High NaCl concentrations induce the resistance to thermal denaturation of an extremely halotolerant (salt-activated) ß-mannanase from Bacillus velezensis H1.

ß-mannanase catalyzes the hydrolysis of mannans ß-1,4-mannosidic linkages to produce industrially relevant oligosaccharides. These enzymes have numerous important applications in the detergent, food, and feed industries, particularly those that are resistant to harsh environmental conditions such as salts and heat. While, moderately salt-tolerant ß-mannanases are already reported, existence of a high halotolerant ß-mannanase is still elusive. This study aims to report the first purification and characterization of ManH1, an extremely halotolerant ß-mannanase from the halotolerant B. velezensis strain H1. Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis revealed a single major peak with a molecular mass of 37.8 kDa demonstrating its purity. The purified enzyme showed a good thermostability as no activity was lost after a 48 h incubation under optimal conditions of 50 °C and pH 5.5. The enzyme's salt activation nature was revealed when its maximum activity was obtained in the presence of 4 M NaCl, it doubled compared to the no-salt condition. Moreover, NaCl strengthens its resistance to thermal denaturation, as its melting temperature (Tm) increased steadily with increasing NaCl concentrations reaching 75.5 °C in the presence of 2.5 M NaCl. The Km and Vmax values were 5.63 mg/mL and 333.33 µmol/min/mL, respectively, using carob galactomannan (CG) as a substrate. The enzyme showed a significant ability to produce manno-oligosaccharides (MOS) from lignocellulosic biomass releasing 13 mg/mL of reducing sugars from olive mill wastes (OMW) after 24 h incubation. The results revealed that this enzyme may have significant commercial values for agro-waste treatment, and other potential applications.

Box-Behnken design optimization of xylanase and cellulase production by Aspergillus fumigatus on Stipa tenacissima biomass.

Optimization of xylanase and cellulase production by a newly isolated Aspergillus fumigatus strain grown on Stipa tenacissima (alfa grass) biomass without pretreatment was carried out using a Box-Behnken design. First, the polysaccharides of dried and ground alfa grass were characterized using chemical methods (strong and diluted acid). The effect of substrate particle size on xylanase and carboxymethylcellulase (CMCase) production by the selected and identified strain was then investigated. Thereafter, experiments were statistically planned with a Box-Behnken design to optimize initial pH, cultivation temperature, moisture content, and incubation period using alfa as sole carbon source. The effect of these parameters on the two enzyme production was evaluated using the response surface method. Analysis of variance was also carried out, and production of the enzymes was expressed using a mathematical equation depending on the influencing factors. The effects of individual, interaction, and square terms on production of both enzymes were represented using the nonlinear regression equations with significant R2 and P-values. Xylanase and CMCase production levels were enhanced by 25% and 27%, respectively. Thus, this study demonstrated for the first time the potential of alfa as a raw material to produce enzymes without any pretreatment. A set of parameter combinations was found to be effective for the production of xylanase and CMCase by A. fumigatus in an alfa-based solid-state fermentation.

Yellow laccase produced by Trametes versicolor K1 on tomato waste: A comparative study with the blue one produced on semi-synthetic medium.

Laccase production by fungal growth on agrifood waste is still poorly studied. Trametes versicolor K1 isolated from palm bark produced a yellow non glycosylated laccase from tomato waste based medium (TMT) and a blue glycosylated laccase on glucose medium (GLU). Lignocellulosic biomass, such as pinecones (PIN), palm leaves (PLM), olive pomace (OLV), and alfa stems (ALF) have also been used as growth medium for T. versicolor K1. In these conditions, very low or no laccase production was observed. When peptone was supplied in TMT medium, the laccase activity increased from 4170 U/L to 8618 U/L. By increasing the culture volume up to 1 L, laccase production on TMT was 9929 U/L. The yellow laccase (TmtLac) was purified from the supernatant TMT medium and has shown similar characteristics with the blue laccase (GluLac) purified from the GLU medium. Their apparent protein size was 63 kDa. Catalytic activities of the yellow form were not very different from those of the blue form, but specific activity of the purified yellow laccase produced on tomato waste was much higher. The Km and Vm values for four substrates, ABTS, DMP, guaiacol, and pyrogallol were almost similar for both isoenzymes. The optimum pH and temperature were respectively 4.0 and 50 °C. Although the level of glycosylation is clearly different, the thermostability of TmtLac and GluLac are quite similar. TmtLac is even slightly more tolerant at 60 °C for 24 h than GluLac. Moreover TmtLac showed greater stability at alkaline pH after 24 h compared to that of GluLac.We demonstrate that activity of the yellow TmtLac is not significantly affected compared to the blue laccase and that tomato waste is a simple and interesting lignocellulosic substrate to the laccase producer Trametes sp.

Production of a halotolerant endo-1,4-ß-glucanase by a newly isolated Bacillus velezensis H1 on olive mill wastes without pretreatment: purification and characterization of the enzyme.

Facing the critical issue of high production costs for cellulase, numerous studies have focused on improving the efficiency of cellulase production by potential cellulolytic microorganisms using agricultural wastes as substrates, extremophilic cellulases, in particular, are crucial in the biorefinery process because they can maintain activity under harsh environmental conditions. This study aims to investigate the ability of a potential carboxymethylcellulose-hydrolyzing bacterial strain H1, isolated from an Algerian saline soil and identified as Bacillus velezensis, to use untreated olive mill wastes as a substrate for the production of an endo-1,4-ß-glucanase. The enzyme was purified 44.9 fold using only two steps: ultrafiltration concentration and ion exchange chromatography, with final recovery of 80%. Its molecular mass was estimated to be 26 kDa by SDS-PAGE. Enzyme identification by LC-MS analysis showed 40% identity with an endo-1,3-1,4-ß-glucanase of GH-16 family. The highest enzymatic activity was significantly measured on barley ß-glucan (604.5 U/mL) followed by lichenan and carboxymethylcellulose as substrates, confirming that the studied enzyme is an endo-1,4-ß-glucanase. Optimal enzymatic activity was at pH 6.0-6.5 and at 60-65 °C. It was fairly thermotolerant, retaining 76.9% of the activity at 70 °C, and halotolerant, retaining 70% of its activity in the presence of 4 M NaCl. The enzyme had a Vmax of 625 U/min/mL and a high affinity with barley ß-glucan resulting a Km of 0.69 mg/mL. It also showed a significant ability to release cello-oligosaccharides. Based on such data, the H1 endo-1,4-ß-glucanase may have significant commercial values for industry, argo-waste treatment, and other biotechnological applications.

Potential of Halophilic Penicillium chrysogenum Isolated from Algerian Saline Soil to Produce Laccase on Olive Oil Wastes.

Enzymes from halophilic fungi offer interesting biotechnological applications, which lead us to explore novel producing strains. 23 fungi were isolated from Algerian saline soil. Among the three strains presenting laccase activities, one exhibited the high decolourising capacity of olive mill wastewaters. Identification showed that the efficient isolate GS15 belongs to Penicillium chrysogenum. This strain achieves optimal growth at 15% NaCl, 25 °C, pH 5, dark, aerobic and static conditions. The selected fungus is capable of producing extracellular enzymes as follows: caseinase, tannase, esterase and lipase. The laccase activities produced by P. chrysogenum on raw olive wastes are being reported here for the first time. GS15 produced 183.0 and 203.0 U/L of laccase activities in 10% and 20% unsupplemented olive mill wastewaters, respectively. The significant enzymatic activities can be correlated to the high ability of GS15 to decolourise industrial wastewater from the olive oil extraction. In these conditions no pre-treatment of olive wastewaters was needed. On the untreated grinded and non-grinded olive pomace, the laccase activity was 5.78 U/g and 5.36 U/g, respectively. Because the halophilic fungus has basic requirement for growth, this fungal strain is promising for saline biotechnological applications.

High yield production in seven days of Coriolopsis gallica 1184 laccase at 50 L scale; enzyme purification and molecular characterization.

The optimization of culture conditions for high yield laccase production by white rot fungi has been extensively studied. However, to achieve short time laccase production remains a major challenge in several cases. The present study investigated an optimal process for production of Coriolopsis gallica 1184 laccase in a high yield of 200 900 Ul(-1) in 7 d by 50 L scale submerged fermentation. Coriolopsis gallica 1184 laccase appeared as a robust enzyme against downstream process; only 13.5 % of laccase activity was lost at the end of downstream procedure. The pure enzyme appeared as a one-species laccase, with a molecular mass of 66 kDa as determined by SDS-PAGE. The pH optimum for 2,2'-azino-bis-[3-ethyltiazoline-6-sulfonate] oxidation ranged between 2.5 and 3.0 in 100 mM tartrate buffer. Optimum temperature for laccase activity was determined to be around 70 °C. The kinetic of laccase was investigated with four phenolic substrates. The lowest Km values (17 and 20 µM) were found for ABTS and guaiacol, respectively. Coriolopsis gallica 1184 laccase was characterized by mass spectrometry and shows that C. gallica 1184_LacI is very likely a new member of the AA1_1 subfamily. Our results clearly show high competitive potential of the robust extracellular C. gallica 1184 laccase to use it in different industrial processes.

Effect of berberine on Staphylococcus epidermidis biofilm formation.

Staphylococcus epidermidis is one of the main causes of medical device-related infections owing to its adhesion and biofilm-forming abilities on biomaterial surfaces. Berberine is an isoquinoline-type alkaloid isolated from Coptidis rhizoma (huang lian in Chinese) and other herbs with many activities against various disorders. Although the inhibitory effects of berberine on planktonic bacteria have been investigated in a few studies, the capacity of berberine to inhibit biofilm formation has not been reported to date. In this study, we observed that berberine is bacteriostatic for S. epidermidis and that sub-minimal inhibitory concentrations of berberine blocked the formation of S.epidermidis biofilm. Using viability assays and berberine uptake testing, berberine at a concentration of 15-30mug/mL was shown to inhibit bacterial metabolism. Data from this study also indicated that modest concentrations of berberine (30-45mug/mL) were sufficient to exhibit an antibacterial effect and to inhibit biofilm formation significantly, as shown by the tissue culture plate (TCP) method, confocal laser scanning microscopy and scanning electron microscopy for both S. epidermidis ATCC 35984 and a clinical isolate strain SE243. Although the mechanisms of bacterial killing and inhibition of biofilm formation are not fully understood, data from this investigation indicated a potential application for berberine as an adjuvant therapeutic agent for the prevention of biofilm-related infections.

Structural and biological characterization of a capsular polysaccharide produced by Staphylococcus haemolyticus.

The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (capABCDEFG(Sh)) showed > or = 57% similarity with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLM(Sh) genes are unique to S. haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: -3-alpha-L-FucNAc-3-(2-NAc-4-N-Asp-2,4,6-trideoxy-beta-D-Glc)-4-alpha-D-GlcNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an N-acyl substituent.

Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A.

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. It has been established that clinical isolates often produce a biofilm, which is involved in adherence to biomaterials and provides enhanced resistance of bacteria against host defenses and antibiotic treatments. It has been thought that the staphylococcal biofilm contains two polysaccharides, one responsible for primary cell adherence to biomaterials (polysaccharide/adhesin [PS/A]) and an antigen that mediates bacterial aggregation (polysaccharide intercellular adhesin [PIA]). In the present paper we present an improved procedure for preparation of PIA that conserves its labile substituents and avoids contamination with by-products. Based on structural analysis of the polysaccharide antigens and a thorough overview of the previously published data, we concluded that PIA from S. epidermidis is structurally identical to the recently described poly-beta-(1-->6)-N-acetylglucosamine from PS/A-overproducing strain S. aureus MN8m. We also show that another carbohydrate-containing polymer, extracellular teichoic acid (EC TA), is an essential component of S. epidermidis RP62A biofilms. We demonstrate that the relative amounts of extracellular PIA and EC TA produced depend on the growth conditions. Moderate shaking or static culture in tryptic soy broth favors PIA production, while more EC TA is produced in brain heart infusion medium.

The Enterococcus faecalis gene encoding the novel general stress protein Gsp62.

The Enterococcus faecalis general stress protein Gsp62 was purified using two-dimensional gel electrophoresis and its 25 N-terminal amino acid sequence determined. Analysis of the corresponding gene revealed that the gsp62 product is a 172 aa protein. Transcriptional analysis of gsp62 gave evidence for a monocistronic mRNA, the synthesis of which was induced at the onset of stationary phase and in response to heat shock, acid pH, detergents (i.e. SDS or bile salts), ethanol, tert-butyl hydroperoxide, sodium chloride and, to a lesser extent, hydrogen peroxide. 5' rapid amplification of cDNA ends by PCR experiments showed that gsp62 transcription initiates 30 nt upstream of the ATG start codon. Although gsp62 expression was induced in response to various stresses, its disruption had no significant effect on the cell survival after each individual stress. Two-dimensional protein gels from wild-type and mutant cells revealed no pleiotropic effect of the mutation on protein synthesis. Transcriptional fusions with the lacL lacM beta-galactosidase genes showed that an inverted repeat located upstream of the promoter is required for transcriptional induction by environmental stresses but not by entrance into stationary phase. Two distinct mechanisms responding to different signals are thus involved in gsp62 induction.

The osmoprotectant glycine betaine inhibits salt-induced cross-tolerance towards lethal treatment in Enterococcus faecalis.

The response of Enterococcus faecalis ATCC 19433 to salt stress has been characterized previously in complex media. In this report, it has been demonstrated that this bacterium actively accumulates the osmoprotectant glycine betaine (GB) from salt-enriched complex medium BHI. To further understand the specific effects of GB and other osmoprotective compounds in salt adaptation and salt-induced cross-tolerance to lethal challenges, a chemically defined medium lacking putative osmoprotectants was used. In this medium, bacterial growth was significantly reduced by increasing concentrations of NaCl. At 0.75 M NaCl, 90% inhibition of the growth rate was observed; GB and its structural analogues restored growth to the non-salt-stressed level. In contrast, proline, pipecolate and ectoine did not allow growth recovery of stressed cells. Kinetic studies showed that the uptake of betaines shows strong structural specificity and occurs through a salt-stress-inducible high-affinity porter [Km = 3.3 microM; Vmax = 130 nmol min(-1) (mg protein)(-1); the uptake activity increased 400-fold in the presence of 0.5 M NaCl]. Moreover, GB and its analogues were accumulated as non-metabolizable cytosolic osmolytes and reached intracellular levels ranging from 1-3 to 1.5 micromol (mg protein)(-1). In contrast to the beneficial effect of GB on the growth of salt-stressed cultures of E. faecalis, its accumulation inhibits the salt-induced cross-tolerance to a heterologous lethal challenge. Indeed, pretreatment of bacterial cells with 0.5 M NaCl induced resistance to 0.3% bile salts (survival of adapted cells increased by a factor of 6800). The presence of GB in the adaptation medium reduced the acquisition of bile salts resistance 680-fold. The synthesis of 11 of the 13 proteins induced during salt adaptation was significantly reduced in the presence of GB. These results raise questions about the actual beneficial effect of GB in natural environments where bacteria are often subjected to various stresses.