Properties of Peanut (KAC431) Protein Hydrolysates and Their Impact on the Quality of Gluten-Free Rice Bread

Protein hydrolysates (PH) with a degree of hydrolysis (DH) of 5%, 10%, and 13% from two varieties of peanut were prepared using two commercial enzymes, Alcalase and Flavourzyme. The content of essential amino acids (30,290 mg/100 g) and hydrophobic amino acids (34,067 mg/100 g) of the peanut variety Kalasin 2 (KAC431) protein was higher than that of a common variety, Kalasin 1 (KAC1) (p < 0.05). The protein molecular weight distributions of the two varieties of peanut detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were similar, ranging from 15 to 75 kDa, with a major protein band at 50–75 kDa. The antioxidant and functional properties of derived PHs were influenced by DH. Although the foaming ability of protein was improved by DH5%, it was obviously decreased upon increasing DH further. The best emulsifying properties were observed in PH with DH5% (p < 0.05). The incorporation of PH with a small DH, especially when produced using Flavourzyme, had a highly positive impact on the specific volume and relative elasticity of gluten-free bread. The effect of PHs on bread quality was highly correlated with their functional properties. This study suggests that partially enzymatically modified proteins are suitable for incorporation in food products such as bread and other gluten-free products.

Cloning, characterization and directed evolution of a xylosidase from Aspergillus niger

β-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant.

Comparative polyphasic characterization of Streptococcus phocae strains with different host origin and description of the subspecies Streptococcus phocae subsp. salmonis subsp. nov.

A polyphasic study was undertaken to clarify the taxonomic position of Streptococcus phocae strains isolated from Atlantic salmon (Salmo salar) cage-farmed in Chile. Four salmon and three seal isolates showed minor differences in the SDS-PAGE protein analysis. Thus, a major protein band present in the salmon isolates, of approximately 22.4 kDa, was absent in the pinniped strains, regardless of the growth media employed. In addition, the pinniped strains showed protein bands with molecular masses of 71.5 and 14.2 kDa, when grown on trypticase soy agar supplemented with 1 % NaCl, or 25.6 kDa, when grown on Columbia blood agar, not present in the Atlantic salmon strains. A high similarity in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS spectra of the strains was observed, although some minor peaks were absent in the fish isolates. Fatty acid methyl esters from isolates with different host origin significantly (P<0.05) differed in the content of C16 : 0, C17 : 0, C18 : 1ω9c, C20 : 4ω6,9,12,15c and summed features 3, 5 and 8. The salmon isolates formed a separate cluster in the phylogenetic analysis of housekeeping genes, separately or as concatenated sequences. Sequence divergences among salmon and seal strains were in the range of inter-subspecies differentiation for groEL (2.5 %), gyrB (1.8 %), recN (2.1 %), rpoB (1.7 %) and sodA (2.0 %) genes. DNA–DNA hybridization results confirmed those of sequencing, showing reassociation values between seal and salmon strains close to the borderline of species definition. Differences in growth at low temperatures and in the haemolytic capacities were also observed between both groups of isolates. On the basis of all these results, the salmon isolates represent a novel subspecies of S. phocae , for which the name Streptococcus phocae subsp. salmonis subsp. nov. is proposed. The type strain is C-4T ( = CECT 7921T = DSM 24768T). The subspecies Streptococcus phocae subsp. phocae subsp. nov. is automatically created. An emended description of S. phocae is also provided.

Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

Revised immunolocalization of the Na+-d-glucose cotransporter SGLT1 in rat organs with an improved antibody

Previously, we characterized localization of Na+-glucose cotransporter SGLT1 ( Slc5a1) in the rat kidney using a polyclonal antibody against the synthetic COOH-terminal peptide of the rat protein (Sabolić I, Škarica M, Gorboulev V, Ljubojević M, Balen D, Herak-Kramberger CM, Koepsell H. Am J Physiol Renal Physiol 290: 913–926, 2006). However, the antibody gave some false-positive reactions in immunochemical studies. Using a shortened peptide for immunization, we have presently generated an improved, more specific anti-rat SGLT1 antibody (rSGLT1-ab), which in immunochemical studies with isolated membranes and tissue cryosections from male (M) and female (F) rats exhibited 1) in kidneys and small intestine, labeling of a major protein band of ∼75 kDa; 2) in kidneys of adult animals, localization of rSGLT1 to the proximal tubule (PT) brush-border membrane (S1 < S2 < S3) and intracellular organelles (S1 > S2 > S3), with zonal (cortex < outer stripe) and sex differences (M < F) in the protein expression, which correlated well with the tissue expression of its mRNA in RT-PCR studies; 3) in kidneys of castrated adult M rats, upregulation of the protein expression; 4) in kidneys of prepubertal rats, weak and sex-independent labeling of the 75-kDa protein band and immunostaining intensity; 5) in small intestine, sex-independent regional differences in protein abundance (jejunum > duodenum = ileum); and 6) thus far unrecognized localization of the transporter in cortical thick ascending limbs of Henle and macula densa in kidney, bile ducts in liver, enteroendocrine cells and myenteric plexus in the small intestine, and initial ducts in the submandibular gland. Our improved rSGLT1-ab may be used to identify novel sites of SGLT1 localization and thus unravel additional physiological functions of this transporter in rat organs.

426. The role of sperm mitochondria in the process of epididymal maturation

On leaving the testis, spermatozoa can neither swim nor achieve fertilisation of the oocyte. These functional properties are acquired as spermatozoa engage a process of post-testicular maturation in the epididymis. Research into the biochemical basis of sperm maturation has revealed that this process is associated with activation of sperm mitochondria. Immature spermatozoa from the caput epididymis displayed a low mitochondrial membrane potential (MMP) whereas mature spermatozoa from the caudal epididymis actively maintained a high MMP. Moreover mitochondrial generation of reactive oxygen species could be triggered by antimycin in mature caudal epididymal spermatozoa but not in immature cells recovered from the caput epididymis. The molecular mechanisms responsible for regulating mitochondrial function were reversible since washing the cells free of epididymal fluid allowed immature spermatozoa to acquire a high MMP while incubating mature caudal cells in caput epididymal fluid, suppressed MMP. These results strongly suggested that fluid from the caput epididymis contains a mitochondrial inhibitor and that activation of mitochondrial activity is due to the removal or inactivation of this inhibitor during epididymal transit. The causative factor is not species specific because incubation of ejaculated human spermatozoa in murine epididymal fluid effectively suppressed their MMP. Caput epididymal fluid was fractionated using FPLC and each of the fractions was tested for their bioactivity. A major protein band with a molecular mass ~150 kDa was present in the active fraction. Further characterisation of this reversible mitochondrial inhibitor may reveal the mechanisms by which epididymal spermatozoa control mitochondrial function during maturation. It may also contribute to our understanding of human male infertility and potentially serve as a novel target for male fertility regulation.

A statherin and calcium enriched layer at the air interface of human parotid saliva

Parotid saliva placed in 35-mm-diameter tissue culture dishes developed increasing surface viscoelasticity at the interface with air. A surface layer became visible with time, and was collected and analysed by protein electrophoresis which indicated that a single protein (pI 4.2; molecular mass approx. 6 kDa) predominated. Western blot analysis demonstrated that the major protein band reacted with an antiserum directed against the C-terminal of the calcium-binding salivary protein statherin. Matrix-assisted laser-desorption ionization–time-of-flight MS analysis gave a molecular mass of 5380 Da for the protein, corresponding to the molecular mass of statherin. Staining of film protein in electrophoresis gels was compared with statherin synthesized on a solid phase, and the mean statherin content of film formed from 1 ml of parotid saliva was measured as 7 nmol. The mean calcium content of the surface layer was 250 nmol. Surface rheology was greatly decreased in the presence of EDTA, whereas surface tension of saliva was unaffected by calcium chelation, suggesting that protein accumulated at the surface was unaffected. The results suggest that a layer rich in statherin forms at the interface of saliva and air, and that the surface rheology developed is dependent upon protein interactions mediated by calcium. The surface layer may enhance the function of saliva as a protective layer on the mucosal surfaces and teeth.

β-1,3-Glucanase Activity in Peanut Seed (Arachis hypogaea) is Induced by Inoculation with Aspergillus flavus and Copurifies with a Conglutin-Like Protein

Infection of peanut (Arachis hypogaea) seed by Aspergillus flavus and A. parasiticus is a serious problem that can result in aflatoxin contamination in the seed. Breeding resistant cultivars would be an effective approach to reduce aflatoxin accumulation. The objective of this study was to investigate the expression of the pathogenesis-related (PR) protein β-1,3-glucanase and the isoform patterns in peanut seed inoculated with A. flavus. Peanut genotypes GT-YY9 and GT-YY20 (both resistant to A. flavus infection) and Georgia Green and A100 (both susceptible to A. flavus infection) were used in this study. The activities of β-1,3-glucanase were similar in the uninfected seed of all genotypes, but increased significantly in the resistant genotypes after inoculation in comparison with the susceptible genotypes. An in-gel (native polyacrylamide gel electrophoresis [PAGE]) enzymatic activity assay of β-1,3-glucanase revealed that there were more protein bands corresponding to β-1,3-glucanase isoforms in the infected seed of resistant genotypes than in the infected seed of susceptible genotypes. Both acidic and basic β-1,3-glucanase isoforms were detected in the isoelectric focusing gels. Thin-layer chromatography analysis of the hydrolytic products from the reaction mixtures of the substrate with the total protein extract or individual band of native PAGE revealed the presence of enzymatic hydrolytic oligomer products. The individual bands corresponding to the bands of β-1,3-glucanase isoforms Glu 1 to 5 were separated on the sodium dodecyl sulfate-PAGE, resulting in two bands of 10 and 13 kDa, respectively. The sequences of fragments of the 13-kDa major protein band showed a high degree of homology to conglutin, a storage protein in peanut seed. Conglutin is reported as a peanut allergen, Ara h2. Our data provide the first evidences for peanut having β-1,3-glucanase activities and the association with the resistance to A. flavus colonization in peanut seed. We have not directly demonstrated that conglutin has β-1,3-glucanase activity.

Secretory PAF-acetylhydrolase of the rat hepatobiliary system: characterization and partial purification

Hepatocytes and Kupffer cells in primary culture both secrete plasma-type platelet-activating factor-acetylhydrolase (pPAF-AH) into serum-free culture medium. The rate of secretion of pPAF-AH by Kupffer cells was 20 to 25 times higher than from hepatocytes, and Kupffer cells expressed a higher level of pPAF-AH mRNA than did hepatocytes. Purified liver cell-secreted pPAF-AH exhibited a major protein band of 65–67 kDa on SDS-PAGE; this was the band predominantly labeled when the enzyme catalytic center was reacted with [3H]diisopropylfluorophosphate ([3H]DFP). Rat bile collected from cannulated bile ducts contained significant PAF-AH activity, and bile samples possessed a prominent band at 30–32 kDa, which was the exclusive target for [3H]DFP. Experiments using tunicamycin, an inhibitor of N-linked glycosylation, and endoglycosidase H suggested that pPAF-AH secreted constitutively by cultured hepatocytes and Kupffer cells is glycosylated. The present study supports the notion that hepatic secretion of pPAF-AH into the blood contributes to the regulation of PAF and oxidized phospholipid levels in the circulation, whereas secretion of PAF-AH into the bile may allow hepatic control of these phospholipid signaling molecules in the gastrointestinal tract.

Survey and Characterization of Viruses in Sweetpotato from Zimbabwe

Thirty-one clones of sweetpotatoes collected from some parts of Zimbabwe were used as inoculum sources to mechanically inoculate 13 experimental hosts: Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Datura stramoniumitalic, Gomphrena globosa, Ipomoea purpurea, I. quamoclit, I. rubrocorulea, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. rustica, and N. tabacum. Systemic vein clearing was observed in N. benthamiana inoculated with buffered sap from nine clones. Purification of the vein clearing inducing agent from one of the sweetpotato clones gave yields ranging from 2 to 17 mg/kg and the A260nm/A280nm was around 1.2. Electron microscopy revealed flexuous filamentous particles with a modal length of 830 nm. Protein analysis of purified virus preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major protein band of 40 kDa, and this was assumed to be the viral coat protein. Minor protein bands of 27, 37, and 46 kDa were also observed. The viral protein degraded upon storage at 4°C over time to yield a protein band of 27 kDa. Polyclonal antiserum was produced against the purified virus. Protein A gold labeling of the purified virus incubated with available antisera; sweetpotato chlorotic stunt virus (SPCSV), sweetpotato feathery mottle virus strain russet crack (SPFMV-RC), sweetpotato feathery mottle virus, sweetpotato mild mottle virus, sweetpotato latent virus, sweetpotato chlorotic fleck virus, and sweetpotato caulimo-like virus resulted in a higher labeling density with the antiserum of SPFMV-RC than with the antiserum of SPCSV, while the other sera did not react. Further characterization of the vein clearing inducing agent was attempted by reverse transcription-polymerase chain reaction amplification of total RNA with degenerate primers for potyviruses and an oligo dT primer and PCR products of correct size were obtained. The nucleotide sequence was determined and the amino acid of the polyprotein deduced. Comparison with other strains of SPFMV showed strong similarity except for an insertion of 22 amino acids at the N-terminus of the coat protein. The coat protein size of 335 amino acids is the biggest SPFMV so far determined.