Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates.

The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.

Lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN--gp120 interaction.

One of the cell types first encountered by human immunodeficiency virus type 1 (HIV-1) following sexual transmission are dendritic cells (DC). DC capture HIV-1 through C-type lectin receptors, of which the best studied example is DC-SIGN, which mediates HIV-1 internalization. DC can keep the virus infectious for several days and are able to transmit HIV-1 to CD4(+) T cells. We tested proteins from milk and serum for their ability to block DC-mediated HIV-1 transmission, of which bovine lactoferrin (bLF) is the most potent inhibitor. bLF binds strongly to DC-SIGN, thus preventing virus capture and subsequent transmission. Interestingly, bLF is a much more efficient inhibitor of transmission than human lactoferrin. Since bLF is nontoxic and easy to purify in large quantities, it is an interesting candidate microbicide against HIV-1. Another advantage of bLF is its ability to block HIV-1 replication in T cells. DC-mediated capture of a bLF-resistant HIV-1 variant that was selected during long-term culturing in T cells could still be blocked by bLF. This underscores the usefulness of bLF as a microbicide drug to prevent HIV-1 transmission.

The antiviral activity of the milk protein lactoferrin against the human immunodeficiency virus type 1.

Milk forms a rich source of biologically interesting components and the protein fraction is known to facilitate many different biological functions. In this manuscript, we review the antiviral properties of the milk protein lactoferrin (LF). In particular, we will describe its antiviral activity against the human immunodeficiency virus type 1 (HIV-1).

Kinetics of enzyme acylation and deacylation in the penicillin acylase-catalyzed synthesis of beta-lactam antibiotics.

Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic beta-lactam antibiotics via formation of a covalent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and d-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acyl-enzyme. The phenylacetyl- and phenylglycyl-enzymes are hydrolysed with rate constants of at least 1000 s-1 and 75 s-1, respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5-[(phenylacetyl)amino]-benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl-enzyme proceeds via a general acid-base mechanism. In agreement with such a mechanism, the proton inventory of the kcat for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate-limiting step. The overall KIE of 2 for the kcat of NIPAOB resulted from an inverse isotope effect at low concentrations of D2O, which is overridden by a large normal isotope effect at large molar fractions of D2O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D2O compared to H2O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The beta-lactam compound 7-aminodesacetoxycephalosporanic acid (7-ADCA) was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysis of the acyl-enzyme upon binding of 7-ADCA. By combining the results of the steady-state, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of beta-lactam antibiotics were obtained.

Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides.

In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition or hydrophobicity profile. In contrast with some chemically modified (strongly negative) milk proteins, unmodified alpha(s2)-, beta- and kappa-casein, as well as several negatively and positively charged fragments thereof, did not show significant inhibition of virus replication. In fact, HIV-1 replication was elevated in the presence of beta-casein or amphiphilic fragments thereof. Bovine lactoferrin (bLF), a milk protein of 80 kDa, showed considerable inhibitory activity against HIV-1 with an IC50 of 0.4 microM. Modest inhibition was obtained with lactoferricin, a highly positively charged loop domain of bLF, indicating that other domains within the native bLF protein may also be required for inhibition. bLF blocked HIV-1 variants that use either the CXCR4 or the CCR5 coreceptor. In order to obtain further insight into the mechanism of action of this antiviral protein, we selected a bLF-resistant HIV-1 variant. The bLF-resistance phenotype is mediated by the viral envelope protein, which contains two interesting mutations that have previously been associated with an altered virus-host interaction and a modified receptor-coreceptor interaction. These results demonstrate that bLF targets the HIV-1 entry process.

The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins.

The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans.

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing beta-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified alpha-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the alpha-amino acid ester hydrolase is a beta-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The alpha-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of beta-lactam antibiotic acylases.