Bacillaceae serine proteases and Streptomyces epsilon-poly-L-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release.

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

Production of epsilon-polylysine in an airlift bioreactor (ABR).

This paper deals with studies on epsilon-poly-L-lysine (epsilon-PL) production in an airlift bioreactor (ABR) using Streptomyces albulus S410 (S410) to minimize the production cost including the downstream processing of epsilon-PL. In a 5-l ABR, 30 g/l of epsilon-PL was produced with a power consumption of 0.3 kW/m3, the production level being similar to that in a 5-l jar fermentor with a power consumption of 8.0 kW/m3. Furthermore, the leakage of intracellular nucleic acid (INA)-related substances into the culture broth in the ABR was less than that in the jar fermentor. Due to the high-level power consumption (8.0 kW/m3) in the jar fermentor, the morphology of the cells changed from the pellet to filament form due to the extensive shear stress arising from continuous agitation, thereby increasing the leakage of the INA-related substances into the culture broth. This suggested that ABR would have an advantage in the low-cost production of epsilon-PL over stirred tank type reactors (STR).

Inhibition of lipase activities by basic polysaccharide.

Basic polysaccharide strongly inhibited the hydrolysis of trioleoylglycerol (TO) emulsified with phosphatidylcholine and taurocholate by either pancreatic lipase or carboxylester lipase. DEAE-Sephadex dose-dependently inhibited the hydrolysis of TO by pancreatic lipase and carboxylester lipase; however, carboxymethyl-Sephadex and Sephadex G-50 did not inhibit the hydrolysis. Polydextrose (PD), a soluble polysaccharide, was a very weak inhibitor of pancreatic lipase. However, when a basic group, a DEAE group, was attached to PD, lipase inhibition by DEAE-PD was increased, and this was dependent on the substitution ratio of DEAE groups. The number of positive charges per PD molecule is important in lipase inhibition. Similar substitution effects were observed with other basic groups, such as piperidinoethyl and 3-triethylamino-2-hydroxypropyl. The natural basic polysaccharide, chitosan, also inhibited pancreatic lipase activity. Gel-filtration experiments suggested that DEAE-PD did not bind strongly to pancreatic lipase. The effect of DEAE-PD on TO hydrolysis by pancreatic lipase was studied using various emulsifiers: DEAE-PD (50 microg/ml) did not inhibit the hydrolysis of TO emulsified with arabic gum, phosphatidylserine, or phosphatidic acid. In vivo, oral administration of DEAE-PD to rats reduced the peak plasma triacylglycerol concentration and increased fecal lipid excretion. These results suggest that basic polysaccharide is able to suppress dietary fat absorption from the small intestine by inhibiting pancreatic lipase activity.

Antiobesity action of epsilon-polylysine, a potent inhibitor of pancreatic lipase.

In vitro, -polylysine (EPL) strongly inhibited the hydrolysis of trioleoylglycerol emulsified with phosphatidylcholine (PC) and taurocholate by either pancreatic lipase or carboxylester lipase. The EPL concentration required for 50% inhibition of pancreatic lipase, 0.12 microM, was eight times lower than the concentration of orlistat required for the same effect. The 50% inhibition concentration by EPL was affected by emulsifier species: it was increased approximately 150 times, 70 times, and 230 times on gum arabic, phosphatidylserine, and phosphatidic acid emulsion, respectively, compared with PC emulsion. The 50% inhibition concentration by orlistat was little changed by emulsifier species. Gel-filtration experiments suggested that EPL did not bind strongly to pancreatic lipase, whereas orlistat did. To test the effect of EPL on obesity, mice were fed a high-fat diet containing 0.1, 0.2, or 0.4% EPL. EPL prevented the high-fat diet-induced increase in body weight and weight of the liver and visceral adipose tissues (epididymal and retroperitoneal). EPL also decreased plasma triacylglycerol and plasma cholesterol concentrations and liver triacylglycerol content after they had been increased by the high-fat diet. The fecal weights of mice were increased by the high-fat diet containing EPL compared with the high-fat diet alone. Fecal lipid was also increased by the diet containing EPL. These data clearly show that EPL has an antiobesity function in mice fed a high-fat diet that acts by inhibiting intestinal absorption of dietary fat.

Biosynthesis of epsilon-poly-L-lysine in a cell-free system of Streptomyces albulus.

epsilon-Poly-L-lysine (epsilon-PL) is a homo-poly-amino acid characterized by a peptide bond between carboxyl and epsilon-amino groups of L-lysine. Here we report the cell-free synthesis of epsilon-PL by a sensitive radioisotopic epsilon-PL assay system. In vitro epsilon-PL synthesis depended on ATP and was not affected by ribonuclease, kanamycin, or chloramphenicol. epsilon-PL synthesizing activity was detected in the membrane fraction. The reaction product, epsilon-PL, from L-lysine was identified by MALDI-TOF MS and the number of lysine residues of the epsilon-PL products was apparently 11-34. These results suggest that the biosynthesis of epsilon-PL is nonribosomal peptide synthesis and is catalyzed by membrane bound enzyme(s). The enzyme preparation showing the epsilon-PL synthesizing activity also catalyzed lysine-dependent AMP production and an ATP-PPi exchange reaction, suggesting that L-lysine is adenylated in the first step of epsilon-PL biosynthesis.

Use of ADME studies to confirm the safety of epsilon-polylysine as a preservative in food.

Epsilon-polylysine is a homopolymer of L-lysine, containing approximately 30 L-lysine subunits, as synthesized in aerobic bacterial fermentation by Streptomyces albulus. epsilon -Polylysine is approved for food use in Japan as an antimicrobial preservative. A series of pharmacokinetic and metabolic profile studies on epsilon -polylysine have been conducted in rats in order to provide a better understanding of the reason for its lack of toxicological effects in subchronic and chronic feeding bioassays using relatively high concentrations in the diet up to 50000 ppm. As reported in this article, epsilon -polylysine was practically non-toxic in an acute oral toxicity study in rats, with no mortality up to 5 g/kg and was not mutagenic in bacterial reversion assays. Absorption, distribution, metabolism and excretion (ADME) studies on 14C-radiolabeled epsilon -polylysine, given in a single dose to fasted male rats at 100mg/kg, revealed low absorption from the gastrointestinal tract. All but trace amounts of the dosed radioactivity was eliminated by excretion within 168 h and over 97% was accounted for in urine (1.2%), feces (92.9%), or expired air (3%) by 48 h. The sum of the cumulative excretion with routes associated with absorption in urine, expired air and carcass was 6.4% of total recovered radioactivity; approximately 94% of the dose of epsilon -polylysine passed unabsorbed through the gastrointestinal tract in the feces. Whole body autoradiography did not show concentration of absorbed epsilon -polylysine in any tissue or organ. Excretion half-lives of epsilon -polylysine equivalents in blood and plasma were 20 and 3.9 days, likely prolonged by the incorporation into protein of cleaved L-lysine. Metabolic profiles by HPLC analysis of plasma samples suggest that L-lysine is the predominant early metabolic by-product, likely from protease activity in the upper GI tract; only 0.2% of the administered parent compound was found in plasma. At 8-72 h, HPLC profiles show diminishing levels of epsilon -polylysine and L-lysine in plasma, accompanied by a shift to larger peaks of homopolymer fragments of varying subunit length, presumably from microbial degradation of epsilon -polylysine in the lower gut. HPLC profiles of urine and feces collected from 0 to 24 h post-dosing revealed three distinct peaks in urine, the first peak likely to be epsilon -polylysine and epsilon -polylysine less a few amino acid subunits, and the second, L-lysine and the third, a metabolite of L-lysine. Radiolabeled L-lysine was reduced from 67.2% of the radioactivity in plasma at 30 min to 7.5% at 4 h, indicating that L-lysine is readily removed from plasma from essential amino acid incorporation into protein. Based on the findings of the ADME studies and lack of toxicity in safety studies, the proposed use of epsilon -polylysine as a preservative in foods is considered to be safe.