Developments and Microbiological applications in African foods: Emphasis on Nigerian Wara cheese

African indigenous foods have received limited research. Most of these indigenous foods are fermented and they form part of the rich nutritional culture of many groups in African countries. The industrialization and commercialisation of these indigenous African fermented foods should be preceded by a thorough scientific knowledge of their processing which can be vital in the elimination of hunger and poverty. This study highlighted emerging developments and the microbiology of cereal-based and cassava-based food products that constitute a major part of the human diet in most African countries. In addition, investigations were also carried out on the coagulant of the Calotropis procera plant used in traditional production of Nigerian Wara cheese and on the effects of adding a nisin producing Lactococcus lactis strain originating from human milk to Nigerian Wara cheese. Fermented cereal-based food such as ogi utilize popular African and readily available grains maize, millet or sorghum as substrates and is popular as a weaning diet in infants. In this study, the bulkiness caused by starch gelatinization was solved by amylase treatments in the investigation on cooked and fermented oat bran porridge. A similar treatment could reduce the viscosity of any cereal porridge. The properties of the Sodom apple leaves (Calotropis procera) extract in cheesemaking were studied. C. procera was affected by monovalent (K+ and Na+) and divalent (Mg2+ and Ca2+) cations during coagulation. The rennet strength of this coagulant was found to be 7 % compared to animal rennet at 35 °C. Increasing the incubation temperature to 70 °C increased the rennet strength 28-fold. The molecular weight of the partially purified protease was determined by SDS-PAGE and was confirmed by Zymography to be approximately 60 kilodaltons. The high proteolytic activity at 70 °C supported the suitability of the protease enzyme as a coagulant in future commercial production of Nigerian Wara cheese. It was also possible to extend the shelf life of Wara cheese by a nisin producing lactic acid bacteria Lactococcus lactis LAC309. The levels of nisin in both whey and curd fractions of Wara were investigated, results showed a 3 log reduction of toxicogenic Bacillus licheniformis spiked on Wara after 3 days. These studies are the first in Finland to promote the advancement of scientific knowledge in African foods. Recognizing these indigenous food products and an efficient transfer of technology from the developed countries to industrialize them are necessary towards a successful realization of the United Nations Millenium Development Program.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

L-rhamnose-1-dehydrogenase gene and L-rhamnose catabolism in the yeast Pichia stipitis

The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.

Seminal plasma proteome of eletroejaculated Bos indicus bulls

The present study describes the seminal plasma proteome of Bos indicus bulls. Fifty-six, 24-month old Australian Brahman sires were evaluated and subjected to electroejaculation. Seminal plasma proteins were separated by 2-D SDS-PAGE and identified by mass spectrometry. The percentage of progressively motile and morphologically normal sperm of the bulls were 70.4±2.3 and 64±3.2%, respectively. A total of 108 spots were identified in the 2-D maps, corresponding to 46 proteins. Binder of sperm proteins accounted for 55.8% of all spots detected in the maps and spermadhesins comprised the second most abundant constituents. Other proteins of the Bos indicus seminal plasma include clusterin, albumin, transferrin, metalloproteinase inhibitor 2, osteopontin, epididymal secretory protein E1, apolipoprotein A-1, heat shock 70kDa protein, glutathione peroxidase 3, cathelicidins, alpha-enolase, tripeptidyl-peptidase 1, zinc-alpha-2-glycoprotein, plasma serine protease inhibitor, beta 2-microglobulin, proteasome subunit beta type-4, actin, cathepsins, nucleobinding-1, protein S100-A9, hemoglobin subunit alpha, cadherin-1, angiogenin-1, fibrinogen alpha and beta chain, ephirin-A1, protein DJ-1, serpin A3-7, alpha-2-macroglobulin, annexin A1, complement factor B, polymeric immunoglobulin receptor, seminal ribonuclease, ribonuclease-4, prostaglandin-H2 D-isomarase, platelet-activating factor acetylhydrolase, and phosphoglycerate kinase In conclusion, this work uniquely portrays the Bos indicus seminal fluid proteome, based on samples from a large set of animals representing the Brahman cattle of the tropical Northern Australia. Based on putative biochemical attributes, seminal proteins act during sperm maturation, protection, capacitation and fertilization.

Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.

Studies on the variants of the protein toxins ricin and abrin

his study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101–109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.

An NAD(P)(+)-dependent secondary-alcohol dehydrogenase of Alcaligenes eutrophus: Purification, characterization and its application for the production of chiral alcohols

A soil micro-organism identified as Alcaligenes eutrophus capable of utilizing nerolidol, a sesquiterpene alcohol as the sole source of carbon, contains an inducible NAD(P)(+)-linked secondary-alcohol dehydrogenase (SADH), The enzyme was purified 252-fold from crude cell-free extract by a combination of salt precipitation, ion-exchange and affinity-matrix chromatography, Native and SDS/PAGE PAGE of the purified enzyme showed a single protein band and the enzyme appears to be a homotetramer having an apparent molecular mass of 139 kDa comprising four identical subunits of 38.5 kDa, The isoelectric point (pi) of SADH was determined to be 6.2, Depending on pH of the reaction media, the enzyme carried out both oxidation and reductions of various terpenoids and steroids, At pH 5.5, the enzyme catalysed the stereospecific reduction of prochiral ketones to optically active (S)-alcohols and the oxidation reaction was predominated over the former at pH 9.5, NADP(+) and NADPH were respectively preferred over NAD(+) and NADH for oxidation and reduction reactions, The K-m values for testosterone, NADP(+) and NAD(+) were 11.8, 55.6, and 122 mu M respectively, Neither enzyme was significantly inhibited by metal-binding agents, but some thiol-blocking compounds inhibited it, SADH tolerates moderate concentrations of water-miscible organic solvents such as ethanol, methanol, acetone and dioxan, Some of the properties of this enzyme were found to be significantly different from those thus far described.

Detection of alpha(2u)-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry

The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha 2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha 2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha 2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats.This novel alpha 2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha 2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha 2u. The present investigation confirms the presence of alpha 2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.Copyright (C) 2010 John Wiley & Sons, Ltd.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Primary Structural Documentation of the Major Urinary Protein of the Indian Commensal Rat (Rattus rattus) Using a Proteomics Platform

Purpose: A number of proteome studies have been performed recently to identify pheromone-related protein expression and their molecular function using genetically modified rodents' urine. However, no such studies have used Indian commensal rodents; interestingly, in a previous investigation, we confirmed the presence of volatile molecules in commensal rodents urine and these molecules seem to be actively involved in pheromonal communication. Therefore, the present study aims to identify the major urinary protein [MUP] present in commensal rat urine, which will help us to understand the protein's expression pattern and intrinsic properties among the rodents globally. Experimental Design: Initially, the total urinary proteins were separated by 1-D and 2-D electrophoresis and then subsequently analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometer (MALDI-TOF/MS). Furthermore, they were then fragmented with the aid of a Tandem Mass Spectrometer (TOF/TOF) and the identified sequences aligned and confirmed using similarity with the deduced primary structures of members of the lipocalin superfamily.Results: The SDS-PAGE protein profiles showed distinct proteins with molecular masses of 15, 22.4, 25, 28, 42, 50, 55, 68, and 91 kDa. Of these proteins, the 22.4 kDa protein was considered to be target candidate. When 2D electrophoresis was carried out, about similar to 50 spots were detected with different masses and various pI ranges. The 22.4 kDa protein was found to have a pI of about 4.9. This 22.4 kDa protein spot was digested and subjected to mass spectrometry; it was identified as rat MUP. The fragmented peptides from the rat MUP at 935, 1026, 1192, and 1303 m/z were further fragmented with the aid of MS/MS and generated de novo sequence and this confirmed this protein to be the MUP present in the urine of commensal rats.Conclusion: The present investigation confirms the presence of MUP with a molecular mass of 22.4 kDa in the urine of commensal rats. This protein may be involved in the binding of volatile molecules and opens up a discussion about how volatile and non-volatile molecules in the commensal rats' urine may contribute chemo-communication.

Characterization of viral proteins ofOryctes baculovirus and comparison between two geographical isolates

BacilliformOryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infectedOryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reportedOryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins stained positive for glycosylation. Comparison between the viral proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

Aspergillus niger -proliiniendopeptidaasi prolamiinien eliminoinnissa

Tutkielman kirjallisuusosassa perehdyttiin vehnän, rukiin ja ohran, eli Triticeaeprolamiinien erityisasemaan keliakianäkökulmasta tarkasteltuna ja prolamiinien hydrolyysiin proliinispesifeillä entsyymeillä. Lisäksi tarkasteltiin prolamiinien immunologisia määritysmenetelmiä. Keliakiassa haitalliset gluteenipeptidit sisältävät runsaasti proliinia ja ovat hankalia pilkkoa muilla kuin proliinispesifeillä peptidaaseilla. Suurin osa immunologisen reaktion aiheuttavista gluteenilähtöisistä peptideistä voidaan pilkkoa idätetyn viljan endogeenisilla entsyymeillä happamissa olosuhteissa, mutta jäljellejäävä prolamiinipitoisuus ylittää edelleen gluteenittomille tuotteille sallitun rajan. Kokeellisen työn tavoitteena oli eliminoida happamalla mallasinkubaatiolla valmistettujen vehnä-, ohra- ja ruismallasautolysaattien sisältämä jäännösprolamiini Aspergillus niger -homeen tuottamalla proliinispesifillä endopeptidaasilla (AN-PEP) siten, että hydrolysaattia voitaisiin käyttää gluteenittomissa leivontasovelluksissa. Proteiinien hydrolyysiä tarkkailtiin kokoekskluusiokromatografialla (SEC), vapaan aminotypen (FAN) muodostumisena ja SDS-PAGE-elektroforeesilla. Jäännösprolamiinien pilkkoutumista seurattiin immunologisella R5-ELISA-menetelmällä. AN-PEP-inkubaatiolla saatiin aikaan voimakasta prolamiinien pilkkoutumista; mallasautolysaattien jäännösprolamiinista pilkkoutui yli 96 %. SEC- ja FAN-analyysien perusteella inkubaatioaikaa kannatti jatkaa yli 4 h, jolloin polypeptidit pilkkoutuivat edelleen pienemmiksi hydrolyysituotteiksi. Vehnä- ja ruismallashydrolysaattien prolamiinipitoisuuden todettiin laskevan 22 h inkubaation aikana alle tason 100 mg/kg R5-ELISA-menetelmällä määritettynä. Matalimmat prolamiinipitoisuudet saavutettiin AN-PEP-pitoisuudella 35 ?l / g mallasautolysaattia. Codex Alimentarius -komission säädöksen mukaan keliakiaruokavalioon soveltuvat ns. erittäin vähägluteeniset tuotteet saavat sisältää gluteenia enintään 100 mg/kg. Erityisesti AN-PEP-käsiteltyä ruismallasraaka-ainetta voitaisiin mahdollisesti käyttää tuomaan rukiista aromia gluteenittomiin leipiin. Ennen kuin mallashydrolysaatit ovat valmiita kaupallisiin sovelluksiin, on tarkasteltava niiden todellisia mahdollisuuksia parantaa elintarvikkeiden makua ja aromia sekä todettava uuden teknologian turvallisuus keliaakikoille.

Isolation and characterization of heat-stable allergens from shrimp (Penaeus indicus)

Shrimp are among the more common causes of immediate hypersensitivity reactions to food. To characterize better the allergenic substances within shrimp, extracts from heated shrimp were systematically examined with solid-phase radioimmunoassay and sera from patients clinically sensitive to shrimp. Two heat-stable protein allergens, designated as Sa-I and Sa-II, were identified from boiled shrimp (Penaeus indicus) extracts. Sa-I was isolated by ultrafiltration, Sephadex G-25, and diethylaminoethyl-Sephacel chromatography, whereas Sa-II, the major allergen, was purified by successive chromatography on diethylaminoethyl-Sephacel, Bio-Gel P-200, and Sepharose 4B columns. Sa-I, which was homogeneous by polyacrylamide gel electrophoresis (PAGE), elicited a single band on sodium dodecyl sulfate-PAGE corresponding to a molecular weight of 8.2 kd. Sa-II was also found to be homogeneous by PAGE, crossed immunoelectrophoresis, and immunoblotting. On sodium dodecyl sulfate-PAGE, it elicited a single band with a molecular weight of 34 kd. Sa-II was found to contain 301 amino acid residues and was particularly rich in glutamate/glutamine and aspartate/asparagine. Solid-phase radioimmunoassay-inhibition studies revealed that Sa-I and Sa-II share 54% of the allergenic epitopes, suggesting that Sa-I may be a fragment of Sa-II.SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MW, Molecular weight; BSA, Bovine serum albumin; DEAE, Diethylaminoethyl; SPRIA, Solid-phase radioimmunoassay; CIE, Crossed immunoelectrophoresis .

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35–40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.

Purification and functional characteristics of an endocellulase from Chaetomium thermophile var. coprophile

An endocellulase (1→4)-β-d-glucan 4-glucanohydrolase was isolated from the culture filtrates of Chaetomium thermophile. The enzyme was homogeneous by PAGE and SDS-PAGE. The molecular weight was 36 000 by SDS-PAGE and 38 000 by gel filtration. It was a glycoprotein. From the amino acid composition, it was found to be rich in glycine, threonine, and aspartic and glutamic acids, but contained only low proportions of histidine and sulfur-containing amino acids. It was optimally active at pH 6 and at 60°. The enzyme did not hydrolyze cellobiose and cellotriose, but hydrolyzed cello-tetraose, -pentaose, and -hexaose at comparable rates. It was specific for molecules containing β-(1→4) linkages. It showed high activity towards amorphous cellulose, and the reaction products contained cellobiose to cellopentaose, showing that it effects random cleavage of cellulose.