Quantification of growth hormone in serum by isotope dilution mass spectrometry

Interassay variation of antibody-based routine tests hampers comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to obtain precise and reliable results that can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole serum tryptic proteolysis and then extracted from the resulting mixture by semipreparative reversed-phase LC followed by strong cation exchange chromatography. Analysis of blank serum spiked with recombinant 22-kDa GH at different concentration levels would result in a mean recovery of 101.6%, a standard deviation (SD) of 2.5%, a combined uncertainty (u_c) of 3.0%, and a limit of quantification (LOQ) of 1.7 μg/L when quantifying T6 as a GH-derived fragment, whereas recovery = 100.7%, SD = 2.4%, u_c = 2.5%, and LOQ = 2.7 μg/L were found with T12. The potential to acquisition of reference values is exemplified by application to serum materials used in a recent quality assessment exercise for routine laboratories.     

Catalytic properties and crystal structure of quinoprotein aldose sugar dehydrogenase from hyperthermophilic archaeon Pyrobaculum aerophilum

We identified a gene encoding a soluble quinoprotein glucose dehydrogenase homologue in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The enzyme was extremely thermostable, and the activity of the pyrroloquinoline quinone (PQQ)-bound holoenzyme was not lost after incubation at 100 °C for 10 min. The crystal structure of the enzyme was determined in both the apoform and as the PQQ-bound holoenzyme. The overall fold of the P. aerophilum enzyme showed significant similarity to that of soluble quinoprotein aldose sugar dehydrogenase (Asd) from E. coli. However, clear topological differences were observed in the two long loops around the PQQ-binding sites of the two enzymes. Structural comparison revealed that the hyperthermostability of the P. aerophilum enzyme is likely attributable to the presence of an extensive aromatic pair network located around a β-sheet involving N- and C-terminal β-strands.     

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k_cat′ for desulfination of l-cysteine sulfinate increased to 0.5 s^− 1 (from 0.05 s^− 1 in wild-type enzyme), whereas k_cat′ for transamination of the same substrate was reduced from 510 s^− 1 to 0.05 s^− 1. Similarly, k_cat′ for β-decarboxylation of l-aspartate increased from < 0.0001 s^− 1 to 0.07 s^− 1, whereas k_cat′ for transamination was reduced from 530 s^− 1 to 0.13 s^− 1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.     

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

Synthetic peptides derived from the C-terminal 6 kDa region of Plasmodium falciparum SERA5 inhibit the enzyme activity and malaria parasite development

Background

Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site.

Methods

In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential.

Results

The final proteolytic step of PfSERA5 involves removal of a C-terminal ~ 6 kDa fragment that results in the generation of a catalytically active ~ 50 kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~ 6 kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5.

Conclusions

Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress.

General significance

These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~ 6 kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.     

P NMR studies of O-acetylserine sulfhydrylase-B from Salmonella typhimurium

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine and bisulfide to l-cysteine and acetate in bacteria and higher plants. Enteric bacteria have two isozymes of OASS, A and B, produced under aerobic and anaerobic growth conditions, respectively, with different substrate specificities. The ³¹P chemical shift of the internal and external Schiff bases of PLP in OASS-B are further downfield compared to OASS-A, suggesting a tighter binding of the cofactor in the B-isozyme. The chemical shift of the internal Schiff base (ISB) of OASS-B is 6.2 ppm, the highest value reported for the ISB of a PLP-dependent enzyme. Considering the similarity in the binding sites of the PLP cofactor for both isozymes, torsional strain of the C5-C5′ bond (O4′-C5′-C5-C4) of the Schiff base is proposed to contribute to the further downfield shift. The chemical shift of the lanthionine external Schiff base (ESB) of OASS-B is 6.0 ppm, upfield from that of unliganded OASS-B, while that of serine ESB is 6.3 ppm. Changes in chemical shift suggest the torsional strain of PLP changes as the reaction proceeds.The apoenzyme of OASS-B was prepared using hydroxylamine as the resolving reagent. Apoenzyme was reconstituted to holoenzyme by addition of PLP. Reconstitution is pseudo-first order and exhibits a final maximum recovery of 81.4%. The apoenzyme shows no visible absorbance, while the reconstituted enzyme has a UV-visible spectrum that is nearly identical to that of the holoenzyme. Steady-state fluorescence spectra gave tryptophan emission of the apoenzyme that is 3.3-fold higher than the emission of either the native or reconstituted enzyme, suggesting that PLP is a potent quencher of tryptophan emission.     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.     

Electrophoresis of periodontal pathogens in poly(ethyleneoxide) solutions with uncoated capillary

Periodontitis is a prevalent inflammatory disease caused by different species of anaerobic bacteria such as Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f). We compared the separation result of DNA ladders in hydroxyethyl cellulose, poly(ethyleneoxide) (PEO), and polyethylene glycol and analyzed the effect of polymer concentration, electric field, and temperature of the background electrolyte on the separation performance. Results demonstrated that there was a linear relationship (R = 0.942) for 100 to 700 bp of DNA and its migration time. Finally, the polymerase chain reaction products of P.g, T.d, and T.f were successfully identified within 8.5 min in 0.5% PEO with uncoated capillary.     

Isolation, affinity purification and biochemical characterization of a lysosomal cathepsin D from the deuterostome Asterias rubens

Cathepsin D (EC 3.4.23.5) is one of the lysosomal enzymes responsible for proteolytic degradation in cells. By virtue of its mannose 6-phosphate residues, shortly after its synthesis, it is recognized by the receptors in the trans-Golgi network that mediate its transport to the lysosomes. The mammalian enzyme has been extensively characterized and several forms of cathepsin have also been identified. Cathepsins have also been isolated from other vertebrates and invertebrates and recent studies suggest that the lysosomal sorting machinery is evolutionarily conserved from fish to mammals. We recently characterized the putative mannose 6-phosphate receptors from the invertebrate starfish (Asterias rubens). In the present study we affinity purified the cathepsin D from this animal and biochemically characterized the same. Purified enzyme migrated as a single band on SDS-PAGE corresponding to a molecular mass of 45 kDa. The protein bound specifically to Con A-Sepharose gel and is glycosylated. The deglycosylated enzyme showed a molecular mass of ~ 40 kDa. Furthermore, an antibody raised for the purified enzyme in a rabbit recognizes the crude, the purified enzyme as well as the deglycosylated product in a western blot experiment. The enzyme in the extracts of different tissues can also be quantified by ELISA. We have further evaluated the binding of purified starfish cathepsin D with its receptor, MPR 300 (mannose 6-phosphate receptor) by immunoprecipitation. Cross-linking experiments using purified cathepsin D and MPR 300 revealed a cross-linked product that migrated with a higher molecular mass (345 kDa) compared to the enzyme (45 kDa). Furthermore the specificity of this interaction was also tested in a ligand blot experiment.     

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3 mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen > dog fibrinogen ≈ human fibrinogen > goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.     

Reductive amination by recombinant Escherichia coli: Whole cell biotransformation of 2-keto-3-methylvalerate to l-isoleucine

A whole cell biotransformation system for reductive amination has been studied in recombinant Escherichia coli cells. Reductive amination of 2-keto-3-methylvalerate to l-isoleucine by a two-enzyme-cascade was achieved by overproduction of endogenous l-alanine dependent transaminase AvtA and heterologous l-alanine dehydrogenase from Bacillus subtilis in recombinant E. coli. Up to 100 mM l-isoleucine were produced from 100 mM 2-keto-3-methylvalerate and 100 mM ammonium sulfate. Regeneration of NADH as cofactor in the whole cell system was driven by glucose catabolism. The effects of defined gene deletions in the central carbon metabolism on biotransformation were tested. Strains lacking the NuoG subunit of NADH:ubiquinone oxidoreductase (complex I) or aceA encoding the glyoxylate cycle enzyme isocitrate lyase exhibited increased biotransformation rates.     

Ginger rhizome as a potential source of milk coagulating cysteine protease

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg²⁺ and Cu²⁺. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α_s-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.     

Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis

Background

Glycogen and starch branching enzymes catalyze the formation of α(1 → 6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1 → 4) linkage and transfer in α(1 → 6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch.

Methods

The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure.

Results

A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3–8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30–70 nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family.

Conclusions

RoBE is able to transfer α(1 → 4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1 → 4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family.

General significance

This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.     

Thermogenic characteristics and evaporative water loss in the tree shrew (Tupaia belangeri)

Thermogenic characteristics and evaporative water loss were measured at different temperatures in Tupaia belangeri. The thermal neutral zone (TNZ) of T. belangeri was 30–35 °C. Mean body temperature was 39.76±0.27 °C and mean body mass was 100.86±9.09 g. Basal metabolic rate (BMR) was 1.38±0.03 ml O₂/g h. Average minimum thermal conductance (C_m) was 0.13±0.01 ml O₂/g h °C. Evaporative water loss in T. belangeri increased when the temperature rose; the maximal evaporative water loss was 3.88±0.41 mg H₂O/g h at 37.5 °C. The results may reflect features of small mammals in the sub-tropical plateau region: T. belangeri had high basal metabolic rate and high total thermal conductance, compared with the predicted values based on their body mass whilst their body temperatures are relatively high; T. belangeri has high levels of evaporative water loss and poor water-retention capacity. Evaporative water loss plays an important role in temperature regulation.     

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 °C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k_cat/K_m = 37.53 mM S⁻¹), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K_i = 0.196 nM), indicating that this protease is a potential target for pest control.     

Cytosolic L-alanine:4,5-dioxovalerate transaminase differs from the mitochondrial form

L-Alanine:4,5-dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic L-alanine: 4,5-dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial L-alanine:4,5-dioxovalerate transaminase. This is the first report of two forms of L-alanine:4,5-dioxovalerate transaminase. The different responses of cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial L-alanine:4,5-dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one-dimensional peptide map of partially digested cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of L-alanine: 4,5-dioxovalerate transaminase is different from the mitochondrial enzyme. Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain-digested cytosolic enzyme 50-kDa fragment.     

Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores

The immobilization of enzymes on edible matrix supports is of great importance for developing stabilized feed enzymes. In this study, probiotic Bacillus spores were explored as a matrix for immobilizing Escherichia coli phytase, a feed enzyme releasing phosphate from phytate. Because Bacillus spore is inherently resistant to heat, solvents and drying, they were expected to be a unique matrix for enzyme immobilization. When mixed with food-grade Bacillus polyfermenticus spores, phytases were adsorbed to their surface and became immobilized. The amount of phytase attached was 28.2 ± 0.7 mg/g spores, corresponding to a calculated activity of 63,960 U/g spores; however, the measured activity was 41,120 ± 990.1 U/g spores, reflecting a loss of activity upon adsorption. Immobilization increased the half life (t_1/2) of the enzyme three- to ten-fold at different temperatures ranging from 60 to 90 °C. Phytase was bound to the spore surface to the extent that ultrasonication treatment was not able to detach phytases from spores. Desorption of spore-immobilized phytase was only achieved by treatment with 1 M NaCl, 10% formic acid in 45% acetonitrile, SDS, or urea, suggesting that adsorption of phytase to the spore might be via hydrophobic and electrostatic interactions. We propose here that Bacillus spore is a novel immobilization matrix for enzymes that displays high binding capacity and provides food-grade safety.     

High level expression of human enteropeptidase light chain in Pichia pastoris

Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4 U/mL of wet matrix.     

Human caspase-3 inhibition by Z-tLeu-Asp-H: tLeu(P2) counterbalances Asp(P4) and Glu(P3) specific inhibitor truncation

Caspase-3 is responsible for the cleavage of several proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Designed on the cleavage site of PARP, Ac-Asp-Glu-Val-Asp-H has been reported as a highly specific inhibitor. To overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of tetra-peptidyl aldehydes, di- and tri-peptidyl caspase-3 inhibitors have been synthesized. Here, the synthesis and the inhibition properties of peptidyl aldehydes Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H are reported. Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H inhibit competitively human caspase-3 activity in vitro with  = 3.6 nM, 18.2 nM, and 109 nM, respectively (pH 7.4 and 25 °C). Moreover, Z-tLeu-Asp-H impairs apoptosis in human DLD-1 colon adenocarcinoma cells without affecting caspase-8. Therefore, Ac-Asp-Glu-Val-Asp-H can be truncated to Z-tLeu-Asp-H retaining nanomolar inhibitory activity in vitro and displaying action in whole cells, these properties reflect the unprecedented introduction of the bulky and lipophilic tLeu residue at the P₂ position.     

Positively Cooperative Binding of Zinc Ions to Bacillus cereus 569/H/9 β-Lactamase II Suggests that the Binuclear Enzyme Is the Only Relevant Form for Catalysis

Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the β-lactamase II from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K₁/K₂ ≥ 5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K₂ < 80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its α-helical content, presumably associated with enhanced flexibility.