Lipase from marine Aspergillus awamori BTMFW032: Production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal- 0.77% (w/v); (NH4)2SO4-0.1 M; KH2PO4-0.05 M; rice bran oil-2% (v/v); CaCl2-0.05 M; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35 8C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6- fold increase in lipase production was achieved. Partial purification by (NH4)2SO4 precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40 8C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.

Extracellular β-glucosidase Production by a Marine Aspergillus sydowii BTMFS 55 under Solid State Fermentation Using Statistical Experimental Design

A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as ^ëéÉêJ Öáääìë=ëóÇçïáá BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of ^ëéÉêÖáääìë=ëóÇçïáá in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH4)2SO4 precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ~95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards éNPG and enzyme has a hã and sã~ñ of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a há of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of p~ÅÅÜ~êçãóÅÉë=ÅÉêÉîáëá~É in presence of cellulase and the purified β-glucosidase of ^ëéÉêÖáääìë=ëóÇçïáá BTMFS 55.

Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11

Bacillus smithii BTMS 11, isolated from marine sediment, produced alkaline and thermostable lipase. The enzyme was purified to homogeneity by ammonium sulfate precipitation and ion exchange chromatography which resulted in 0.51 % final yield and a 4.33 fold of purification. The purified enzyme was found to have a specific activity of 360 IU/mg protein. SDS-PAGE analyses, under non-reducing and reducing conditions, yielded a single band of 45 kDa indicating the single polypeptide nature of the enzyme and zymogram analysis using methylumbelliferyl butyrate as substrate confirmed the lipolytic activity of the protein band. The enzyme was found to have 50 C and pH 8.0 as optimum conditions for maximal activity. However, the enzyme was active over wide range of temperatures (30–80 C) and pH (7.0–10.0). Effect of a number of metal salts, solvents, surfactants, and other typical enzyme inhibitors on lipase activity was studied to determine the novel characteristics of the enzyme. More than 90 % of the enzyme activity was observed even after 3 h of incubation in the presence of commercial detergents Surf, Sunlight, Ariel, Henko, Tide and Ujala indicating the detergent compatibility of B. smithii lipase. The enzyme was also found to be efficient in stain removal from cotton cloths. Further it was observed that the enzyme could catalyse ester synthesis between fatty acids of varying carbon chain lengths and methanol with high preference for medium to long chain fatty acids showing 70 % of esterification. Results of the study indicated scope for application of this marine bacterial lipase in various industries

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion-exchange chromatography, could be determined accurately since, xylem-specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.

Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)2SO4 precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS–PAGE it was found to be a monomeric protein with molecular mass 72±2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 μM) and highest specificity constant (Vmax/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 μM and Ki=205 μM, respectively).

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin I cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins. (C) 2009 Elsevier Inc. All rights reserved.

Galactooligosaccharides (GOS) inhibit Vibrio cholerae toxin binding to its GM1 receptor

It is widely reported that cholera toxin (Ctx) remains a significant cause of gastrointestinal disease globally, particularly in developing countries where access to clean drinking water is at a premium. Vaccines are prohibitively expensive and have shown only short-term protection. Consequently, there is scope for continued development of novel treatment strategies. One example is the use of galactooligosaccharides (GOS) as functional mimics for the cell-surface toxin receptor (GM1). In this study, GOS fractions were fractionated using cation exchange chromatography followed by structural characterization using a combination of hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) such that their molecular weight profiles were known. Each profile was correlated against biological activity measured using a competitive inhibitory GM1-linked ELISA. GOS fractions containing > 5% hexasaccharides (DP6) exhibited > 90% binding, with EC50 values between 29.27 and 56.04 mg/mL. Inhibition by GOS DP6, was dose dependent, with an EC50 value of 5.10 mg/mL (5.15 mu M MW of 990 Da). In removing low molecular weight carbohydrates that do possess prebiotic, nutraceutical, and/or biological properties and concentrating GOS DP5 and/or DP6, Ctx antiadhesive activity per unit of (dry) weight was improved. This could be advantageous in the manufacture of pharmaceutical or nutraceutical formulations for the treatment or prevention of an acute or chronic disease associated with or caused by the adhesion and/or uptake of a Ctx or HLT.

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillits phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5. a K-m of 14 mM with alpha-D-Manp-(1 -> 6)-D-Manp as substrate. It was strongly inhibited by Mn2+ and did not need Ca2+ or any other metal cofactor of those tested. The enzyme cleaves specifically (1 -> 6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocus sing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1 -> 3)-D-Manp 10 times faster than alpha-D-Manp-(1 -> 6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1 -> 2)-D-Manp. The activity towards (1 -> 3)-linked mannobiose is strongly activated by 1 mM Ca2+ and inhibited by 10 mM EDTA, while (1 -> 6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance

The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR). Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry. Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively. A significant correlation was observed (r(2) = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A range of proteins (n = 20) previously associated with the mar locus in E. coli were also found including the key MAR effectors AcrA, TolC and OmpF.

The p38-MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs).

SB203580 is a recognised inhibitor of p38-MAPKs. Here, we investigated the effects of SB203580 on cardiac SAPKs/JNKs. The IC50 for inhibition of p38-MAPK stimulation of MAPKAPK2 was approximately 0.07 microM, whereas that for total SAPK/JNK activity was 3-10 microM. SB203580 did not inhibit immunoprecipitated JNK1 isoforms. Three peaks of SAPK/JNK activity were separated by anion exchange chromatography, eluting in the isocratic wash (44 kDa), and at 0.08 M (46 and 52 kDa) and 0.15 M NaCl (54 kDa). SB203580 (10 microM) completely inhibited the 0.15 M NaCl activity and partially inhibited the 0.08 M NaCl activity. Since JNK1 antibodies immunoprecipitate the 46 kDa activity, this indicates that SB203580 selectively inhibits 52 and 54 kDa SAPKs/JNKs.

Expression and purification of a recombinant adenovirus fiber knob in a baculovirus system

Objectives: To construct a recombinant baculovirus expressing the fiber knob domain of human adenovirus type 2 modified by the insertion of a foreign peptide, purify this protein after its production in insect cells, and to test its properties. Methods: Recombinant baculoviruses expressing the fiber knob were produced in Sf9 cells. The recombinant fiber knob was recovered from culture supernatants of infected cells and purified by a combination of Ni-NTA and ion-exchange chromatography. Results: Fiber knob was recovered from the culture media as a soluble protein. In the system used, the fiber knob is expressed fused with the V5 epitope and a histidine tag, which allowed purification by Ni-NTA chromatography. The protein was further purified by ion-exchange chromatography. We show that the recombinant fiber knob produced, with 31 extra amino acids in the C-terminus, can oligomerize and bind to the adenovirus receptor CAR, as it can block the infection of a recombinant type 5 adenovirus. Conclusions: The modified form of the fiber knob, produced in insect cells and purified by Ni-NTA and ion-exchange chromatography, retains the properties of oligomerization and binding to the fiber natural receptor, CAR. This construct has the potential to be a new adjuvant. Copyright (C) 2008 S. Karger AG, Basel.

Fluorimetric determination of intra- and extracellular free amino acids in the microalgae Tetraselmis gracilis (Prasinophyceae) using monolithic column in reversed phase mode

This paper describes the development and application of an RP HPLC method using a C(18) monolithic stationary phase for the separation and quantification of extra- and intracellular amino acids in a batch cultivation of the marine alga Tetraselmis gracilis. Fluorimetric detection was made after separation of the o-phthaldialdehyde 2-mercaptoethanol (OPA-2MCE) derivatives using a binary gradient elution. Separation of 19 amino acids was achieved with resolution >1.5 in about 39 min at a flow rate of 1.5 mL/min. RSD of analyses in seawater medium ranged from 0.36% for Orn (0.50 mu mol/L) to 12% for Ile (0.10 mu mol/L). The main constituents of the intracellular dissolved free amino acids (DFAAs) in the exponential growth phase were arginine (Arg), asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), glutamine (Gln), and leucine (Leu). The major amino acids excreted to the media were valine (Val), Ala, Ser, and Gly. The monolithic phase facilitates the analysis by shortening the separation time and saving solvents and instrumentation costs (indeed conventional HPLC instrumentation can be used, running at lower pressures than those ones used with packed particle columns).

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k(cat)/K(m) values of 20.0 +/- 1.1 mu M(-1) s(-1) and 30.0 +/- 0.5 mu M(-1) s(-1), respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane. (C) 2011 Elsevier Ltd. All rights reserved.

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, A beta NA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3. Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. (c) 2010 Elsevier Inc. All rights reserved.