Identification of bioactive peptide from Oreochromis niloticus skin gelatin.

Fish skin, one type of wastes generated from Nile tilapia processing, is still a good source of collagen and gelatin. Bioactive peptides can be obtained from Nile tilapia skin gelatin by trypsin digestion. Trypsin hydrolysate was subsequently purified by gel filtration chromatography. Trypsin A fraction showed the greatest reducing power (5.138 ± 1.060 µM trolox/mg peptide) among all hydrolysate fractions, while trypsin B fraction from gel filtration column was found to exhibit the best radical scavenging and angiotensin-I-converting enzyme (ACE) inhibitory activities 8.16 ± 2.18 µg trolox/mg peptide and 59.32 ± 9.97 % inhibition, respectively. The most active fraction was subjected to MALDI-TOF/TOF MS/MS. After annotation by Mascot sequence matching software (Matrix Science) with Ludwig NR Database, two peptide sequences were identified; GPEGPAGAR (MW 810.87 Da) and GETGPAGPAGAAGPAGPR (MW 1490.61 Da). The docking analysis suggested that the shape of the shorter peptide may be slightly more proper, to fit into the binding cleft of the ACE. However, the binding affinities calculated from the docking showed no significant difference between the two peptides. In good agreement with the in silico data, results from the in vitro ACE inhibitory activity with synthetic peptides also showed no significant difference. Both peptides are thus interesting novel candidates suitable for further development as ACE inhibitory and antioxidant agents from the natural source.

Antioxidant and antihypertensive activity of gelatin hydrolysate from Nile tilapia skin.

Fish skin, a by-product from fish processing industries, still contains a significant amount of protein-rich material. Gelatin was extracted from Nile tilapia skin with the yield 20.77 ± 0.80 % wet weight. Gelatin was then separately hydrolyzed by proteases, including bromelain, papain, trypsin, flavourzyme, alcalase and neutrase. Low molecular weight gelatin hydrolysate (<10 kDa) has a great potential as an antioxidant agent. Flavourzyme hydrolysate has potent activity on ABTS radical scavenging (1,413.61 ± 88.74 µg trolox/mg protein) and also inhibits the oxidation of linoleic acid at a high level (59.74 ± 16.57 % inhibition). The greatest reducing power is in alcalase hydrolysate (4.951 ± 1.577 mM trolox/mg protein). While, bromelain hydrolysate has the highest ferrous ion chelating activity (86.895 ± 0.061 %). Evaluation of the angiotensin-I-converting enzyme's inhibitory activity indicates that all hydrolysates have great potency as an antihypertensive agent. All studied tilapia skin gelatin hydrolysates contain potent antioxidant and anti-hypertensive effects.

Proteomic Analysis of Isogenic Rice Reveals Proteins Correlated with Aroma Compound Biosynthesis at Different Developmental Stages.

Fragrant rice has a potent flavor compound, 2-acetyl-1-pyrroline (2AP). A better understanding of the 2AP biosynthetic pathway is gained by proteomic analysis of two isogenic lines of Thai jasmine rice, Oryza sativa L. cv. Khao Dawk Mali 105, which differ only in the aromatic gene Os2AP. The protein profiles of two lines, from six growth stages, seedling to grain filling, had 41 identifiable protein spots. Four of these spots were betaine aldehyde dehydrogenase, a key enzyme responsible for 2AP production. This enzyme occurred in every growth stage of the non-aromatic rice line except smaller amount detected in the hard grain-filling stage of the aromatic line. Glyceraldehyde 3-phosphate dehydrogenase and aspartate aminotransferase, observed in the aromatic line, may involve in the metabolism of precursors for 2AP biosynthesis. In addition, glutamine synthetase and 1-cys peroxiredoxin A which function in ammonia reassimilation and hydrogen peroxide detoxification were unique in the aromatic line. However, proteins that correspond to photosynthesis and the nutrient reservoir were only detected in lower abundances. This possibly explains why the aroma rice grain weight is low. Our study proposed the possible role of these remarkable proteins which involved in 2AP biosynthesis in jasmine rice.

Transcriptional response elements in the promoter of CYP6B1, an insect P450 gene regulated by plant chemicals.

Papilio polyxenes, a lepidopteran continually exposed to toxic furanocoumarins in its hostplants, owes its tolerance to these compounds to the transcriptional induction of the CYP6B1 gene encoding a P450 capable of metabolizing linear furanocoumarins, such as xanthotoxin, at high rates. Transient expression of various lengths of wild-type and mutant CYP6B1v3 promoter in lepidopteran Sf9 cells defines a positive element (XRE-xan) from -136 to -119 required for both basal and xanthotoxin-inducible transcription and a negative element from -228 to -146 that represses basal transcription. Fusion of the CYP6B1v3 XRE-xan element to the Drosophila melanogaster Eip28/29 core promoter indicates that the XRE-xan functions in conjunction with its own core promoter but not with a heterologous core promoter. Sequence searches of the CYP6B1v3 proximal promoter region revealed a number of putative elements (XRE-AhR, ARE, OCT-1, EcRE, C/EBP, Inr) sharing sequence similarity with those in other regulated vertebrate and insect promoters. Mutation of TGAC nucleotides shared by the overlapping EcRE/ARE/XRE-xan indicates that this sequence is essential for basal and regulated transcription of this gene. Mutagenesis in the non-overlapping region of the EcRE indicates it modulates basal transcription. These findings are incorporated into a working model for regulation of this toxin-inducible promoter.

Evaluation of sample preparation methods from rice seeds and seedlings suitable for two-dimensional gel electrophoresis.

In a proteomic study, sample preparation is very important because it affects the quality of protein profiles on two-dimensional gel electrophoresis (2-DE). This study investigated the suitability of four protein extraction methods-direct lysis buffer extraction, trichloroacetic acid (TCA)/acetone precipitation, phenol extraction, and polyethylene glycol (PEG) fractionation-from rice seeds and seedlings (Oryza sativa L. ssp. indica cv. Khao Dawk Mali 105). The effectiveness of these methods was evaluated by the protein quantity and the 2-DE profiling quality. This included the number of protein spots, the consistency and uniqueness of protein spots, and their distribution in different ranges of pI and molecular weight (M r ). For protein quantity, the phenol and direct lysis extraction methods gave the highest protein yield in rice seeds and rice seedlings, respectively. However, in terms of the quality of 2-DE profiles, samples prepared by the TCA/acetone and phenol methods exhibited higher protein resolution and more spots than the protein profile derived from direct lysis extract. In addition, TCA/acetone method had the efficiency for high M r protein detection. PEG fractionation provided the best 2-DE pattern in terms of resolution, number of spots, minimal streaking, and reproducibility. Moreover, PEG fractionation was better for determining low M r basic proteins.

Improvement of myrosinase activity of Aspergillus sp. NR4617 by chemical mutagenesis

A myrosinase (thioglucoside glucohydrolase or thioglucosidase, EC 3.2.3.147) producing fungus, Aspergillus sp. NR4617, was newly isolated from decayed soil sample obtained in Thailand and was subjected to single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Its myrosinase production was selected on low cost medium prepared from mustard seed cake (Brassica juncea). Studies of production and stability of the enzyme showed that EMS mutagenesis increased myrosinase activity. Aspergillus sp. NR4617E1 produced myrosinase 1.90 U ml-1 at 36 hrs of the cultivation equivalent to 171 percent of the enzyme production in wild-type. The stability studies revealed that myrosinase from the mutant strains retained activity similar to wild-type at 30ºC. Aspergillus sp. NR4617E1 degraded 10 mM of glucosinolate completely in 36 hrs. Enhanced myrosinase production and high yields of products (allylisothiocyanate) demonstrated that this mutant could be a new found candidate for feed detoxification and industrial allylisothiocyanate production.

Screening of filamentous fungi for production of myrosinase

A linhagem Aspergillus sp. NR46FB, isolada de solo através da técnica do ágar sulfato de bário-sinigrina, foi testada quanto à producão de mirosinase. O fungo degradou completamente o glicosinolato e produziu 3,19 U.mL-1 de mirosinase, após 48 h de cultivo. Devido à alta producão de mirosinase, esse novo isolado é um potente candidato para aplicacões industriais.