Enzymatic Hydrolysis of Black Liquor Xylan by a Novel Xylose-Tolerant,Thermostable β-Xylosidase from a Tropical Strain of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> CBS 135684

From three cell-associated β-xylosidases produced by Aureobasidium pullulans CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be active at high temperatures (60–70 °C) over a pH range of 5–9 with a specific activity of 163.3 units (U) mg^?1. The enzyme was thermostable, retaining over 80% of its initial activity after a 12-h incubation at 60 °C, with half-lives of 38, 22, and 10 h at 60, 65, and 70 °C, respectively. Moreover, it was tolerant to xylose inhibition with a K _i value of 18 mM. The K _m and V _max values against p-nitrophenyl-β-d-xylopyranoside were 5.57 ± 0.27 mM and 137.0 ± 4.8 μmol min^?1 mg^?1 protein, respectively. When combining this β-xylosidase with xylanase from the same A. pullulans strain, the rate of black liquor xylan hydrolysis was significantly improved by up to 1.6-fold. The maximum xylose yield (0.812 ± 0.015 g g^?1 dry weight) was obtained from a reaction mixture containing 10% (w/v) black liquor xylan, 6 U g^?1 β-xylosidase and 16 U g^?1 xylanase after incubation for 4 h at 70 °C and pH 6.0.     

Production and Biotechnological Application of Extracellular Alkalophilic Lipase from Marine Macroalga-Associated <Emphasis Type="Italic">Shewanella algae</Emphasis> to Produce Enriched C<Subscript>20-22</Subscript><Emphasis Type="Italic">n</Emphasis>-3 Polyunsaturated Fatty Acid Concentrate

An extracellular alkalophilic lipase was partially purified from heterotrophic Shewanella algae (KX 272637) associated with marine macroalgae Padina gymnospora. The enzyme possessed a molecular mass of 20 kD, and was purified 60-fold with a specific activity of 36.33 U/mg. The enzyme exhibited V_max and K_m of 1000 mM/mg/min and 157 mM, respectively, with an optimum activity at 55 °C and pH 10.0. The catalytic activity of the enzyme was improved by Ca²⁺ and Mg²⁺ ions, and the enzyme showed a good tolerance towards organic solvents, such as methanol, isopropanol, and ethanol. The purified lipase hydrolyzed the refined liver oil from leafscale gulper shark Centrophorus squamosus, yielding a total C_20-22 n-3 PUFA concentration of 34.99% with EPA + DHA accounting the major share (34% TFA), after 3 h of hydrolysis. This study recognized the industrial applicability of the thermostable and alkalophilic lipase from marine macroalga-associated bacterium Shewanella algae to produce enriched C_20-22 n-3 polyunsaturated fatty acid concentrate.     

Green Synthesis of Silver Nanoparticles Using <Emphasis Type="Italic">Commiphora caudata</Emphasis> Leaves Extract and the Study of Bactericidal Efficiency

The present study reports the synthesis of silver nanoparticles (Ag NPs) from silver nitrate solution using leaf extracts of Commiphora caudata. The formation of Ag NPs in the colloidal solution is confirmed by UV–Vis spectroscopy analysis. The identification of biomolecules is analyzed through fourier transform infrared spectroscopy. X-ray diffraction pattern shows that an average particle size of the synthesized nanoparticles are in the range of 40–24 nm. Field emission scanning electron microscopy and transmission electron microscopy confirm the formation Ag NPs in spherical shape. The photoluminescence study of the synthesized Ag NPs interprets the influence of C caudata leaf concentrations on emission behavior. Zeta potential measurement is carried out to determine the stability of synthesized Ag NPs. GC–MS analysis revealed that the C. caudata contained 11 compounds, such as Stigmasterol (24.14 %), Hexacosanoic acid, methyl ester (15.13 %) and 2-bromophenyl morpholine-4-carboxylate (11.71 %). The antibacterial activity of Ag NPs shows that these bio capped Ag NPs have higher inhibitory action for Escherichia coli, Klebsiella pheumoniea, Micrococcus flavus, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus.     

Biosynthesis of Gold Nanoparticles by Flavonoids from <Emphasis Type="Italic">Lilium casa blanca</Emphasis>

Biosynthesis of gold nanoparticles (GNPs) by flavonoids from Lilium casa blanca has been developed. Several parameters such as pH, reaction temperature, reaction time and concentration of flavonoids were explored to control the formation of the GNPs. The synthesized GNPs were characterized by UV–Vis spectroscopy, transmission electron microscopy and X-ray diffraction. Stability and catalytic activity of the synthesized GNPs were also discussed. The results showed that the synthesized GNPs were in spherical, about 2.6 nm, with a face centered cubic structure. Synthesized GNPs showed good catalytic activity in the reduction of p-nitrophenol (p-NP) to p-aminphenol (p-AP). This method for synthesis of GNPs is simple, economic, nontoxic and efficient.     

Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from <Emphasis Type="Italic">Hypocrea orientalis</Emphasis>

A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7–22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19–29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K _m and V _max values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.     

Cotton Stalk Pretreatment Using <Emphasis Type="Italic">Daedalea flavida</Emphasis>, <Emphasis Type="Italic">Phlebia radiata</Emphasis>, and <Emphasis Type="Italic">Flavodon flavus</Emphasis>: Lignin Degradation,Cellulose Recovery,and Enzymatic Saccharification

Lignocellulolytic enzyme activities of selective fungi Daedalea flavida MTCC 145 (DF-2), Phlebia radiata MTCC 2791 (PR), and non-selective fungus Flavodon flavus MTCC 168 (FF) were studied for pretreatment of cotton stalks. Simultaneous productions of high LiP and laccase activities by DF-2 during early phase of growth were effective for lignin degradation 27.83 ± 1.25 % (w/w of lignin) in 20-day pretreatment. Production of high MnP activity without laccase in the early growth phase of PR was ineffective and delayed lignin degradation 24.93 ± 1.53 % in 25 days due to laccase production at later phase. With no LiP activity, low activities of MnP and laccase by FF yielded poor lignin degradation 15.09 ± 0.6 % in 20 days. Xylanase was predominant cellulolytic enzyme produced by DF-2, resulting hemicellulose as main carbon and energy source with 83 % of cellulose recovery after 40 days of pretreatment. The glucose yield improved more than two fold from 20-day DF-2 pretreated cotton stalks after enzymatic saccharification.     

Biosynthesis,characterization, and antimicrobial effect of silver nanoparticles obtained using <Emphasis Type="Italic">Lavandula</Emphasis> × <Emphasis Type="Italic">intermedia</Emphasis>

The use..... of aqueous leaf extract of Lavandula × intermedia for biosynthesis of silver nanoparticles (AgNPs) is presented. The plant extract was obtained by boiling dried leaves and using the obtained filtrate for the synthesis of AgNPs. The study was conducted to investigate an ecofriendly approach to metal nanoparticle synthesis and to evaluate the antimicrobial potential of both the aqueous plant extract and resulting silver nanoparticles against different microbes using the disc diffusion method. The synthesis of silver nanoparticles was monitored using ultraviolet–visible (UV–v is) spectroscopy, which showed a localized surface plasmon resonance band at 411 nm and a shift of the band to higher wavenumber of 422 nm after 90 min of reaction. Powder X-ray diffraction analysis and transmission electron microscopy of the obtained AgNPs revealed their crystalline nature, with average size of 12.6 nm. Presence of elemental silver was further confirmed by energy-dispersive X-ray spectroscopy. Fourier-transform infrared spectroscopy confirmed presence of phytochemicals from Lavandula × intermedia leaf extract on the AgNPs. The AgNPs showed good antimicrobial activity with inhibition zone ranging from 10 to 23 mm; the largest inhibition zone (23 mm) occurred against Escherichia coli. Generally, the AgNPs displayed more antimicrobial activity against all investigated pathogens compared with Lavandula × intermedia leaf extract, and were also more active than streptomycin against Klebsiella oxytoca and E. coli at the same concentration. The silver nanoparticles showed prominent antimicrobial activity with a lowest minimum inhibitory concentration (MIC) value of 15 μg/mL against E. coli, K. oxytoca, and Candida albicans.     

Heterologous Expression of Aldehyde Dehydrogenase in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> for Acetaldehyde Detoxification at Low pH

Aldehyde dehydrogenase (E.C. 1.2.1.x) can catalyze detoxification of acetaldehydes. A novel acetaldehyde dehydrogenase (istALDH) from the non-Saccharomyces yeast Issatchenkia terricola strain XJ-2 has been previously characterized. In this work, Lactococcus lactis with the NIsin Controlled Expression (NICE) System was applied to express the aldehyde dehydrogenase gene (istALDH) in order to catalyze oxidation of acetaldehyde at low pH. A recombinant L. lactis NZ3900 was obtained and applied for the detoxification of acetaldehyde as whole-cell biocatalysts. The activity of IstALDH in L. lactis NZ3900 (pNZ8148-istALDH) reached 36.4 U mL^?1 when the recombinant cells were induced with 50 ng mL^?1 nisin at 20 °C for 2 h. The IstALDH activity of recombinant L. lactis cells showed higher stability at 37 °C and pH 4.0 compared with the crude enzyme. L. lactis NZ3900 (pNZ8148-istALDH) could convert acetaldehyde at pH 2.0 while the crude enzyme could not. Moreover, the resting cells of L. lactis NZ3900 (pNZ8148-istALDH) showed a 2.5-fold higher activity and better stability in catalyzing oxidation of acetaldehyde at pH 2.0 compared with that of Escherichia coli expressing the IstALDH. Taken together, the L. lactis cells expressing recombinant IstALDH are potential whole-cell biocatalysts that can be applied in the detoxification of aldehydes.     

Purification and Characterisation of a Thermostable β-Xylosidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> van Tieghem of Potential Application in Lignocellulosic Bioethanol Production

A locally isolated strain of Aspergillus niger van Tieghem was found to produce thermostable β-xylosidase activity. The enzyme was purified by cation and anion exchange and hydrophobic interaction chromatography. Maximum activity was observed at 70–75 °C and pH 4.5. The enzyme was found to be thermostable retaining 91 and 87% of its original activity after incubation for 72 h at 60 and 65 °C, respectively, with 52% residual activity detected after 18 h at 70 °C. Available data indicates that the purified β-xylosidase is more thermostable over industrially relevant prolonged periods at high temperature than those reported from other A. niger strains. Maximum activity was observed on p-nitrophenyl-β-d-xylopyranoside and the enzyme also hydrolysed p-nitrophenyl β-d-glucopyranoside and p-nitrophenyl α-l-arabinofuranoside. The purified enzyme acted synergistically with A. niger endo-1,4-β-xylanase in the hydrolysis of beechwood xylan at 65 °C. During hydrolysis of pretreated straw lignocellulose at 70 °C using a commercial lignocellulosic enzyme cocktail, inclusion of the purified enzyme resulted in a 19-fold increase in the amount of xylose produced after 6 h. The results observed indicate potential suitability for industrial application in the production of lignocellulosic bioethanol where thermostable β-xylosidase activity is of growing interest to maximise the enzymatic hydrolysis of lignocellulose.     

Purification and Characterization of Agarase from Marine Bacteria <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>

Acinetobacter strain PS12B was isolated from marine sediment and was found to be a good candidate to degrade agar and produce agarase enzyme. The extracellular agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the crude enzyme which was 1.52 U increased to 45.76 U, after two-stage purification, with an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24 kDa. The optimum pH and temperature for activity of purified agarase were found to be 8.0 and 40 °C, respectively. The K_m and V_max values for agarase were 4.69 mg/ml and 0.5 μmol/min, respectively. Treatment with EDTA reduced the agarase activity by 58% at 5 mM concentration. The enzyme activity was stimulated by the presence of Fe²⁺, Mn²⁺, and Ca2⁺ ions while reducing reagents (β-mercaptoethanol and dithiothreitol, DTT) enhanced its activity by 30–40%. The purified agarase exhibited tolerance to both detergents and organic solvents. Major hydrolysis products of agar were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to produce seaweed hydrolysate having health benefits as well as the industrial application like the production of biofuels.     

Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant,High-Thermostable,and Salt-Tolerant Phenylalanine Dehydrogenase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis>

As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60 °C and pH 7.0, and it showed better stability in hot environment (40–70 °C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2 h in 3 M NaCl at 60 °C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.     

<Emphasis Type="Italic">Stachys lavandulifolia</Emphasis> and <Emphasis Type="Italic">Lathyrus</Emphasis> sp. Mediated for Green Synthesis of Silver Nanoparticles and Evaluation Its Antifungal Activity Against <Emphasis Type="Italic">Dothiorella sarmentorum</Emphasis>

In this study, silver nanoparticles (AgNPs) were biosynthesized using Stachys lavandulifolia and Lathyrus sp. The first sign of the reduction of silver ions to AgNPs was the change in color of S. lavandulifolia and Lathyrus sp. extracts changed into dark brown and auburn after treating with silver nitrate, respectively. The UV–Vis spectroscopy of reaction mixture (extract+silver nitrate) produced by S. lavandulifolia and Lathyrus sp. showed the strong adsorption peaks at ?440 and 420 nm, respectively. The transmission electron microscope images showed the synthesis of AgNPs using S. lavandulifolia and Lathyrus sp. with an average size of 7 and 11 nm, respectively. The result of X-ray diffraction pattern showed four diffraction peaks at 38°, 44°, 64°, and 77° for both types of biosynthesized AgNPs. Fourier transform infrared spectroscopy showed the possible role of involved proteins and polyhydroxyl functional groups in the synthesis process of AgNPs. Inductively coupled plasma analysis determined the conversion rate (percentage) of silver ions to silver nanoparticles in reaction mixtures of S. lavandulifolia and Lathyrus sp. 99.73 and 99.67 %, respectively. In addition, antifungal effect of AgNPs, synthesized by both extracts, was studied separately on mycelial growth of Dothiorella sarmentorum, in a completely randomized design on potato dextrose agar (PDA) medium. The inhibition rate of mycelial growth was strongly depended on the density of AgNPs and it strongly increased with increasing the density of AgNPs in the PDA medium. AgNPs more than 90 % of them inhibited from the mycelia growth of the fungus at the concentration of 40 µg/mL and higher.     

Production of <Emphasis Type="Italic">R</Emphasis>-Mandelic Acid Using Nitrilase from Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Cells Immobilized with Tris(Hydroxymethyl)Phosphine

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L^?1 day^?1 and the space-time productivity of 143.2 mmol L^?1 h^?1 g^?1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.     

Recovery of Phenolic Acid and Enzyme Production from Corn Silage Biologically Treated by <Emphasis Type="Italic">Trametes versicolor</Emphasis>

Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm³), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm³). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45–70 °C with the maximal activity at pH = 4.5.     

Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.     

Europium and terbium Schiff base peptide complexes as potential antimicrobial agents against <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Eu(III) and Tb(III) Schiff base complexes are applicable in various fields such as sensing, assays, screening protocols in vitro, and imaging studies in vitro or in vivo. Fluorescent europium and terbium complexes and their interaction with cell penetrating peptide (KKKRKC) can represent an excellent key for understanding pathway of peptide transportation though cell membrane and the application of Schiff base complexes as potential antibacterial drugs. The Schiff base–metal complexes and its conjugates with peptide were tested for their antibacterial activity against Pseudomonas aeruginosa and Salmonella typhimurium. Schiff base–metal complexes conjugated with peptide show minor toxicity in normal human PNT1A cells and high antibacterial activity against P. aeruginosa and S. typhimurium, where IC50 down to 125.9 and/or 36.1 µM were found for P. aeruginosa and S. typhimurium, respectively. Our results strongly suggest that Schiff base–metal complexes conjugated with peptide have great potential to be developed into highly effective antibacterial drug.     

Facile Biosynthesis of Silver Nanoparticles Using <Emphasis Type="Italic">Descurainia sophia</Emphasis> and Evaluation of Their Antibacterial and Antifungal Properties

The objective of the present study was to evaluate efficiency of silver nanoparticles (Ag-NPs) biosynthesis using Descurainia sophia as a novel biological resource. The resulting synthesized Ag-NPs were characterized using UV visible spectroscopy, X-ray diffraction, transmission electron microscopy and dynamic light scattering (DLS). The UV–Vis spectra gave surface plasmon resonance at ~420 nm. TEM images revealed formation spherical shaped Ag-NPs with size ranged from to 1–35 nm. DLS confirmed uniformity of the synthesized Ag-NPs with an average size of ~30 nm. Following, the antibacterial and antifungal activities of the synthesized Ag-NPs were investigated. The concentration 25 µg/ml of the Ag-NPs showed maximum inhibitory effect on mycelium growth of Rhizoctonia solani (More than 86 % inhibition), followed by 15 µg/ml (55 % inhibition) and 10 µg/ml (63 % inhibition). The minimum inhibitory concentration and minimum bactericidal concentration of Ag-NPs against Agrobacterium tumefaciens (strain GV3850) and A, rhizogenes (strain 15843) were 4 and 8 µg/ml, respectively. The Ag-NPs were stable in vitro for 3 months without any precipitation or decrease of antifungal effects. Finally, it could be concluded that D. sophia can be used as an effective method for biosynthesis of nanoparticles, especially Ag-NPs.     

Engineering <Emphasis Type="Italic">Pichia pastoris</Emphasis> for Efficient Production of a Novel Bifunctional <Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis> Invertebrate-Type Lysozyme

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of >?99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na⁺. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.     

Purification and characterization of an extracellular lipolytic enzyme from the fermented fish-originated halotolerant bacterium, <Emphasis Type="Italic">Virgibacillus alimentarius</Emphasis> LBU20907

A halotolerant Virgibacillus alimentarius LBU20907 isolated from fermented fish (Budu) was found to be an efficient producer of extracellular halophilic lipase enzyme. The enzyme was purified 5.99-fold with a 0.15% final yield to homogeneity by ammonium sulfate precipitation, followed by dialysis, Toyopearl DEAE-650 M ion exchange chromatography, Toyopearl butyl-650 M hydrophobic interaction chromatography, and Toyopearl-HW 55 F gel filtration chromatography. SDS-PAGE of purified lipase exhibited a homogenous single band with a very high molecular weight of 100 kDa. The properties of purified lipase revealed maximum activity at pH 7.0 and 40 °C. It was also highly stable in a pH range of 6.0–7.0, retaining more than 90% activity for 24 h. It was stable at the temperature of 30–50 °C and maintained more than 80% activity for 16 h. The purified lipase performing the maximal activity in the presence of 20.0% NaCl indicated halophilic enzyme properties. Its lipolytic activity was highest against p-nitrophenyl palmitate. The lipase activity was found to be enhanced in hexane. The enzyme activity was stimulated in the presence of Zn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺; while, it was completely inhibited by Ba²⁺ and Co²⁺. The enzyme had a K _m and V _max of 108.0 mg and 79.1 U mL^?1, respectively.     

Study of the growth of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>, <Emphasis Type="Italic">Escherichia coli</Emphasis> and their mixtures by microcalorimetry

In a majority of environments, microbes live as interacting communities. Microbial communities are composed of a mix of microbes with often unknown functions. Polymicrobial diseases represent the clinical and pathological manifestations induced by the presence of multiple infectious agents. These diseases are difficult to diagnose and treat and usually are more severe than monomicrobial infections. The interaction relationship between Enterococcus faecalis and Escherichia coli was researched using a Calvet calorimeter. Three mixtures of both bacteria were prepared in the following proportions: 20 + 80 % (0.2 mL E. faecalis + 0.8 mL E. coli), 50 + 50 % (0.5 mL E. faecalis + 0.5 mL E. coli) and 80 + 20 % (0.8 mL E. faecalis + 0.2 mL E. coli). Experiments were carried out at concentration of 10⁶ CFU mL^?1 and a constant temperature of 309.65 K. The differences in shape of graph of E. faecalis, E. coli and their mixtures were compared. Also, the thermokinetic parameters such as detection time (t _d), growth constant (k), generation time (G) and the amount of heat released (Q) were calculated.