Among the isolates, D6-O-isobutyryl-acarviostatin II03 (3) and D6-O-acetyl-acarviostatin II03 (8), sharing acarviostatin II03-type structure, showed the most potent α-glucosidase and lipase inhibitory effects, far stronger than the antidiabetic acarbose towards α-glucosidase and almost equal to the anti-obesity orlistat towards lipase in vitro. This is the first report on inhibitory activities against the two major digestive enzymes for acylated aminooligosaccharides. The results from our investigation highlight the potential of acylated aminooligosaccharides for the future development of multi-target anti-diabetic drug.

Characterization and engineering control of the effects of reactive oxygen species on the conversion of sterols to steroid synthons in Mycobacterium neoaurum.

The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism. CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.

Enhanced conversion of sterols to steroid synthons by augmenting the peptidoglycan synthesis gene pbpB in Mycobacterium neoaurum.

Some species of mycobacteria have been modified to transform sterols to valuable steroid synthons. The unique cell wall of mycobacteria has been recognized as an important organelle to absorb sterols. Some cell wall inhibitors (e.g., vancomycin and glycine) have been validated to enhance sterol conversion by interfering with transpeptidation in peptidoglycan biosynthesis. Therefore, two transpeptidase genes, pbpA and pbpB, were selected to rationally modify the cell wall to simulate the enhancement effect of vancomycin and glycine on sterol conversion in a 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain (WIII). Unexpectedly, the pbpA or pbpB gene augmentation was conducive to the utilization of sterols. The pbpB augmentation strain WIII-pbpB was further investigated for its better performance. Compared to WIII, the morphology of WIII-pbpB was markedly changed from oval to spindle, indicating alterations of the cell wall. Biochemical analysis indicated that the altered cell wall properties of WIII-pbpB might contribute to the positive effect on sterol utilization. The productivity of 4-HBC was enhanced by 28% in the WIII-pbpB strain compared to that of WIII. These results demonstrated that the modification of peptidoglycan synthesis can improve the conversion of sterols to steroid synthons in mycobacteria.

Construction and characterization of the recombinant immunotoxin RTA-4D5-KDEL targeting HER2/neu-positive cancer cells and locating the endoplasmic reticulum.

The specific targeting of immunotoxins enables their wide application in cancer therapy. The A-chain of the ricin protein (RTA) is an N-glycosidase that catalyzes the removal of adenine from the 28S rRNA, preventing protein translation and leading to cell death. Ricin is highly toxic but can only exert its toxic effects from within the cytoplasm. In this study, we linked the anti-HER2 single-chain variable fragment 4D5 scFv and the endoplasmic reticulum-targeting peptide KDEL to the C-terminal of the RTA to construct immunotoxin RTA-4D5-KDEL. In vitro experiments showed that the anticancer effect of RTA-4D5-KDEL towards ovarian cancer cells SKOV-3 increased 440-fold and 28-fold relative to RTA and RTA-4D5, respectively. RTA-4D5-KDEL had a strong inhibitory effect on HER2-overexpressing SKOV-3 cells and caused little damage to normal HEK-293 cells and H460 lung cancer cells. Immunofluorescence experiments showed that the immunotoxin RTA-4D5 could specifically bind to SKOV-3 cells, but not to normal cells HEK-293. The immunotoxin RTA-4D5-KDEL could rapidly localize the recombinant protein to the endoplasmic reticulum. These results suggest that the recombinant immunotoxin RTA-4D5-KDEL has a strong inhibitory effect on ovarian cancer cells that overexpress HER2 but little harm to the normal cells.

Establishment of a low-dosage-IPTG inducible expression system construction method in Escherichia coli.

The lac operon is a delicate inducible gene expression element in bacteria. To efficiently induce gene expression, a sufficient dosage of an inducer, usually that of 500-1000 µM isopropyl ß-D-1-thiogalactopyranoside (IPTG), is required to keep repressor LacI from its binding sites, which is a heavy cost burden in low-value-added products. So we propose a strategy to reduce the required dosage of IPTG by restricting LacI expression. To test this strategy, we employed a reconstructed IPTG inducible expression system based on lac operon, Promoter(lacO)-target gene-PtacL-lacI, where a modified promoter, Ptac, with a random synthetic library (PtacL) to instead of PlacI to optimize LacI expression in Escherichia coli. Finally, the PtacL mutant, PtacL4, which could maintain the same repression effect as the original PlacI while reducing the required dosage of IPTG from 500 to 20 µM, was selected. This method is simple and efficient and can be of a good reference point for attempts to reduce inducer concentration in the IPTG or similar inducible expression systems.

Enhanced itaconic acid production by self-assembly of two biosynthetic enzymes in Escherichia coli.

Here, we described a novel strategy for the production of itaconic acid in Escherichia coli by self-assembly of aconitase (ACO) and cis-aconitate decarboxylase (CAD) existing in the metabolic pathway of itaconic acid via the protein-peptide interactions of PDZ domain and PDZ ligand. Co-expression of ACO and CAD in E. coli (uCA) resulted in low levels of itaconate (117.25 mg/L) after 48 h fermentation while the itaconate titre was significantly improved up to 222.15 mg/L by self-assembly of ACO-PDZ (APd) and CAD-PDZlig (CPl) in E. coli (sPP) under the same conditions. To further confirm the effect of self-assembly, the itaconate catalyzed from sodium citrate was determined. The sPP was extra efficacious in the early catalytic period, showing approximately threefold itaconate yields increased after 2 h catalysis, when compared to uCA. Furthermore, the itaconate production of sPP was increased from 5 to 8.7 g/L after 30 h of reaction compared to uCA. This self-assembly strategy showed remarkable potential for the further improvement of itaconate production. Biotechnol. Bioeng. 2017;114: 457-462. © 2016 Wiley Periodicals, Inc.

Characterization of a novel nitrilase, BGC4, from Paraburkholderia graminis showing wide-spectrum substrate specificity, a potential versatile biocatalyst for the degradation of nitriles.

OBJECTIVE: To investigate the biodegradation of nitriles via the nitrilase-mediated pathway. RESULTS: A novel nitrilase, BGC4, was identified from proteobacteria Paraburkholderia graminis CD41M and its potential for use in biodegradation of toxic nitriles in industrial effluents was studied. BGC4 was overexpressed in Escherichia coli BL21 (DE3), the recombinant protein was purified and its enzymatic properties analysed. Maximum activity of BGC4 nitrilase was at 30 °C and pH 7.6. BGC4 has a broad substrate activity towards aliphatic, heterocyclic, and aromatic nitriles, as well as arylacetonitriles. Iminodiacetonitrile, an aliphatic nitrile, was the optimal substrate but comparable activities were also observed with phenylacetonitrile and indole-3-acetonitrile. BGC4-expressing cells degraded industrial nitriles, such as acrylonitrile, adiponitrile, benzonitrile, mandelonitrile, and 3-cyanopyridine, showing good tolerance and conversion rates. CONCLUSION: BGC4 nitrilase has wide-spectrum substrate specificity and is suitable for efficient biodegradation of toxic nitriles.

A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production.

A novel aliphatic nitrilase, REH16, was found in Ralstonia eutropha H16 and overexpressed in Escherichia coli BL21(DE3), and its enzymatic properties were studied. The temperature and pH optima were 37 °C and 6.6, respectively, and the best thermostability of the nitrilase was observed at 25 °C, which preserved 95% of activity after 120 h of incubation. REH16 has a broad hydrolytic activity toward aliphatic and heterocyclic nitriles and showed high tolerance of 3-cyanopyridine; this enzyme could hydrolyze as high as 100 mM 3-cyanopyridine completely. To improve the 3-cyanopyridine conversion efficiency in an aqueous reaction system, water-miscible organic solvents were tested, and ethanol (10% v/v) was chosen as the optimal co-solvent. Finally, under optimized conditions, using the fed-batch reaction mode, total of 1050 mM 3-cyanopyridine was hydrolyzed completely in 20.8 h with eight substrate feedings, yielding 129.2 g/L production of nicotinic acid and thus showing a potential for industrial application.