Screening, Production and Partial Characterization of a Thermostable Laccase from Trametes sp. Isolate B7 with Biotechnological Potentials

The search for efficient and green oxidation technologies has increased interest in utilization of laccases in non conventional methods. Laccases catalyze a wide range of substrates due to low substrate specificity and strong oxidative potentials. Challenges to large-scale enzyme utilization include, low enzyme activity and instability which restrict use in many areas of biotechnology. In the study, 59 fungi comprising Aspergillus niger (40%), Trichoderma harzianum (31%), Aspergillus flavus (9.0%), Trichoderma viride (5.0%), Fusarium oxysporum (5.0%), Rhizopus stolonifer (5.0%), Trametes sp. (3.0%) and Aspergillus nidulans (2.0%) were isolated and screened for laccase production. Plate screening test showed 57.5%, 34.0% and 8.5% of fungi were laccase-positive on ABTS, Guaiacol, and α-naphthol agar respectively. Isolates were further screened in liquid cultures, and the highest laccase producer identified molecularly. Trametes sp isolate B7 was selected for solid state fermentation (SSF). Laccase production in SSF was highest at pH 5.0 (2356 U/mL). The purified laccase showed high activity (pH 3.0 - 6.0) and stability (pH 3.0 - 8.5) using ABTS. It was active (20 - 80°C) and thermostable (30 - 80°C) with optimum stability at 70°C (100% for 1 hour). The percentage decolourization of Phenol red were 28% and 36% using 1000 U/mL and 2000 U/mL crude laccases respectively. Similarly, RBBR (100%), Congo red (75%) and Malachite green (62%), and 77.4%, 64% and 28% were decolourized using 1000 U/mL and 2000 U/mL crude laccases respectively. ABTS agar was very reliable in large-scale screening for laccase which possessed thermostable property and degraded synthetic dyes without use of enzyme mediators. These attribute made the enzyme suitable for application in industry and biotechnology.

Alanine mother liquor as a nitrogen source for docosahexaenoic acid production by Schizochytrium sp. B4D1

Alanine mother liquor, a type of industrial waste from alanine fermentation, was used as a nitrogen source to produce docosahexaenoic acid (DHA) by Schizochytrium sp. B4D1. The results indicated that yeast extract could trigger the utilization of the alanine mother liquor. Additionally, the alanine can be quenched during the culture, which aids in DHA accumulation. The medium components were optimized via response surface methodology as follows: 99.98-g/L glucose, 0.05-g/L yeast extract and a 183.17 dilution factor of the alanine mother liquid (v/v, with an alanine content of 0.72 g/L) and 17.98% inoculum concentration (v/v). Finally, in a 50-mL shake-flask fermentation, the DHA yield was 2.29 g/L.

Effects of Mycobacterium tuberculosis heat-resistant antigen on secretion of SP-B and apoptosis in A549 cells / 安徽医科大学学报

Objective To investigate secreting pulmonary surfactant-associated protein B (SP-B) and apoptosis in human lung adenocarcinoma cell line (A549) cells,treated with heat-resistant antigen of Mycobacterium tuberculosis (Mtb-HAg).Methods Different concentrations of Mtb-HAg were used to culture A549 cells for 24 h and 48 h respectively,and the blank control groups(control group 1 and control group 2) and positive control groups [lipopolysaccharide (LPS) group and curcumin group] were set up.SP-B in the culture supernatant,was assessed by ELISA in control group 1,LPS group and experimental group 1 (A549 cells were respectively induced by 2 μg/ml,3 μg/ml Mtb-HAg to culture for 24 h and 48 h).For control group 1,LPS group and experimental group 1,the relative expression of SFTPB gene was quantified with quantitative real-time fluorescence quantitative PCR (qRT-PCR).The apoptosis rate of control group 2,curcumin group and experimental group 2(A549 cells were respectively induced by 2 μg/ml,3 μg/ml Mtb-HAg to culture for 24 h) in A549 cells were detected by flow cytometry.Results SP-B expression in the experimental group 1 was significantly lower than the control group 1,the difference was statistically significant (P ＜ 0.05).The difference of SP-B expression in the experimental group 1 was not obvious with the prolongation of the same concentration.At the same incubation time,the expression of SFTPB in the experimental group 1 decreased obviously with the increasing concentration of Mtb-HAg,the difference was statistically significant (P ＜ 0.05).The change with time was not significant.The apoptosis rate of curcumin group and experimental group 2 were significantly higher than that in control group 2 in A549 cells (P ＜ 0.05).Conclusion Mtb-HAg inhibited the expression of SP-B in A549 cells significantly,and induced apoptosis of A549 cells.

In vitro activity of the clinical pulmonary surfactant Surfacen® against Leishmania amazonensis / Actividad in vitro de surfactante pulmonar clínico Surfacen® frente a Leishmania amazonensis

Surfacen® is an exogenous natural lung surfactant, composed by phospholipids and hydrophobic proteins, which is applied successfully in Newborn Respiratory Distress Syndrome. In this paper, in vitro activity of Surfacen® against Leishmania amazonensis is described. The product showed activity against the amastigote form found in peritoneal macrophages from BALB/c mice, with an IC50 value of 17.9 ± 3.0 µg/mL; while no toxic effect on host cell was observed up to 200 µg/mL. This is the first report about the antileishmanial activity of Surfacen®.

Surfacen® es un surfactante natural exógeno extraído del pulmón, formado por fosfolípidos y proteínas hidrofóbicas, el cual es aplicado con éxito en el Síndrome de Distrés Respiratorio en Niños Recién Nacidos. En este trabajo, se describe la actividad in vitro del Surfacen® contra Leishmania amazonensis. El producto mostró actividad frente a amastigotes que se encuentran en macrófagos peritoneales de ratón BALB/c, con una CI50 de 17.9 ± 3.0 µg/mL, mientras no se observaron efectos tóxicos sobre la célula hospedera hasta 200 µg/mL. Este estudio constituye el primer reporte sobre la actividad antileishmania del Surfacen®.

Construction of PLAGL2 siRNA Expression DNA Plasmid and the Interfering Effect on Gene Expression in H441 Cells / 华中科技大学学报(医学版)

Objective To construct human PLAGL2 siRNA expression DNA plasmid and study the interfering effect on gene expression in H441 cells. Methods H441 cells were transfected with PLAGL2 siRNA expression DNA plasmid with Fu-gene6. Real time PCR and Western blot were employed to detect the interfering effect on the expression of PLAGL2.SP-CSP-B mRNA and protein in wildtype and PLAGL2 siRNA expression DNA plasmid transfected H441 cells respectively. Results Real time PCR revealed that,as compared with wildtype H441 cells, the expression level of PLAGL2 mRNA in PLAGL2 siRNA plasmid transfected H441 cells was significantly reduced(P<0. 05) ,but that of SP-C mRNA in PLAGL2 siRNA plasmid transfected H441 cells was significantly increased(P<0. 05). Western blot showed that,as compared with wildtype H441 cells, the expression level of PLAGL2 protein in PLAGL2 siRNA DNA plasmid transfected H441 cells was significantly reduced(P< 0. 05). Conclusion Human PLAGL2 siRNA expression DNA plasmid was constructed successfully,and its transfection into the H441 cells could effectively inhibit the PLAGL2 expression,and simultaneously promote the expression of SP-C.

A Study of Surface Physical Properties of New Surfactant Using Synthetic Peptides of Surfactant Protein-B

PURPOSE: To produce a new generation of artificial pulmonary surfactant(PS), surfactant protein (SP)-B from human PSwas isolated, and the amino acid sequences of these proteins were studied. Artificial peptides of human SP-B were synthesized. New artificial PS preparations which were cornposed of phospholopids and two artificial synthetic SP-B peptides were made, and the surface physical properties of these new PS preparations were tested. METHODS: The purities of SP-B were assessed by SDS-polyacrylamide gel and the amino acid sequences of these proteins were determined. We synthetized two peptides SP-1 and SP-2 and the amino acid sequences were as follows,' SP-1: RMLPQLVCRLVLRCSMD, SP-2: RMLP- QLVCRLVLRCSM. Surface physical properties of newly artificial PSs, which were composed of a mixture of phospholipid(PL) and SP-1 or SP-2(sample A; PL+SP-1, sample B; PL+SP-2), were measured by surface spreading, adsorption rate, and surface tension-area diagram. RESULTS: The amino acid sequence of human SP-B was obtained. We produced the artificial peptides of SP-B and prepared the new generation PS(sample A and sample B). The order of the superiority of spreading and adsorption rate was Surfacten

Purification and Analysis of Amino Acid Sequences of Pulmonary Surfactant Proteins

PURPOSE: For the synthesis of surfactant protein(SP) peptides and production of next generation artificial pulmonary surfactant(PS), we have isolated SP-B, C from bovine PS, and studied the biochemical properties and amino acid sequences of these protein-peptides. METHODS: Crude surfactant and purified surfactant were isolated from materials extracted from the bovine lung lavage. The hydrophobic SP-B, C were purified by Sephadex LH 60 column chromatography from PS. The purities of SP-B, C were assessed by tricine buffer SDS-polyacrylamide gel electrophoresis and the amino acid sequences of these proteins were determined using Beckman PI-2090. RESULTS: The molecular weights of SP-B, C shown in SDS-polyacrylamide gel electrophoresis were as follows; 15,000-18,000(oligomer) Da for SP-B, 3,500-5,000 Da for SP-C. The amino acid sequences were; FPIPLPYCWL LRTLIKKIQA VIPKGVLAMT VAQCHVVPL LVGGIQQLV IEYSVILLTD TLLGRLPNLV CGLRLRCSG in SP-B, LIPCCPVNIK RLLIVVVVVV LLVVVIVGAL LMGL in SP-C, respectively. These results indicated that the amino acid sequences of bovine SPs were different. CONCLUSION: The SP-B, C were purified from bovine PS, and amino acid sequences of SP-B, C were determined. Further studies are needed for the development and use of next generations of exogenous PS preparation based on synthetic SP-peptides for the treatment of neonatal RDS in the future.

Preparation and in vitro Physical Activities of Crude Natural Surfactant and Artificial Pulmonary Surfactant Containing Synthetic Peptide and Phospholipid Mixtures

PURPOSE: In this study, natural pulmonary surfactant was extracted from bovine lung lavage and its surface activity was determined. To investigate the usefulness of synthetic peptides reconstituted with phospholipid as artificial surfactant, truncated peptides from surfactant protein (SP)-B were synthesized and restored the surface tension lowering activities when appropriately recombined with phospholipid. METHODS: Crude natural surfactant (CNS) was isolated from lung lavage by centrifugation and organic solvent for the extraction of pulmonary surfactant was selected to satisfy the in vitro physical properties. Two truncated peptides derived from C-terminal end of bovine SP-B hydrophobic protein were selected and synthesized. To prepare artificial surfactant, synthetic peptides was added to the phospholipid mixture. The various surfactant mixtures were assayed for in vitro physical activity with the Wilhemly plate method and were determined by surface spreading rate, surface adsorption rate and surface tension-area diagram. RESULTS: CNS-chloroform methanol (CM) displayed efficient surface activity compared with clinically used Surfacten but CNS-BuOH did not. The artificial surfactants containing phospholipid mixture and synthetic peptide were analyzed for their surface activities and displayed significant surfactant properties. CONCLUSION: 1-Butanol or CM (3:1) was used as an extraction solvent for CNS. CNS-CM showed more efficient surface activity than CNS-BuOH. Two synthetic peptides composing artificial pulmonary surfactant were designed and mixing ratio of peptide and phospholipid was established. Artificial surfactant dispalyed weaker surface activity than natural surfactant but significant surfactant activity.