Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

NLRC5 potentiates anti-tumor CD8+ T cells responses by activating interferon-β in endometrial cancer

ObjectivesNLR family CARD domain containing 5 (NLRC5) could promote major histocompatibility complex class I (MHC-I)-dependent CD8⁺ T cell-mediated anticancer immunity. In this study, the immunosurveillance role and underlying mechanisms of NLRC5 in endometrial cancer (EC) were characterized.MethodsCD8⁺ T cells were separated from healthy women's peripheral blood by using magnetic beads. The effect of NLRC5 and interferon-β (IFN-β) on immunosurveillance of EC were examined through a mouse tumor model and a CD8⁺ T cell-EC cell coculture system after NLRC5 overexpression and IFN-β overexpression or depletion. The effect of NLRC5 on IFN-β expression was examined with gain- and loss-of-function experiments.ResultsNLRC5 overexpression in the EC cell and CD8⁺ T cell coculture system inhibited EC cell proliferation and migration and promoted EC cell apoptosis and CD8⁺ T cell proliferation. In vivo, NLRC5 overexpression increased the proportion of CD8⁺ T cells and inhibited EC progression. Furthermore, IFN-β overexpression in the EC cell and CD8⁺ T cell coculture system activated CD8⁺ T cell proliferation; however, genetic depletion of IFN-β exerted the opposite effects. In addition, NLRC5 could negatively regulate IFN-β expression in EC cells. Mechanistically, NLRC5 potentiated the antitumor responses of CD8⁺ T cells to EC by activating IFN-β.ConclusionsTaken together, our findings demonstrated that NLRC5 potentiates anti-tumor CD8⁺ T cells responses by activating interferon-β in EC, suggesting that genetically escalated NLRC5 and IFN-β may act as potential candidates for the clinical translation of adjuvant immunotherapies to patients with EC.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Identification of a highly thermostable mannitol 2-dehydrogenase from Caldicellulosiruptor morganii Rt8.B8 and its application for the preparation of D-mannitol

D-mannitol is a kind of hexitols widely applied in the food and medicinal fields due to its numerous benefits. Mannitol 2-dehydrogenase (MDH, EC 1.1.1.67) is a kind of oxidoreductase playing a pivotal part in the production of d-mannitol from d-fructose. In this work, we identified a highly thermostable d-mannitol-producing MDH from a thermo-tolerant bacterium, Caldicellulosiruptor morganii Rt8.B8. When using d-fructose as the substrate, the recombinant MDH was activated obviously in the presence of Mn²⁺ with an optimal pH as 8.0 and temperature at 75 °C. The specific activity, Michaelis-Menten constant (K_m) and catalytic efficiency (k_cat/K_m) for d-fructose were determined as 115 U mg⁻¹, 18 mM and 8.5 s^-1 mM⁻¹. Moreover, the half-life (t_1/2) of recombinant MDH at 75, 85 and 95 °C was 19 h, 3.5 h and 1.62 h respectively, which was much higher than that of most MDHs. The optimal condition for the production of d-mannitol was determined to be pH at 7.5, the temperature at 70 °C, and 2:1 ratio of C. morganii MDH and Ogataea parapolymorpha formate dehydrogenase (FDH, EC 1.2.1.2). Meanwhile, approximately 80 % d-mannitol was generated by two enzymes after a 50 h reaction from 400 mM d-fructose, indicating a great potentiality in the industrial preparation of d-mannitol.     

The photosynthetic irradiance-response of Norway spruce exposed to a long-term elevation of CO2 concentration

During an open-top chamber experiment performed in a mountain stand of young (12-year-old) Norway spruce (Picea abies [L.] Karst.), the trees were exposed to one of two CO² concentrations (ambient CO², AC, or AC + 350 μmol mol-1 = elevated CO₂, EC) continuously over three growing seasons. To evaluate the EC influence, measurements of the relations between the rate of net CO₂ uptake (P _N ) and incidental photosynthetically active photon flux density (PPFD), as well as the content of photosynthetic pigments and chlorophyll (Chl) a fluorescence were taken in the third growing season. The short-term response to EC was evident mainly on ribulose-1,5-bisphosphate carboxylase/oxygenase kinetics without any significant change to the utilization of radiant energy. The long-term effect of EC was responsible for a decrease in P _N , content of Chl a + b, Fv/Fm ratio, quantum yield of fluorescence, and photochemical quenching. Changes of stoichiometry between the electron transport, Calvin cycle and the end-product synthesis were confirmed for responses to the long-term import of EC and led to a definition of the photosynthetic acclimation to EC in Norway spruce. This revised version was published online in August 2006 with corrections to the Cover Date.     

Preparation of [U-14C]-labelled glycogen, maltosaccharides, maltose, and D-glucose by photoassimilation of 14CO2 in Anacystis nidulans and selective enzymic degradation.

A procedure is described for the isolation from the phototrophic procaryole Anacystis nidulans of [U-¹⁴C]-labelled glycogen, with high specific radioactivity,formed when NaH¹⁴CO₃ was added to non-dividing cells that continued to photoassimilate CO₂. [U-¹⁴C]-Labelled glycogen was then treated with isoamylase (EC 3.2.1.68), isoamylase plus beta-amylase (EC 3.2.1.2), or glucoamylase (EC 3.2.1.3) to give [U-¹⁴C]-labelled maltosaccharides, maltose-U-¹⁴C, or d-glucose-U-¹⁴C, respectively.     

Characterization and directed evolution of BliGO,a novel glycine oxidase from Bacillus licheniformis

Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (K_m, k_cat and k_cat/K_m) of BliGO were 11.22 mM, 0.08 s⁻¹, and 0.01 mM⁻¹ s⁻¹, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM⁻¹ s⁻¹) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.     

Purification of a trifunctional enzyme, catalysing three steps of the histidine pathway, from Neurospora crassa

1. A procedure is described for the purification of an enzyme from Neurospora crassa that has three catalytic functions. These are 1-N-(5′-phosphoribosyl)-ATP pyrophosphohydrolase, 1-N-(5′-phosphoribosyl)-AMP cyclohydrolase and histidinol dehydrogenase (l-histidinol–NAD oxidoreductase, EC 1.1.1.23), and are responsible for the catalysis of reactions 2, 3 and 10 in the histidine pathway. The ratio of these three catalytic activities remains approximately the same throughout the purification procedure. Evidence is presented that the purified preparations contain a single protein exhibiting association–dissociation equilibria.     

Enhanced Amino Acid Selection in Fully Evolved Tryptophanyl-tRNA Synthetase,Relative to Its Urzyme,Requires Domain Motion Sensed by the D1 Switch,a Remote Dynamic Packing Motif

We previously showed (Li, L., and Carter, C. W., Jr. (2013) J. Biol. Chem. 288, 34736–34745) that increased specificity for tryptophan versus tyrosine by contemporary Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) over that of TrpRS Urzyme results entirely from coupling between the anticodon-binding domain and an insertion into the Rossmann-fold known as Connecting Peptide 1. We show that this effect is closely related to a long range catalytic effect, in which side chain repacking in a region called the D1 Switch, accounts fully for the entire catalytic contribution of the catalytic Mg²⁺ ion. We report intrinsic and higher order interaction effects on the specificity ratio, (k_cat/K_m)_Trp/(k_cat/K_m)_Tyr, of 15 combinatorial mutants from a previous study (Weinreb, V., Li, L., and Carter, C. W., Jr. (2012) Structure 20, 128–138) of the catalytic role of the D1 Switch. Unexpectedly, the same four-way interaction both activates catalytic assist by Mg²⁺ ion and contributes −4.4 kcal/mol to the free energy of the specificity ratio. A minimum action path computed for the induced-fit and catalytic conformation changes shows that repacking of the four residues precedes a decrease in the volume of the tryptophan-binding pocket. We suggest that previous efforts to alter amino acid specificities of TrpRS and glutaminyl-tRNA synthetase (GlnRS) by mutagenesis without extensive, modular substitution failed because mutations were incompatible with interdomain motions required for catalysis.     

The Oligomerization of Phage T4 DNA-(Adenine-N6)-Methyltransferase and Its Effect on the Catalytic Characteristics of the Enzyme

The structural and catalytic properties of the phage T4 DNA-(adenine-N ⁶)-methyltransferase (EC 2.1.1.72) were studied at different enzyme–substrate concentration ratios by chemical crosslinking of the protein subunits and by measuring the presteady state kinetics of the reactions. Various structural states of the methyltransferase were correlated with its catalytic activity, and it was shown that the oligomeric forms of the enzyme are catalytically active but are characterized by the reaction parameters different from those of the monomer.     

Identification, expression, and characterization of a novel bacterial RGI lyase enzyme for the production of bio-functional fibers

A gene encoding a putative rhamnogalacturonan I (RGI) Lyase (EC 4.2.2.-) from Bacillus licheniformis (DSM13) was selected after a homology search and phylogenetic analysis and optimized with respect to codon usage. The designed gene was transformed into Pichia pastoris and the enzyme was produced in the eukaryotic host with a high titer in a 5 l bioreactor. The RGI Lyase was purified by Cu²⁺ affinity chromatography and 1.1 g pure enzyme was achieved pr. L. When the denatured protein was deglycosylated with EndoH, the molecular weight of the protein decreased to 65 kDa, which correlated with the predicted molecular weight of the mature RGI Lyase of 596 amino acids. By use of a statistical design approach, with potato rhamnogalacturonan as the substrate, the optimal reaction conditions for the RGI Lyase were established to be: 61 °C, pH 8.1, and 2 mM of both Ca²⁺ and Mn²⁺ (specific activity 18.4 U/mg; K_M 1.2 mg/ml). The addition of both Ca²⁺ and Mn²⁺ was essential for enzyme activity. The enzyme retained its catalytic activity at higher temperatures and the enzyme has a half life at 61 °C of 15 min. The work thus demonstrated the workability of in silico based screening coupled with a synthetic biology approach for gene synthesis for identification and production of a thermostable enzyme.     

Cloning,sequencing, and overexpression in Escherichia coli of a sarcosine oxidase-encoding gene linked to the Bacillus creatinase gene

Bacillus sp. B-0618 produces both creatinase (Cre; creatine amidinohydrolase; EC 3.5.3.3) and sarcosine oxidase (Sox; EC 1.5.3.1) enzymes when grown in the presence of an inducer, choline chloride. A genomic library of Bacillus sp. B-0618, prepared in the plasmid vector pACYC184, was screened to obtain a gene (sox) encoding Sox by a convenient colorimetric assay. A plasmid, pOXI101, isolated from a sox-positive clone, contained a 14.2-kb insert of Bacillus DNA. The nucleotide sequence of a 1.7-kb segment containing the sox gene was determined, and it was found that an open reading frame encoding a protein consisting of 390 amino acids was located upstream from the cre structural gene cloned previously. When a 1.6-kb XhoI-BglII fragment of pOXI101 was inserted into the pUC118 vector and introduced into Escherichia coli, transformants cultured in the absence of the inducer produced Sox about 50-fold more than Bacillus sp. B-0618 cultured in the presence of the inducer. The Bacillus Sox had the -¹¹Gly-X-¹³Gly-X-X-¹⁶Gly- sequence motif that is highly conserved in flavoproteins. We created an FAD-free Sox by changing ¹³Gly to Asp in the motif of the parental Sox by oligodeoxynucleotide-directed mutagenesis. The mutant protein no longer expressed the Sox activity, even on the addition of FAD.     

Characterization of EstB,a novel cold-active and organic solvent-tolerant esterase from marine microorganism Alcanivorax dieselolei B-5(T)

A novel esterase gene, estB, was cloned from the marine microorganism Alcanivorax dieselolei B-5(T) and overexpressed in E. coli DE3 (BL21). The expressed protein EstB with a predicted molecular weight of 45.1 kDa had a distinct catalytic triad (Ser²¹¹-Trp³⁵³-Gln³⁸⁵) and the classical consensus motif conserved in most lipases and esterases Gly²⁰⁹-X-Ser²¹¹-X-Gly²¹³. EstB showed very low similarity to any known proteins and displayed the highest similarity to the hypothetical protein (46 %) from Rhodococcus jostii RHA1. EstB showed the optimal activity around pH 8.5 and 20 °C and was identified to be extremely cold-adaptative retaining more than 95 % activity between 0 and 10 °C. The values of kinetic parameters on p-NP caproate (K _m, K _cat and K _cat/K _m) were 0.15 mM, 0.54 × 10³ s^?1 and 3.6 × 10³ s^?1 mM^?1, respectively. In addition, EstB showed remarkable stability in several studied organic solvents and detergents of high concentrations with the retention of more than 70 % activity after treatment for 30 min. The cold activity and its tolerance towards organic solvents made it a promising biocatalyst for industrial applications under extreme conditions.     

Molecular evolution of SPARC: absence of the acidic module and expression in the endoderm of the starlet sea anemone, Nematostella vectensis

The matricellular glycoprotein SPARC is composed of three functional domains that are evolutionarily conserved in organisms ranging from nematodes to mammals: a Ca²⁺-binding glutamic acid-rich acidic domain at the N-terminus (domain I), a follistatin-like module (domain II), and an extracellular Ca²⁺-binding (EC) module that contains two EF-hands and two collagen-binding epitopes (domain III). We report that four SPARC orthologs (designated nvSPARC1-4) are expressed by the genome of the starlet anemone Nematostella vectensis, a diploblastic basal cnidarian composed of an ectoderm and endoderm separated by collagen-based mesoglea. We also report that domain I is absent from all N. vectensis SPARC orthologs. In situ hybridization data indicate that N. vectensis SPARC mRNAs are restricted to the endoderm during post-gastrula development. The absence of the Ca²⁺-binding N-terminal domain in cnidarians and conservation of collagen-binding epitopes suggests that SPARC first evolved as a collagen-binding matricellular glycoprotein, an interaction likely to be dependent on the binding of Ca²⁺-ions to the two EF-hands in the EC domain. We propose that further Ca²⁺-dependent activities emerged with the acquisition of an acidic N-terminal module in triplobastic organisms.     

The identity of a new copper(II) electron paramagnetic resonance signal in cytochrome c oxidase

In reoxidation experiments with cytochrome c oxidase (EC 1.9.3.1) in the presence of both reducing substrate and molecular oxygen, a new EPR signal from Cu²⁺ has been observed. The new signal corresponds to 0.45 Cu per functional unit. It is concluded that the new EPR signal originates from Cu²⁺_B, the copper which is EPR-nondetectable in the resting enzyme.Optical absorption changes in the 500–700 nm region accompanies the decay of the new Cu²⁺ EPR signal.Based on the results in this investigation a catalytic cycle for cytochrome oxidase is proposed.     

Effect of 24-epibrassinolide on growth, protein content and antioxidative defense system of Brassica juncea L. subjected to cobalt ion toxicity

Brassica juncea L. plants were subjected to cobalt (Co) ion (0, 5?×?10^?4, 10^?3, 1.5?×?10^?3 and 2?×?10^?3?M) toxicity and were sprayed with different concentrations of 24-epibrassinolide (24-EBL) (0, 10^?10, 10^?8 and 10^?6?M) at 15-day stage after sowing. They were sampled at 30 and 60?days after sowing and analyzed for growth parameters in terms of shoot length and number of leaves. Thereafter, leaves were excised and content of proteins and the activities of antioxidative enzymes (superoxide dismutase (SOD) (EC 1.15.1.1) catalase (CAT) (EC 1.11.1.6), ascorbate peroxidase (APOX) (EC 1.11.1.11), guaiacol peroxidase (POD) (EC 1.11.1.7) glutathione reductase (GR) (EC 1.6.4.2), monodehydroascorbate reductase (MDHAR) (EC 1.1.5.4) and dehydroascorbate reductase (DHAR) (EC 1.8.5.1)) were analyzed. The plants exposed to cobalt ion exhibited a significant decline in growth in terms of shoot length and number of leaves. However, foliar spray treatment with 24-EBL was able to alleviate the stress generated by cobalt ion and significantly improved the above parameters. The activities of antioxidative enzymes (SOD, CAT, POD, GR, APOX, MDHAR and DHAR) and protein content were also regulated considerably in leaves of plants treated with 24-EBL alone, 10^?8?M concentration being the most effective. The activities of antioxidative enzymes also increased in leaves of B. juncea plants by the application of cobalt ion to soil and consequently sprayed with 24-EBL. Similarly, the protein content was also regulated in leaves of B. juncea plants treated with 24-EBL as compared to untreated control plants, thereby revealing stress-protective properties of 24-EBL.     

Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps

Gaussia luciferase secreted by the copepod Gaussia princeps catalyzes the oxidation of coelenterazine to produce blue light. The primary structure of Gaussia luciferase deduced from the cDNA sequence shows two repeat sequences of 71 amino acid residues, suggesting the luciferase consists of two structural domains. Two domains in Gaussia luciferase were expressed independently in Escherichia coli cells, purified and characterized. We found that both domains have luminescence activity with coelenterazine, and the catalytic properties including luminescence spectrum, optimal pH, substrate specificity and luminescence stimulation by halogen ions (Cl⁻, Br⁻ and I⁻) are identical to intact Gaussia luciferase. Thus, Gaussia luciferase has two catalytic domains for the luminescence reaction.     

Aidi injection,a traditional Chinese biomedical preparation for gynecologic tumors: a systematic review and PRISMA-compliant meta-analysis

Aidi injection (ADI), a traditional Chinese biomedical preparation, is a promising adjuvant therapy for gynecologic tumors (GTs), including cervical cancer (CC), endometrial cancer (EC), and ovarian cancer (OC). Although studies have reported positively on ADI therapy, its exact effects and safety in GT patients remain controversial. Therefore, a wide-ranging systematic search of electronic databases was performed for this meta-analysis. Data from 38 trials including 3309 GT patients were analyzed. The results indicated that the combination of conventional treatment and ADI markedly improved the patients’ overall response rate (P<0.00001), disease control rate (P<0.00001), and quality of life (P<0.05) compared with conventional treatment alone. Furthermore, patient immunity was enhanced with combined treatment, as indicated by significantly increased percentages of CD3⁺ (P=0.005) and CD4⁺ (P<0.00001) and increased CD4⁺/CD8⁺ ratio (P=0.001). Most of the adverse events caused by radiochemotherapy such as gastrointestinal issues, leukopenia, thrombocytopenia, and hepatotoxicity, (P<0.05 for all) were significantly alleviated when ADI was used in the GT patients. However, other adverse events such as nephrotoxicity, diarrhea, alopecia, and neurotoxicity did not significantly differ between the two groups. Overall, these results suggest that the combination of conventional and ADI treatment is more effective than conventional treatment alone.     

Pseudomonas aeruginosa transhydrogenase: affinity of substrates for the regulatory site and possible hysteretic behavior.

The polymeric enzyme transhydrogenase from Pseudomonas aeruginosa (NADPH:NAD⁺ oxidoreductase; EC 1.6.1.1) has been shown to possess distinct catalytic and regulatory sites, despite the close structural relationship between substrates and effectors. The present report substantiates the previous conclusions from kinetic and affinity chromatography studies, which have suggested that the substrates NADPH and oxidized thionicotinamide ademine dinucleotide phosphate could bind to both catalytic and regulatory sites. In addition, the allosteric R form of the enzyme appears now to be stabilized against high dilution inactivation.The oxidized substrate thionicotinamide adenine dinucleotide forms a dead end complex on binding to the catalytic site in the T form. The process is slow, and can be termed hysteretic, as defined by Frieden (J. Biol. Chem. (1970), 245, 5788–5799).     

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [³H]glucose + [¹⁴C]-uridine or [¹⁴C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.