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A histidine acid phosphatase (HAP) (PhySc) with 99.50% protein sequence similarity with PHO5 from Saccharomyces cerevisiae was expressed functionally with the molecular mass of ∼110 kDa through co-expression along with the set of molecular chaperones dnaK, dnaJ, GroESL. The purified HAP illustrated the optimum activity of 28.75 ± 0.39 U/mg at pH 5.5 and 40 ˚C. The Km and Kcat values towards calcium phytate were 0.608 ± 0.09 mM and 650.89 ± 3.6 s− 1. The half-lives (T1/2) at 55 and 60 ˚C were 2.75 min and 55 s, respectively. The circular dichroism (CD) demonstrated that PhySc includes 30.5, 28.1, 21.3, and 20.1% of random coils, α-Helix, β-Turns, and β-Sheet, respectively. The Tm recorded by CD for PhySc was 56.5 ± 0.34˚C. The molecular docking illustrated that His59 and Asp322 act as catalytic residues in the PhySc. MD simulation showed that PhySc at 40 ˚C has higher structural stability over those of the temperatures 60 and 80 ˚C that support the thermodynamic in vitro investigations. Secondary structure content results obtained from MD simulation indicated that PhySc consists of 34.03, 33.09, 17.5, 12.31, and 3.05% of coil, helix, turn, sheet, and helix310, respectively, which is almost consistent with the experimental results. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) are protein modifications that occur after protein biosynthesis, playing a crucial role in regulating protein function. They are involved in the functional expression of G-protein-coupled receptors (GPCRs), as well as intracellular and secretory protein signaling. Here, we aimed to investigate the PTMs of the apelin receptor (APLNR), a GPCR and their potential influence on the receptor's function. In an in vitro experiment using HEK cells, we only observed glycosylation as a PTM of the APLNR and ineffective receptor signaling by the agonist, (Pyr1)-apelin-13. In contrast, when analyzing mouse spinal cord, we detected glycosylation and other PTMs, excluding isopeptidation. This suggests that additional PTMs are involved in the functional expression of the APLNR in vitro. In summary, these findings suggest that the APLNR in vivo requires multiple PTMs for functional expression. To comprehensively understand the pharmacological effects of the APLNR, it is essential to establish an in vitro system that adequately replicates the receptor's PTM profile. Nonetheless, it is crucial to overcome the challenge of heat-sensitive proteolysis in APLNR studies. By elucidating the regulation of PTMs, further research has the potential to advance the analysis and pharmacological studies of both the apelin/APLNR system and GPCR signal modulation. [ABSTRACT FROM AUTHOR]

Farnesyl/geranylgeranyl diphosphate synthases (FPPS/GGPPS) as the short‐chain prenyltransferases catalyse the formation of the acyclic precursors (E)‐FPP and (E)‐GGPP for isoprenoid biosynthesis. Here, we first cloned the cDNAs encoding FPPS and GGPPS in the vetch aphid Megoura viciae (designated as MvFPPS and MvGGPPS). They had an open reading frame of 1185 and 930 bp in length, encoding 395 and 309 amino acids, with a theoretical isoelectric point of 6.52 and 6.21, respectively. Sequence alignment and phylogenetic analysis showed that MvFPPS and MvGGPPS shared the conserved aspartate‐rich motifs characterized by all prenyltransferases identified to date and were clustered with their homologues in two large clades. RNA interference (RNAi) combined with gas chromatography/mass spectrometry (GC–MS) analysis showed that both MvFPPS and MvGGPPS were involved in the biosynthesis of alarm pheromone. Spatiotemporal expression profiling showed that the expression of MvFPPS and MvGGPPS was significantly higher in embryos than in other tissues. RNAi and GC–MS performed specifically in embryos corroborated the function of MvFPPS and MvGGPPS. In vitro, enzymatic activity assay and product analysis demonstrated that MvFPPS could catalysed the formation of (E)‐FPP using DMAPP or (E)‐GPP as the allylic cosubstrates in the presence of IPP, while MvGGPPS could only use (E)‐GPP or (E)‐FPP as cosubstrates. Functional interaction analysis using RNAi revealed that MvGGPPS exerts unidirectional functional compensation for MvFPPS. Moreover, it can regulate the biosynthesis of alarm pheromone by imposing a negative feedback regulation on MvFPPS. Our study helps to understand the molecular regulatory mechanism of terpenoid biosynthesis in the aphid. [ABSTRACT FROM AUTHOR]

A new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant known as Omicron has caused a rapid increase in recent global patients with coronavirus infectious disease 2019 (COVID-19). To overcome the COVID-19 Omicron variant, production of a recombinant spike receptor binding domain (RBD) is vital for developing a subunit vaccine or a neutralizing antibody. Although bacterial expression has many advantages in the production of recombinant proteins, the spike RBD expressed in a bacterial system experiences a folding problem related to disulfide bond formation. In this study, the soluble Omicron RBD was obtained by a disulfide isomerase-assisted periplasmic expression system in Escherichia coli. The Omicron RBD purified from E. coli was very well recognized by anti-SARS-CoV-2 antibodies, sotrovimab (S309), and CR3022, which were previously reported to bind to various SARS-CoV-2 variants. In addition, the kinetic parameters of the purified Omicron RBD upon binding to the human angiotensin-converting enzyme 2 (ACE2) were similar to those of the Omicron RBD produced in the mammalian expression system. These results suggest that an E. coli expression system would be suitable to produce functional and correctly folded spike RBDs of the next emerging SARS-CoV-2 variants quickly and inexpensively. [ABSTRACT FROM AUTHOR]

Background: This study aimed to analyze the in vitro effect of micro-strain stress on the proliferation and functional marker expression in chondrocytes isolated from human osteoarthritis cartilage samples. Methods: Chondrocytes isolated from human osteoarthritis cartilage samples were subjected to loading with different types of micro-strain stress. The proliferation activity was assessed by flow cytometry, and the functional expression of chondrocyte markers was detected by qRT-PCR and western blot. Results: Flow cytometry results showed stimulation of proliferation of human osteoarthritic chondrocytes when an adequate micro-strain stress was applied. qRT-PCR and western blot results showed that micro-strain stress promotes human osteoarthritic chondrocyte functional marker expression. These features coincide with the upregulation of multiple proteins and genes affecting cell proliferation and functional chondrocyte marker expression, including cyclin D1, collagen II, and Rock. Conclusion: Adequate micro-strain stress could activate the Rho/Rock signaling pathway in osteoarthritic chondrocytes, thus transmitting mechanical signals to the cytoskeleton. This process leads to cytoskeleton reorganization, and transmission of the mechanical signals to the downstream effectors to promote proliferation and functional marker expression of osteoarthritic chondrocytes. [ABSTRACT FROM AUTHOR]

This study focuses on an overlooked but critical issue: the composition and functional expression of microbial communities on the concrete surface in different areas of sewer pipes. Three immersion conditions were applied to simulate the duration of concrete in different areas ofsewers exposed to sewage, including short-term (L1), long-term (L2) and permanent immersion (L3). The properties of concrete under different immersion conditions and the bacterial diversity and functional capabilities in biofilms on the concrete surface were analyzed. Results showed that the L1 group was dominated by Halothiobacillus , whereas Desulfomicrobium was prominent in the L3 group. Significant differences in the predominant functional microbial communities and metabolic functional genes further confirmed the strong impact of immersion time on the pathways of microbial sulfur metabolism and concrete performance in sewer environment. Compared with the L2 and L3 groups, the decreased sewage immersion time resulted in an increase in the abundance and metabolic activity of sulfur-oxidizing bacteria in the L1 group. Hence, greater mass loss and gypsum production of concrete was found in the L1 group. The structural and functional differentiation of bacterial communities on the concrete surface observed in this study contributes to a better understanding of the uneven corrosion in real sewer pipes. [Display omitted] • Immersion conditions alter bacterial population involved in sulfur metabolism. • Sulfur oxidation functional genes are dominant under short-term immersion condition. • Biological sulfur oxidation can cause significant corrosion on concrete surfaces. [ABSTRACT FROM AUTHOR]

A high serum uric acid (SUA) concentration is associated with hyperuricemia (HUA) and gout. In order to obtain long-acting therapeutic effect, correction of purine metabolism at genetic level is advantageous. For this purpose, we expressed three "human-like" urate oxidases in human hepatocytes (HL-7702) by lentivirus-mediated transduction. Enzymatic assay revealed that the recombinant urate oxidases expressed in HL-7702 cells were functionally active. Electron microscopy study showed that the recombinant enzymes were localized to peroxisome and formed distinct crystalloid core structures as in other mammal cells. Although similar rate of uric acid degradation was observed for all recombinant urate oxidases, HL-7702-pLVX-UOX 83 cells and HL-7702-pLVX-UOX 214/217 cells retained more cell viability compared with HL-7702-pLVX-UOX PBC at high uric acid level. This study provides a new direction for the treatment of gout and hyperuricemia. [ABSTRACT FROM AUTHOR]

Summary: Black pepper (Piper nigrum L.) is known for its high content of piperine, a cinnamoyl amide derivative regarded as largely responsible for the pungent taste of this widely used spice. Despite its long history and worldwide use, the biosynthesis of piperine and related amides has been enigmatic up to now. In this report we describe a specific piperic acid CoA ligase from immature green fruits of P. nigrum. The corresponding enzyme was cloned and functionally expressed in E. coli. The recombinant enzyme displays a high specificity for piperic acid and does not accept the structurally related feruperic acid characterized by a similar C‐2 extension of the general C6–C3 phenylpropanoid structure. The enzyme is also inactive with the standard set of hydroxycinnamic acids tested including caffeic acid, 4‐coumaric acid, ferulic acid, and sinapic acid. Substrate specificity is corroborated by in silico modelling that suggests a perfect fit for the substrate piperic acid to the active site of the piperic acid CoA ligase. The CoA ligase gene shows its highest expression levels in immature green fruits, is also expressed in leaves and flowers, but not in roots. Virus‐induced gene silencing provided some preliminary indications that the production of piperoyl‐CoA is required for the biosynthesis of piperine in black pepper fruits. Significance Statement: In this report we provide biochemical and molecular evidence for a piperic acid CoA ligase from black pepper fruits, capable to produce piperoyl‐CoA from piperic acid, which both are considered precursors in the biosynthesis of piperine and related piperamides, the pungent principle of the Piperaceae. [ABSTRACT FROM AUTHOR]

An integrated approach for real‐time oscillatory stability assessment (OSA) based on mutual information theory is proposed in this study. An advanced maximum‐relevance minimum‐redundancy (MRMR) ensemble scheme is designed to explore the internal relations between operation variables and the oscillatory stability margin (OSM). Multiple MRMR procedures are generated in parallel to select multiple different feature subsets, in which each feature presents a relevant and complementary description of OSM. The functional expression of the relationships is obtained by curve fitting. The 21‐bus system and 1648‐bus system are implemented to test the performance of the proposed approach. A compared investigation is made with some other data mining methods. The impacts of the number of feature sets, size of feature sets, size of training set, invalid data and computation time are studied. Experimental results reveal that the proposed approach provides faster and more accurate assessment results and is a real‐time adaptive approach for OSA. [ABSTRACT FROM AUTHOR]

Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2, and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result. [ABSTRACT FROM AUTHOR]

The aim of this study was to establish a system for the functional expression, purification and identification of new subtypes of modified antioxidant peptides from Pinctada fucata muscle (named MMP). After molecular modification, MMP had a primary coil spatial conformation that resembled an α-helix and improved the functional activity of MMP. The MMP DNA sequence was modified and codon optimized based on the amino acid sequence of the natural antioxidant peptide (named MP), which was isolated and purified from Pinctada fucata muscle by enzymatic hydrolysis. Antioxidant activity assay results showed that the IC50 values for DPPH-radical scavenging activity, superoxide-radical scavenging activity and hydroxyl-radical scavenging activity of MMP were 0.056, 0.310 and 0.126 mg/ml, respectively, which were higher than the antioxidant activities of enzymatically digested or chemically synthesized MMP (p < 0.05). In addition, this protocol exhibited great advantage in purity, speed, cost, and degree of automation for preparing MMP compared with enzymatic hydrolysis or chemical synthesis. Thus, the proposed method can be used to obtain MMP with high antioxidant activity and at a high yield. In addition, this method provided test data and a theoretical basis for high-value application of polypeptides from marine sources. [ABSTRACT FROM AUTHOR]

The human sense of taste is devoted to the analysis of the chemical composition of food prior to ingestion. Among the five basic taste qualities bitter taste perception is believed to avoid ingestion of potentially toxic substances. The receptors facilitating the detection of hundreds of chemically different bitter compounds belong to the taste 2 receptor (TAS2R) family, which are part of the G protein-coupled superfamily. Although the chemical classes of bitter compounds that have been identified as agonists of one of the 25 potentially functional human bitter taste receptors cover an enormous chemical space, one distinct group of bitter compounds, the bitter salts have not been assigned to any bitter taste receptor. To close this gap, we screened the entire human bitter taste receptor repertoire by functional calcium mobilization assays with the most famous bitter salt, magnesium sulfate, also known as Epsom salt. Although the profound pharmacological activity and the bitter taste of spring water containing magnesium sulfate has been known since 1697, the molecular basis for its taste has not been elucidated until now. Our screening resulted in the identification of a single receptor, the TAS2R7, responding to magnesium sulfate at concentrations humans perceive this salt as bitter. Subsequently, TAS2R7 was stimulated with other salts and it was found that this receptor also responds to manganese2+ and iron2+ ions, but not to potassium ions. Magnesium sulfate is known to exert a number of beneficial effects on the human body and thus, has been used as medicine against premature uterine contractions, as anti-arrhythmic drug and as laxative, however, magnesium sulfate overdosage can result in cardiac arrest and thus have fatal consequences. Therefore, it appears reasonable that nature placed TAS2R7 as sentinel for high concentrations of bitter salts on our tongues. • The human bitter taste receptor TAS2R7 responds to bitter salts. • Bitter salts have profound pharmacological activity and thus, require oral monitoring. • Magnesium sulfate concentrations activating TAS2R7 match human perception. • Manganese and iron ions also activate TAS2R7, however, potassium ions do not. • The anions sulfate and chloride do not contribute to TAS2R7 activation. [ABSTRACT FROM AUTHOR]

The present study aimed to evaluate the functional expression of tree sinusiae in three sectors of a forest fragment in relation to different land use histories. The study recorded the number of individuals, number of individuals with multiple stems and number of tillers, calculated the percentages of tillered individuals and species, and then classified the species into regeneration and dispersal guilds. The most anthropised sector of the forest showed higher tillering and a larger proportion of pioneer and light-demanding climax species. The sector with moderate anthropisation presented intermediate tillering and mixed occurrence of light-demanding and shade-tolerant guilds. The non-anthropised sector showed lower tillering and a higher number of shade-tolerant species. The distribution of zoochorous individuals was not significant. In conclusion, the different levels of anthropisation provided distinct patterns of tillering and different compositions in regeneration guilds, highlighting their influence on the ecology of tropical forests. [ABSTRACT FROM AUTHOR]

Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni 2+ -NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies. [ABSTRACT FROM AUTHOR]

We investigated the molecular and biochemical properties of two acetylcholinesterases (FoAChE1 and FoAChE2) from the Western flower thrips, Frankliniella occidentalis. Polyacrylamide gel electrophoresis and western blotting confirmed the membrane-anchored nature of both FoAChE1 and FoAChE2, which was further supported by hydrophobicity and glycophosphatidylinositol anchor predictions. High expression levels of both enzymes were observed in the head, indicating their predominant distribution in neuronal tissues. FoAChE1 exhibited significantly higher expression levels in all examined tissues compared to FoAChE2, suggesting its major role as a synaptic enzyme. Nonetheless, both recombinant enzymes displayed robust catalytic activity toward acetylthiocholine iodide, and FoAChE1 demonstrated nearly identical catalytic efficiency compared to FoAChE2. FoAChE1 exhibited slightly lower sensitivities to the cholinesterase inhibitors tested, including organophosphates (OPs) and carbamates (CBs), compared to FoAChE2. Field populations of F. occidentalis exhibited polymorphism of alanine vs. serine at position 197 of FoAChE1 within the conserved oxyanion hole. Contrary to common belief, however, functional analysis using recombinant enzymes revealed that neither A197 nor S197 residue was associated with FoAChE1 insensitivity to OPs and CBs. FoAChE2 did not exhibit any polymorphic amino acid substitutions at the positions known to be associated with resistance. Due to the absence of apparent resistance-associated mutations in field populations of F. occidentalis , the judicious use of some OPs or CBs can be suggested for controlling the highly resistant populations to other insecticides. Overall, our findings highlight the significance of both FoAChE1 and FoAChE2 as targets for toxicity assessment, while the specific contribution of each enzyme to toxicity remains unclear. [Display omitted] • Both FoAChE1 and FoAChE2 exist in membrane anchored forms. • FoAChE1 is the main synaptic enzyme. • The catalytic activity might be mostly originated from FoAChE1. • FoAChE1 showed slightly lower sensitivities to cholinesterase inhibitors compared to FoAChE2. • Neither the A197 nor S197 residue was associated with FoAChE1 insensitivity to OPs and CBs. [ABSTRACT FROM AUTHOR]

Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications. [ABSTRACT FROM AUTHOR]

Versatile peroxidase (VP) secreted by white-rot fungi is involved in the degradation of lignin within land ecosystems. With a broad substrate scope and minor requirements, VP is an extremely attractive blueprint to be designed by the directed evolution tool-box. In recent years, improved VP variants have been generated that meet industrial requirements in terms of improved heterologous functional expression and activity, as well as stability at high temperatures or in the presence of strong inhibitors. This review describes the making of VP by directed evolution, addressing the most important findings and challenges that were faced, along with the future prospects for this research field. [ABSTRACT FROM PUBLISHER]

Cytochrome P450s (CYPs or P450s) have been long recognized as very important enzymes in the metabolism of xenobiotic and endogenous compounds, but only a few CYPs have been functionally characterized in insects. The effort in functional characterization of insect P450s is heavily hindered by technical difficulties in preparing active, individual P450 enzymes directly from the target insect. In this paper, we describe the functional expression of two additional pyrethroid resistance-associated CYP9A genes (CYP9A12 and CYP9A17) from the polyphagous pest Helicoverpa armigera in the facile Escherichia coli. The functionality of E. coli produced CYP9A12, CYP9A14, and CYP9A17 was investigated and activities of these CYP9As were compared against threeprobe substrates after reconstitutionwithNADPH-dependent cytochrome P450 reductase. The results showedthat active forms ofCYP9A12 andCYP9A17were expressed in E. coli with a content ofabout 1.0-1.5 nmolmg--1 protein inmembranepreparations. In vitro assays showed that CYP9A14was capable of catalyzingO-dealkylation ofmethoxyresorufin (MROD), ethoxyresorufin (EROD), and benzyloxyresorufin (BROD), while CYP9A12 and CYP9A17 exhibited onlyMRODand ERODactivities. Kinetic studies demonstrated that CYP9A14 had the greatest kcat/Km value forMROD, and CYP9A17 for EROD, while the lowest kcat/Km values for both MROD and EROD were observed for CYP9A12.The distinct biochemical traits suggest that the three paralogousCYP9Asmay play different roles in xenobiotic metabolism in this important pest. [ABSTRACT FROM AUTHOR]

CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus. This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p-nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat) 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin. [ABSTRACT FROM AUTHOR]

[Objectives] Aquaporin, a member of the major intrinsic protein superfamily, functions mainly for transporting water and small molecule compounds across cell membrane, and the aim of this study was to analyze the function of the aquaporin and its possible contribution to insecticide metabolism in an important agricultural pest Laodelphax striatellus. [Methods] In this study, aquaporin genes in L. striatellus were cloned based on the transcriptome data and with PCR and RACE cloning techniques. The cloned genes were subjected to phylogenetic relationship analysis by constructing phylogenetic tree with previously reported insect aquaporin genes, tests of the expression level in the susceptible and resistant strains by real-time quantitative PCR, and functional verification for their transporting capability by combinant expression in vitro. [Results] Six full length aquaporin genes were cloned from L. striatellus and designated as LsAQP1, LsAQP2, LsAQP3, LsAQP4, LsAQP5 and LsAQP6, respectively. Among them, LsAQP6 was found expressed with higher level in all three resistant strains tested as compared to the susceptible strain. Furthermore, functional expression showed that LsAQP6 had strong transport capacity for water and significant transport capacity for methomyl, but not for glycerol and bisultap. [Conclusions] Thus, it is speculated that LsAQP6 plays a significant role in transporting water and some small molecule insecticides in L. striatellus. [ABSTRACT FROM AUTHOR]

Phytocystatins play multiple roles in plant growth, development and resistance to pests and other environmental stresses. A ramie (Boehmeria nivea L.) phytocystatin gene, designated as BnCPI, was isolated from a ramie cDNA library and its full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence (691 bp) consisted of a 303 bp open reading frame (ORF) encoding a protein of 100 amino acids with deduced molecular mass of 11.06 kDa and a theoretical isoelectric point (pI) of 6.0. The alignment of genome DNA (accession No. MF153097) and cDNA sequences of BnCPI showed that an intron (~104 bp) exists in the coding region. The BnCPI protein contains most of the highly conserved blocks including Gly5-Gly6 at the N-terminal, the reactive site motif QxVxG (Q49V50V51S52G53), the L79-W80 block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (L22G23R24 F25A26V27 D28D29H30 N31) block that is common among plant cystatins. BLAST analysis indicated that BnCPI is similar to cystatins from Glycine max (77%), Glycine soja (76%), Hevea brasiliensis (75%) and Ricinus communis (75%). The BnCPI was subcloned into expression vector pSmart-I and then overexpressed in Escherichia coli BL21 (DE3) as a His-tagged recombinant protein. The purified reBnCPI has a molecular mass of 11.4 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS--PAGE). Purified reBnCPI can efficiently inhibit the protease activity of papain and ficin toward BANA (Nα-benzoyl-L-arginine-2-naphthyamide), as well as the mycelium growth of some important plant pathogenic fungi. The data further contribute to our understanding of the molecular functions of BnCPI. [ABSTRACT FROM AUTHOR]

The gene encoding a novel acidic lipoxygenase from Myxococcus xanthus DK1622 (accession: WP_011551853.1) was cloned into vector pET-28a and expressed in Escherichia coli BL21(DE3). The recombinant enzyme (rMxLOX), with a molecular weight of approximately 80 kDa, was purified to homogeneity using one-step nickel-affinity chromatography and showed an activity of 5.6 × 10 4 U/mg. The optimum pH and temperature for rMxLOX activity were found to be 3.0 and 30 °C, respectively. Purified rMxLOX exhibited activity towards linoleic acid and arachidonic acid as substrates, with linoleic acid being the better substrate (K m and k cat values of 0.048 mM and 13.3/s, respectively). The synthetic dye aniline blue was decolorized 69.7 ± 3.5%, following incubation with rMxLOX for 35 min. These results reveal the potential for the use of rMxLOX in the pulp, textile, and wastewater treatment industries. [ABSTRACT FROM AUTHOR]

The TRPV1 receptor plays a significant role in many biological processes, such as perception of external temperature (above 43°C), inflammation development, and thermoregulation. Activation of TRPV1 leads to the pain occurrence and decrease in the body temperature, while inhibition of this receptor can lead to an increase in the temperature. The TRPV1 peptide modulators from sea anemone Heteractis crispa extract (APHC1 and APHC3) have been previously characterized as molecules, which generated a pronounced analgesic effect and a decrease in the body temperature in experimental animals. Using the combined APHC1 and APHC3 amino acid sequences, we have prepared a hybrid peptide molecule named A13 that contains all residues potentially important for the activity of the peptide precursors. Biological tests on animals have shown that the hybrid molecule not only combines the analgesic properties of both peptides but, unlike the peptide precursors, also raises the body temperature of experimental animals. [ABSTRACT FROM AUTHOR]

Nitrile hydratase (NHase) has attracted substantial attention for industrial applications to produce large-scale amides. Several NHases have been investigated for functional expression in Escherichia coli ( E. coli ). A Fe-type NHase was obtained from an acetamiprid-degrading bacterium, Pseudoxanthomonas sp. AAP-7 and functionally expressed in E. coli BL21 (DE3). No significant NHase activity was detected from the E. coli expressing either the NHase gene alone or NHase and P46K genes transcribed as one unit. Purified recombinant NHase, co-expressed with P46K on two separate plasmids, exhibited the maximal enzyme activity. Furthermore, a GST tag attached to the N-terminus of α subunit resulted in a slight increase in the solubility and stability of NHase compared with a His tag at the C-terminus of β subunit. When co-expressed with the chaperones GroEL-GroES, the yield of the soluble recombinant NHase was improved substantially, while a small decrease in NHase activity was observed. The putative activator P46K was strictly required for production of the recombinant NHase for full enzyme activity, although the chaperones GroEL-GroES appeared to assist NHase to fold properly. This study of the expression of a fully active Fe-type NHase would provide another example to enhance our understanding of NHase biosynthesis. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome. [ABSTRACT FROM AUTHOR]

A rubber-degrading bacterial consortium named H2DA was obtained from an enrichment culture with natural rubber latex and rubber-processing factory waste in Vietnam. Gel permeation chromatography analysis revealed that only the strain NVL3 degraded synthetic poly( cis -1,4-isoprene) into low-molecular-weight intermediates among the three strains found in the H2DA. The 16S-rRNA gene sequence of NVL3 showed the highest identity with that of Nocardia farcinica DSM 43665 T . NVL3 accumulated aldehyde intermediates from synthetic poly( cis -1,4-isoprene) on a rubber-overlay plate as indicated by Schiff's staining. NVL3 also degraded deproteinized natural rubber into low-molecular-weight aldehyde intermediates. A latex-clearing protein ( lcp ) gene ortholog was identified within the genome sequence of NVL3, and it showed a moderate amino-acid identity (54–75%) with the lcp genes from previously reported rubber degraders. The heterologous expression of the NVL3 lcp in Escherichia coli BL21(DE3) allowed us to purify the 46.8-kDa His-tagged lcp gene product (His-Lcp). His-Lcp degraded synthetic poly( cis -1,4-isoprene) and accumulated aldehyde intermediates from deproteinized natural rubber suggesting the functional expression of the lcp gene from a Nocardia degrader in E. coli . Quantitative reverse transcription PCR analysis indicated the strong transcriptional induction of the lcp gene in NVL3 in the presence of synthetic poly( cis -1,4-isoprene). These results suggest the involvement of the lcp gene in rubber degradation in NVL3. [ABSTRACT FROM AUTHOR]

The American horseshoe crab, Limulus polyphemus, excretes nitrogenous waste in the form of toxic ammonia across their book gills. The mechanism of this branchial excretion is yet unknown. In the current study, two isoforms of a novel ammonia transporter, LpHIAT1α and LpHIAT1β, have been identified in L. polyphemus. Both isoforms have 12 predicted transmembrane regions and share 82.7% of amino acid identity to each other, and 77–86% amino acid homology to HIAT1 found in fish and crustaceans. In L. polyphemus , both isoforms were expressed in the gills, coxal glands, and brain. Slightly higher mRNA expression levels of LpHIAT1α were observed in the peripheral mitochondria-poor region of the gill (PMPA), central mitochondria-rich region of the gill (CMRA), and brain compared to the LpHIAT1β isoform. A functional expression analysis of LpHIAT1α and LpHIAT1β in Xenopus laevis oocytes resulted in a significantly lower uptake of the radiolabeled ammonia analogue 3H-methylamine when compared to controls, indicating an ammonia excretory function of the proteins. Exposure to elevated environmental ammonia (HEA, 1 mmol l−1 NH 4 Cl) caused an increase in mRNA expression of LpHIAT1β in the ion-conductive ventral gill half. High mRNA expression of both isoforms in the brain, and the observation that LpHIAT1α and LpHIAT1β likely mediate cellular ammonia excretion, suggests that these highly conserved ammonia transporters have an important housekeeping function in cellular ammonia elimination. [Display omitted] • First invertebrate study on 2 isoforms of a novel ammonia transporter, HIAT1α and HIAT1ß. • HIAT1 is highly conserved within the animal kingdom. • HIATα and HIAT1ß show highest mRNA expression levels in brain tissue. • In gills and brain HIAT1α is higher expressed when compared to HIAT1ß. • Both HIATα and HIAT1ß promote methylamine excretion when expressed in frog oocytes. [ABSTRACT FROM AUTHOR]

The pullulanase gene (pulPL), encoding a novel type I pullulanase (PulPL), was obtained from a Paenibacillus lautus DSM3035 isolate. The gene has an open reading frame of 2355 bp, After optimizing induction conditions in Escherichia coli, we overexpressed recombinant PulPL, purified this enzyme, and assayed its function. The level of functional PulPL-like protein reached its maximum (about 0.28 mg/mL, 15% of total protein) after induction for 16 h at 20°C. Under these optimized harvesting conditions, PulPL activity was 11.1U/mL. The purified recombinant enzyme with an apparent molecular mass of about 87.9 kDa was able to specifically attack the a-1,6 linkages in pullulan to generate maltotriose as the major product. The purified PulPL exhibited optimal activity at pH 7.0 and 40°C. The PulPL hydrolyzed pullulan, amylopectin, starch, and glycogen, but not amylose. Substrate specificity observations and reaction products identifications indicate that the purified pullulanase from P. lautus DSM3035 is a type I pullulanase. The present report, to our knowledge, the first to identify type I pullulanase in P.lautus, and detail the enzymatic properties of this enzyme after heterologous expression. [ABSTRACT FROM AUTHOR]

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate “revertants” viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria. [ABSTRACT FROM AUTHOR]

PH20 is known as sperm adhesion molecule 1 (SPAM1) and also has hyaluronidase function to preferentially hydrolyze the glycosidic linkage of hyaluronic acid (HA). A DNA fragment containing core domain of human PH20 gene was cloned into a constitutive expression plasmid (pGAPZαC) of Pichia pastoris to produce a fusion protein with α factor signal in the N-terminus and 6 × His as well as c-Myc tags in the C-terminus. The resulting plasmid pGAPZαC-PH20 was integrated into the genome of P. pastoris strain GS115. Functional recombinant human PH20 (rHuPH20) was successfully expressed and secreted by the recombinant P. pastoris transformant. Highest hyaluronidase activity of 2 mU/mL could be obtained at 3 day in an YPD culture. After purified by phenylboronic acid resin adsorption, rHuPH20 with a specific activity of 230 mU/mg was obtained. Via periodic acid-Schiff staining and zymogram analysis, the partially purified rHuPH20 was determined to be highly glycosylated to various extents with molecular mass in the range of 100–300 kDa. The enzyme showed a maximal activity at pH 5.0 but no appreciable activity at pH ≤3 and pH ≥8. The hyaluronidase activity could be stably maintained at 4°C but lost 40% after incubating at 30°C for 4 h. Both N -acetyl cysteine and glutathione showed a half maximal inhibitory concentration (IC 50 ) of 8 mM against rHuPH20. [ABSTRACT FROM AUTHOR]

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides. [ABSTRACT FROM AUTHOR]

The biogenic amine octopamine plays a critical role in the regulation of many physiological processes in insects. Octopamine transmits its action through a set of specific G-protein coupled receptors (GPCRs), namely octopamine receptors. Here, we report on a β-adrenergic-like octopamine receptor gene (BdOctβR1) from the oriental fruit fly, Bactrocera dorsalis (Hendel), a destructive agricultural pest that occurs in North America and the Asia-Pacific region. As indicated by RT-qPCR, BdOctβR1 was highly expressed in the central nervous system (CNS) and Malpighian tubules (MT) in the adult flies, suggesting it may undertake important roles in neural signaling in the CNS as well as physiological functions in the MT of this fly. Furthermore, its ligand specificities were tested in a heterologous expression system where BdOctβR1 was expressed in HEK-293 cells. Based on cyclic AMP response assays, we found that BdOctβR1 could be activated by octopamine in a concentration-dependent manner, confirming that this receptor was functional, while tyramine and dopamine had much less potency than octopamine. Naphazoline possessed the highest agonistic activity among the tested agonists. In antagonistic assays, mianserin had the strongest activity and was followed by phentolamine and chlorpromazine. Furthermore, when the flies were kept under starvation, there was a corresponding increase in the transcript level of BdOctβR1, while high or low temperature stress could not induce significant expression changes. The above results suggest that BdOctβR1 may be involved in the regulation of feeding processes in Bactrocera dorsalis and may provide new potential insecticide leads targeting octopamine receptors. [ABSTRACT FROM AUTHOR]

Recombinant goat chymosin (RGC), a promising alternate of bovine/calf chymosin, has better catalytic, proteolytic and thermostable characteristics. Limited information exists about its expression in different hosts. Pichia pastoris ( P . pastoris ) has ability to grow to very high cell densities and improved protein production has been demonstrated in scale up studies. A modified methanol feeding strategy based on flask level optimizations was successfully used for expressing the goat prochymosin in recombinant methylotrophic yeast P . pastoris Mut + bioreactor cultures. Additionally, a comparative analysis of RGC expression in flask versus bioreactor cultures was performed. Bioreactor cultures yielded an approximate 8 times increase in cell density compared to flask cultures prior to induction and an increase of 24% in expression level of chymosin was also recorded. Functional characterization demonstrated that the enzyme produced even at early stages was found to be correctly folded, intact and functional. The recombinant enzyme preparation has 42.4% residual activity at 60 °C and retained 69% activity at pH 6.5. An expression strategy for scale up production of RGC, which can act as a lead for production of industrially viable enzyme preparation, is outlined in the present study. [ABSTRACT FROM AUTHOR]

Background: Pullulanase is an important debranching enzyme and has been widely utilized to hydrolyse the α-1,6 glucosidic linkages in starch/sugar industry. Selecting new bacterial strains or improving bacterial strains is a prerequisite and effective solution in industrial applications. Although many pullulanase genes have been cloned and sequenced, there is no report of P. polymyxa type I pullulanase gene or the recombinant strain. Meanwhile most of the type I pullulanase investigated exhibit thermophilic or mesophilic properties. There are just few reports of cold-adapted pullulanases, which have optimum activity at moderate temperature and exhibit rather high catalytic activity at cold. Previously, six strains showing distinct pullulan degradation ability were isolated using enrichment procedures. As containing novel bacterium resource and significant pullulanase activity, strain Nws-pp2 was selected for in-depth study. Methods: In this study, a type I pullulanase gene (pulN) was obtained from the strain P. polymyxa Nws-pp2 by degenerate primers. Through optimization of induced conditions, the recombinant PulN achieved functional soluble expression by low temperature induction. The enzyme characterizations including the enzyme activity/stability, optimum temperature, optimum pH and substrate specificity were also described through protein purification. Results: The pullulanase gene (named pulN), encoding a novel cold-adapted type I pullulanase (named PulN), was obtained from isolated strain Paenibacillus polymyxa Nws-pp2. The gene had an open reading frame of 2532-bp and was functionally expressed in Escherichia coli through optimization of induced conditions. The level of functional PulN-like protein reached the maximum after induction for 16 h at 20 °C and reached about 0.34 mg/ml (about 20 % of total protein) with an activity of 6.49 U/ml. The purified recombinant enzyme with an apparent molecular mass of about 96 kDa was able to attack specifically the α-1,6 linkages in pullulan to generate maltotriose as the major product. The purified PulN showed optimal activity at pH 6.0 and 35 °C, and retained more than 40 % of the maximum activity at 10 °C (showing cold-adapted). The pullulanase activity was significantly enhanced by Co2+ and Mn2+, meanwhile Cu2+ and SDS inhibited pullulanase activity completely. The Km and Vmax values of purified PulN were 15.25 mg/ml and 20.1 U/mg, respectively. The PulN hydrolyzed pullulan, amylopectin, starch, and glycogen, but not amylose. Substrate specificity and products analysis proved that the purified pullulanase from Paenibacillus polymyxa Nws-pp2 belong to a type I pullulanase. Conclusions: This report of the novel type I pullulanase in Paenibacillus polymyxa would contribute to pullulanase research from Paenibacillus spp. significantly. Also, the cold-adapted pullulanase produced in recombinant strain shows the potential application. [ABSTRACT FROM AUTHOR]

While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase (designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa, respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species. Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins, showed that only simultaneous incubation of the membrane-bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levels of the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19- E -vallesamine, that accumulated in these plant parts. These results suggested that CYP97C27 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus . [ABSTRACT FROM AUTHOR]

The substrate recognition mechanism of phospholipase D and enzymatic measurement of choline plasmalogen were investigated. A phospholipase D (PLD 684 ) from Streptomyces sp. strain NA684 was purified 184-fold from the culture supernatant with 23.7% recovery. Maximum activity for l -α-lysophosphatidylcholine (LPC) hydrolysis was found at pH 5.0 and 80°C. The hydrolytic activity was remarkably affected by the concentration of Triton X-100 in the reaction mixture. In the presence of 0.05−0.5% and 0.1−0.2% (wt/vol) Triton X-100, 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) and choline plasmalogen were efficiently hydrolyzed by PLD 684 , respectively. Hydrolysis of LPC and choline lysoplasmalogen did not require Triton X-100; rather, the hydrolytic activity was inhibited by more than 0.05% (wt/vol) Triton X-100. The enzyme preferred mixed micelle substrates to liposomal substrates and hydrolyzed 98.4% of mixed micelle POPC in 1 h. Kinetic analysis showed that the rate-limiting steps of hydrolysis of mixed micelle POPC and emulsified LPC by PLD 684 were the bulk step and the surface step, respectively. These results suggest that PLD 684 has at least two substrate recognition mechanisms to recognize various phospholipids that have considerably different physical properties derived from their head and tail groups. Understanding of how PLD 684 recognizes substrate forms will be useful for elucidating roles of lipolytic proteins in nature. Moreover, we report an enzymatic measurement of choline plasmalogen using PLD 684 and phospholipase B. This is the first enzymatic method for measuring choline plasmalogen. [ABSTRACT FROM AUTHOR]

ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts. [ABSTRACT FROM AUTHOR]

Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae , the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (∼8 mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217 mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production. [ABSTRACT FROM AUTHOR]

The hERG potassium channel is essential for repolarization of the cardiac action potential. Due to this vital function, absence of unintended and potentially life-threatening interactions with hERG is required for approval of new drugs. The structure of hERG is therefore one of the most sought-after. To provide purified hERG for structural studies and new hERG biomimetic platforms for detection of undesirable interactions, we have developed a hERG expression platform generating unprecedented amounts of purified and functional hERG channels. Full-length hERG, with or without a C-terminally fused green fluorescent protein (GFP) His8-tag was produced from a codon-optimized hERG cDNA in Saccharomyces cerevisiae. Both constructs complemented the high potassium requirement of a knock-out Saccharomyces cerevisiae strain, indicating correct tetramer assembly in vivo. Functionality was further demonstrated by Astemizole binding to membrane embedded hERG-GFP-His8 with a stoichiometry corresponding to tetramer assembly. The 156 kDa hERG-GFP protein accumulated to a membrane density of 1.6%. Fluorescence size exclusion chromatography of hERG-GFP-His8 solubilized in Fos-Choline-12 supplemented with cholesteryl-hemisuccinate and Astemizole resulted in a monodisperse elution profile demonstrating a high quality of the hERG channels. hERG-GFP-His8 purified by Ni-affinity chromatography maintained the ability to bind Astemizole with the correct stoichiometry indicating that the native, tetrameric structure was preserved. To our knowledge this is the first reported high-yield production and purification of full length, tetrameric and functional hERG. This significant breakthrough will be paramount in obtaining hERG crystal structures, and in establishment of new high-throughput hERG drug safety screening assays. [ABSTRACT FROM AUTHOR]

Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate “green catalysts”. This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure–function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts. [ABSTRACT FROM AUTHOR]

S100A4 protein is associated with Ca2+-dependent regulation of intracellular activities and is significant in invasion, growth and metastasis of cancer. In order to express rat S100A4 functionally and identify its biological activity following purification, an S100A4 gene fragment was optimized and fully synthesized via overlapping polymerase chain reaction. The gene was inserted into the prokaryotic expression vector, pBV220, with phage λ PRPL promoters following confirmation by DNA sequencing. The pBV220-S100A4 plasmid was constructed and transformed into Escherichia coli DH5α. Following temperature induction, rat S100A4 overexpressed and the protein was observed to be located the supernatant of the lysates, which was ~30-40% of the protein within the host. The protein was isolated and purified by metal-chelate affinity chromatography. High purity protein (>98% purity) was obtained and in vitro western blot analysis identified that the recombinant S100A4 was able to bind the antibody against wild-type S100A4. The bioactivity of recombinant protein was detected via Transwell migration and invasion assays. The polyclonal antibody of rat S100A4 protein was prepared for rabbit immunization and exhibited similar efficacies when compared with commercial S100A4. Therefore, rat S100A4 was functionally expressed in E. thus, the production of active recombinant S100A4 protein E. coli may further aid with the investigation and application of S100A4. [ABSTRACT FROM AUTHOR]

Highlights: [•] Functional expression of Pleurotus sapidus lipoxygenase (Lox1) in Pichia pastoris. [•] Secretion of Lox1 using different well-proven expression vector constructs failed. [•] Replacing the α-factor signal sequence by a second Kozak sequence resulted in protein expression. [•] Wild-type and recombinant enzyme showed similar v max (111Umg−1). [ABSTRACT FROM AUTHOR]

Extracellular cellulase free xylanase from Thermomyceslanuginosus sp. SS-8, isolated from self heating plant wreckage material was identified as β-1,4-endo-xylanase precursor, a monomer of 21.3 kDa with no carbohydrate residue. This xylanase retained 80 % activity at 60 °C for 96 h, was active at a wide pH range of 3–11 and uniquely hydrolyzed xylan to xylose without production of xylo-oligosaccharides. Gene xynSS8 encoding xylanase from T. lanuginosus SS-8 was cloned and functionally expressed in Escherichia coli XL1 Blue using pTZ57R/T plasmid and xynSS8/pQE-9 expression vector construct respectively. Gene xynSS8 was of 777 bp and deduced amino acid sequence was a mature xylanase of 258 amino acids. XynSS8 has extra 33 amino acids compared to its nearest homolog and was thermo-alkali tolerant as that of native protein. The xylanase could degrade pulp and release substantial chromophoric materials and lignin derived compounds indicating its effective utility in pulp bleaching. Novel characteristics of the enzyme may contribute to its wide industrial usage. This is first report of cloning and functional expression of the novel xylanase from T. lanuginosus SS-8. [ABSTRACT FROM AUTHOR]

Abstract: Structure–function relationships underlying laccases properties are very limited that makes these enzymes interesting for protein engineering approaches. Therefore in the current study, a thermostable laccase that was isolated from Bacillus sp. HR03 with the ability of bilirubin oxidation besides its laccase and tyrosinase activity is used. The extensive application of this enzyme is limited by its low expression level in Escherichia coli. Based on sequence alignments and structural studies, three single amino acid substitutions, D500G, D500E, D500S and a glycine insertion, are introduced using site-directed mutagenesis to evaluate the role of Asp500 located in the C-terminal segment close to the T1 copper center. Substitution of aspartic acid with less sterically hindered, conserved residue such as glycine increase k cat (2.3 fold) and total activity (7.3 fold) which is accompanied by a significant increase in the expression level up to 3 fold. Biochemical characterization and structural studies using far-UV CD and fluorescence spectroscopy reveal the importance of C-terminal copper-binding loop in the laccase functional expression and catalytic efficiency. Kinetic characterization of the purified mutants toward 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ) and bilirubin, shows that substrate specificity is left unchanged. [Copyright &y& Elsevier]

Abstract: Bumblebee males (Hymenoptera) produce species-specific labial gland secretions called marking pheromones (MPs). MPs generally consist of terpenoids and fatty-acid-derived aliphatic compounds with various chain lengths predominantly containing one or no double bonds. The unsaturated fatty-acid-derived MP components were hypothesized to be produced by fatty acid desaturases (FADs) that exhibit diverse substrate specificities. To address this hypothesis, we isolated and functionally characterized FADs from three bumblebee species: Bombus lucorum, Bombus terrestris, and Bombus lapidarius. By employing RNA sequencing of the male labial glands and fat bodies of B. lucorum and B. terrestris, we identified five paralogous FAD-like sequences but only two FAD lineages were abundant and differentially expressed in the labial glands. We found that abundant FAD lineages were also expressed in the labial gland and fat body of Bombus lapidarius. Functional characterization of FADs in a yeast expression system confirmed that Δ4-FADs exhibited a unique Δ4-desaturase activity exclusively on 14-carbon fatty acyls and Δ9-FADs displayed Δ9-desaturase activity on 14- to 18-carbon fatty acyls. These results indicate that Δ9-FADs are involved in the biosynthesis of major unsaturated components of MPs in B. lucorum and B. lapidarius despite the diverse MP composition of these bumblebee species. The contribution of lipases, acyltransferases, esterases, and fatty acid reductases to production of the species-specific MP composition is also discussed in light of the transcriptomic data obtained in this study. [Copyright &y& Elsevier]

Background: Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory. Results: A blood-active laccase (ChU-B mutant) fused to the native/evolved а-factor prepro-leader was cloned under the control of two different promoters (PAOX1 and PGAP) and expressed in Pichia pastoris. The most active construct, which contained the PAOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the а-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers. Conclusions: The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications. [ABSTRACT FROM AUTHOR]

Abstract: The chicken (Gallus gallus) is one of the most economically important domestic animals and also an avian model species. Chickens have two CYP1A genes (CYP1A4 and CYP1A5) which are orthologous to mammalian CYP1A1 and CYP1A2. Although the importance of chicken CYP1As in metabolism of endogenous compounds and xenobiotics is well recognized, their enzymatic properties, substrate preference and inhibitor selectivity remain poorly understood. In this study, functional enzymes of chicken CYP1A4 and CYP1A5 were successfully produced in Escherichia coli (E. coli). The substrate preference and inhibitor specificity of the two chicken CYP1As were compared. Kinetic results showed that the enzymatic parameters (Km, Vmax, Vmax/Km) for ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-debenzylase (BROD) differed between CYP1A4 and CYP1A5, while no significant difference was observed for methoxyresorufin O-demethylase (MROD). Lower Km of CYP1A4 for BROD suggests that CYP1A4 has a greater binding affinity to benzyloxyresorufin than either ethoxyresorufin or methoxyresorufin. The highest Vmax/Km ratio was seen in BROD activity for CYP1A4 and in MROD for CYP1A5 respectively. These results indicate that substrate preference of chicken CYP1As is more notably distinguished by BROD activity and CYP1A5 prefers shorter alkoxyresorufins resembling its mammalian ortholog CYP1A2. Differential patterns of MROD inhibition were observed between CYP1As and among the five CYP inhibitors (α-naphthoflavone, furafylline, piperonyl butoxide, erythromycin and ketoconazole). α-Naphthoflavone was determined to be a potent MROD inhibitor of both CYP1A4 and CYP1A5. In contrast, no or only a trace inhibitory effect (<15%) was observed by erythromycin at a concentration of 500μM. Stronger inhibition of MROD activity was found in CYP1A5 than CYP1A4 by relatively small molecules α-naphthoflavone, piperonyl butoxide and furafylline. AROD kinetics and inhibition profiles between chicken CYP1A4 and CYP1A5 demonstrate that the two paralogous members of the CYP1A subfamily have distinct enzymatic properties, reflecting differences in the active site geometry between CYP1A4 and CYP1A5. These findings suggest that CYP1A4 and CYP1A5 play partially overlapping but distinctly different physiological and toxicological roles in the chicken. [Copyright &y& Elsevier]

Oxylipins generated by the lipoxygenase (LOX) pathway play an important role in plant defense against biotic and abiotic stress. In chitosan-treated Adelostemma gracillimum seedlings, obvious accumulation of 9-LOX-derived oxylipins, namely 9,10,11-trihydroxy-12-octadecenoic acid, was detected. Using degenerate primers, a LOX-specific fragment putatively encoding LOX was obtained by RT-PCR, and a 2.9-kb full-length cDNA named AgLOX1 was isolated by RACE from chitosan-induced A. gracillimum seedlings. Genomic Southern analysis implied that there was only one copy of AgLOX1 in the A. gracillimum genome. AgLOX1 was expressed in Escherichia coli and the recombinant protein was partially purified. The enzyme converted linoleic and linolenic acids almost exclusively to their 9-hydroperoxides. AgLOX1 encoded a 9-lipoxygenase. Northern blot analysis indicated that chitosan-induced AgLOX1 transcript accumulation peaked at 8 h after initiation of treatment, whereas trihydroxy derivatives accumulation was highest at 24 h after elicitation. Results showed that chitosan-induced AgLOX1 encoded a 9-lipoxygenase potentially involved in the defense response through 9-LOX pathway leading to biosynthesis of antimicrobial compounds in A. gracillimum seedlings. [ABSTRACT FROM AUTHOR]

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys–Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys–Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82–1.6-fold for benzaldehyde and 0.9–1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core. [ABSTRACT FROM AUTHOR]

Abstract: The Wood-Ljungdahl pathway is responsible for acetyl-CoA biosynthesis and used as a major mean of generating energy for growth in some anaerobic microbes. Series of genes, from the anaerobic human pathogen Clostridium difficile, have been identified that show striking similarity to the genes involved in this pathway including methyltetrahydrofolate- and corrinoid-dependent methyltransferase. This methyltransferase plays a central role in this pathway that transfers the methyl group from methyltetrahydrofolate to a cob(I)amide center in the corrinoid iron–sulfur protein. In this study, we developed two efficient expression and purification methods for methyltransferase from C. difficile for the first time with two expression vectors MBPHT-mCherry2 and pETDuet-1, respectively. Using the latter vector, more than 50mg MeTr was produced per liter Luria–Bertani broth media. The recombinant methyltransferase was well characterized by SDS–PAGE, gel filtration chromatography, enzyme assay and far-UV circular dichroism (CD). Furthermore, a highly effective approach was established for determining the methyl transfer activity of the methyltetrahydrofolate- and cobalamin-dependent methyltransferase using exogenous cobalamin as a substrate by stopped-flow method. These results will provide a solid basis for further study of the methyltransferase and the Wood-Ljungdahl pathway. [Copyright &y& Elsevier]