Nutritional Profiles of Yoom Noon Rice from Royal Initiative of Southern Thailand: A Comparison of White Rice, Brown Rice, and Germinated Brown Rice.

For long-term food sustainability and security, it is crucial to recognize and preserve Indigenous rice varieties and their diversity. Yoom Noon is one of the non-glutinous rice (Oryza sativa L.) varieties being conserved as part of the Phanang Basin Area Development Project, which is administered by the Royal Initiative of Nakhon Si Thammarat in Southern Thailand. The goal of this research was to compare the nutritional profiles of Yoom Noon white rice, brown rice, and germinated brown rice. The results indicated that carbohydrate content was found to be the most plentiful macronutrient in all processed Yoom Noon rice types, accounting for 67.1 to 81.5% of the total. White rice had the highest carbohydrate content (p < 0.05), followed by brown rice and germinated brown rice. Brown rice had more protein and fat than white rice (p < 0.05). The maximum protein, dietary fiber, and ash content were found in germinated brown rice, followed by brown rice and white rice (p < 0.05). White rice had the highest amylose content, around 24% (p < 0.05), followed by brown rice (22%), and germinated brown rice (20%). Mg levels in all white, brown, and germinated brown rice ranged from 6.59 to 10.59 mg/100 g, which was shown to be the highest among the minerals studied (p < 0.05). Zn (4.10-6.18 mg/100 g) was the second most abundant mineral, followed by Fe (3.45-4.92 mg/100 g), K (2.61-3.81 mg/100 g), Mn (1.20-4.48 mg/100 g), Ca (1.14-1.66 mg/100 g), and Cu (0.16-0.23 mg/100 g). Se was not found in any processed Yoom Noon rice. Overall, brown rice had the highest content of macro- and micronutrients (p < 0.05). In all processed rice, thiamin was found in the highest amount (56-85 mg/100 g), followed by pyridoxine (18-44 g/100 g) and nicotinamide (4-45 g/100 g) (p < 0.05). Riboflavin was not identified in any of the three types of processed Yoom Noon rice. Individual vitamin concentrations varied among processed rice, with germinated brown rice having the highest thiamine content by around 1.5 and 1.3 folds compared to white and brown rice, respectively. The GABA level was the highest in germinated rice (585 mg/kg), which was around three times higher than in brown rice (p < 0.05), whereas GABA was not detectable in white rice. The greatest total extractable flavonoid level was found in brown rice (495 mg rutin equivalent (RE)/100 g), followed by germinated brown rice (232 mg RE/100 g), while white rice had no detectable total extractable flavonoid. Brown rice had the highest phytic acid level (11.2 mg/100 g), which was 1.2 times higher than germinated brown rice (p < 0.05). However, phytic acid was not detected in white rice. White rice (10.25 mg/100 g) and brown rice (10.04 mg/100 g) had the highest non-significant rapidly available glucose (RAG) values, while germinated brown rice had the lowest (5.33 mg/100 g). In contrast, germinated brown rice had the highest slowly available glucose (SAG) value (9.19 mg/100 g), followed by brown rice (3.58 mg/100 g) and white rice (1.61 mg/100 g) (p < 0.05).

Effects of Signal Peptide and Chaperone Co-Expression on Heterologous Protein Production in Escherichia coli.

Various host systems have been employed to increase the yield of recombinant proteins. However, some recombinant proteins were successfully produced at high yields but with no functional activities. To achieve both high protein yield and high activities, molecular biological strategies have been continuously developed. This work describes the effect of signal peptide (SP) and co-expression of molecular chaperones on the production of active recombinant protein in Escherichia coli. Extracellular enzymes from Bacillus subtilis, including ß-1,4-xylanase, ß-1,4-glucanase, and ß-mannanase constructed with and without their signal peptides and intracellular enzymes from Pseudomonas stutzeri ST201, including benzoylformate decarboxylase (BFDC), benzaldehyde dehydrogenase (BADH), and d-phenylglycine aminotransferase (d-PhgAT) were cloned and overexpressed in E. coli BL21(DE3). Co-expression of molecular chaperones with all enzymes studied was also investigated. Yields of ß-1,4-xylanase (Xyn), ß-1,4-glucanase (Cel), and ß-mannanase (Man), when constructed without their N-terminal signal peptides, increased 1112.61-, 1.75-, and 1.12-fold, respectively, compared to those of spXyn, spCel, and spMan, when constructed with their signal peptides. For the natural intracellular enzymes, the chaperones, GroEL-GroES complex, increased yields of active BFDC, BADH, and d-PhgAT, up to 1.31-, 4.94- and 37.93-fold, respectively, and also increased yields of Man and Xyn up to 1.53- and 3.46-fold, respectively, while other chaperones including DnaK-DnaJ-GrpE and Trigger factor (Tf) showed variable effects with these enzymes. This study successfully cloned and overexpressed extracellular and intracellular enzymes in E. coli BL21(DE3). When the signal peptide regions of the secretory enzymes were removed, yields of active enzymes were higher than those with intact signal peptides. In addition, a higher yield of active enzymes was obtained, in general, when these enzymes were co-expressed with appropriate chaperones. Therefore, E. coli can produce cytoplasmic and secretory enzymes effectively if only the enzyme coding sequence without its signal peptide is used and appropriate chaperones are co-expressed to assist in correct folding.

Comparative Analysis of Antioxidant Compounds and Antioxidative Properties of Thai Indigenous Rice: Effects of Rice Variety and Processing Condition.

Indigenous southern Thai non-glutinous rice varieties Kaab Dum, Khai Mod Rin, Yar Ko, Yoom Noon, and Look Lai made under four different processing conditions, white rice, brown rice, germinated brown rice, and rice grass, were assessed for antioxidant components and in vitro antioxidative activities. According to the findings, rice's antioxidant components and antioxidant activity were considerably impacted by both variety and processing. High levels of total extractable phenolic compounds (164−314 mg gallic acid equivalent (GAE)/kg, dry weight (dw)) and carotenoid (0.92−8.65 mg/100 g, dw) were found in all rice varieties, especially in rice grass and germinated brown rice, indicating that milling to generate white rice had an adverse effect on those components. Additionally, after germination, a higher γ-oryzanol concentration (9−14 mg/100 g, dw) was found. All rice varieties had higher ascorbic acid, phenolic compound, and carotenoid contents after sprouting. Overall, Yoom Noon rice grass had the highest total extractable phenolic content (p < 0.05). The rice grass from Yoom Noon/Look Lai/Kaab Dum had the highest ascorbic acid content (p < 0.05). The total carotenoid concentration of Look Lai rice grass was the highest, and Yoom Noon's germinated brown rice had the highest γ-oryzanol content (p < 0.05). All rice varieties' aqueous extracts had remarkable ABTS free radical scavenging activity, with Khai Mod Rin reaching the highest maximum value of 42.56 mmol Trolox equivalent/kg dw. Other antioxidant mechanisms, however, were quite low. Compared to germinated brown rice, brown rice, and white rice, rice grass often tended to have stronger antioxidant activity. Yar Ko rice grass was found to have the highest DPPH free radical scavenging activity (3.8 mmol Trolox equivalent/kg dw) and ferric reducing antioxidant power (FRAP) (4.6 mmol Trolox equivalent/kg dw) (p < 0.05). Khai Mod Rice grass had the most pronounced metal chelation activity (1.14 mmol EDTA equivalent/kg dw) (p < 0.05). The rice variety and processing conditions, therefore, influenced the antioxidant compounds and antioxidative properties of Thai indigenous rice. The results can be used as a guide to select the optimal rice variety and primary processing in order to satisfy the needs of farmers who want to produce rice as a functional ingredient and to promote the consumption of indigenous rice by health-conscious consumers.

Insights into the effects of dietary supplements on the nutritional composition and growth performance of sago palm weevil (Rhynchophorus ferrugineus) larvae.

Nutritional composition and growth performance of sago palm weevil larvae (SPWL) fed with ground sago palm trunk mixed with different supplements including commercial pig feed, rice bran, cornmeal, soybean meal, and perilla seed were evaluated. SPWL fed with supplemented diets were richer in protein, lipid, and mineral contents (p < 0.05). Marked increases in polyunsaturated fatty acids (10.75-fold) and omega-3 fatty acids (25.42-fold) with the lowest n-6:n-3 ratio, atherogenicity index, and thrombogenicity index were found in SPWL fed with perilla seed (p < 0.05). Perilla seed, cornmeal, and soybean meal improved essential amino acid content and essential amino acid index of SPWL. Growth performance varied, depending on feed compositions, where a comparable or even greater effect was observed in SPWL fed with supplemented diets compared to control. Therefore, plant-based supplements, especially perilla seed, efficiently improved nutritional quality of SPWL, making them more attractive in terms of nutritional and economical value.

Alternative Sigma Factor B in Bovine Mastitis-Causing Staphylococcus aureus: Characterization of Its Role in Biofilm Formation, Resistance to Hydrogen Peroxide Stress, Regulon Members.

This study examines treatments of the bacterial pathogen Staphylococcus aureus, namely, in the context of its being a major cause of subclinical bovine mastitis. Such infections caused by S. aureus among dairy cows are difficult to detect and can easily become chronic, leading to reduced productivity and large losses for dairy manufacturers. In this study, the role of alternative sigma factor B (σB), which has been shown to be a global regulator for S. aureus infections, was explored in a mastitis-causing S. aureus strain, RF122. For comparison with the wild-type strain, a sigB null (ΔsigB) mutant was constructed and analyzed for its phenotypes and transcriptome. Our study found that σB is essential for biofilm formation as the ΔsigB mutant strain produced significantly less biofilm than did the wild-type strain at 48 h. σB is involved in response to H2O2 stress. However, σB plays a minor or no role in resistance to antiseptics (e.g., povidone-iodine and chlorhexidine), resistance to tested antibiotics, hemolysin activity, and invasion ability. RNA sequencing identified 225 σB-dependent genes, of which 171 are positively regulated and 54 are negatively regulated. The identified genes are involved in stress response, pathogenesis, and metabolic mechanisms. Quantitative TaqMan RT-PCR was performed to verify the RNA sequencing results; i.e., σB is a positive regulator for asp23, sarA, katA, yabJ, sodA, SAB2006c, and nrdD expressions. In the RF122 strain, σB plays a role in biofilm formation, general stress response (e.g., H2O2), and regulation of virulence factors and virulence-associated genes.

A kinetic spectrophotometric method for the determination of pyridoxal-5'-phosphate based on coenzyme activation of apo-d-phenylglycine aminotransferase.

A new PLP assay method based on the coenzyme activation of apo-d-phenylglycine aminotransferase (apo-d-PhgAT) is reported. The assay process is comprised of two steps. First, PLP present in plasma samples is allowed to reconstitute apo-d-PhgAT, forming active holo-d-PhgAT. In the second step, the enzymatic activity of reconstituted d-PhgAT is determined using d-4-OH-phenylglycine as the amino donor substrate with 4-OH-benzoylformate (OH-BZF) as the reaction product. OH-BZF absorbs UV light strongly at 334 nm (molar absorption coefficient = 25.4 × 103 M-1cm-1) and its rate of formation is monitored spectrophotometrically. The rate of the transamination reaction catalyzed by the reconstituted d-PhgAT is directly proportional to the amount of PLP in the sample. The method is applicable for determining PLP in the concentration range from 5.2 to 250 nM and requires 50 µL of plasma sample. The mean within- and between-run coefficient of variations (CVs) were 8.1% and 12.4%, respectively. Analytical recoveries ranged from 98 to 108%. The assay was specific and showed good correlation with the established method (CDC, Method No: 4002.05). The assay requires one reaction catalyzed by a single enzyme, does not require a radioactive substrate, and a derivatization reagent is not needed. This PLP determination process is relatively simple to perform and can be completed using common laboratory equipment.

Improvement of extracellular bacterial protein production in Pichia pastoris by co-expression of endoplasmic reticulum residing GroEL-GroES.

Pichia pastoris is an established host system for heterologous protein expression. However, the potential productivity of this system can be limited. In this study, the Escherichia coli chaperones (GroES-GroEL) were expressed from the PGAP promoter and targeted to the secretory pathway through the endoplasmic reticulum (ER). The ability of the ER targeted chaperones to improve production of bacterial protein in P. pastoris was evaluated. The chaperones tagged with α-factor secretion- and ER retention-signal sequences were co-expressed with either extracellularly secreted phytase or intracellular d-phenylglycine aminotransferase (D-PhgAT) enzymes. The ER residing GroEL-GroES successfully increased the levels of active phytase extracellularly, 1.5-2.3-fold higher than the phytase expression alone, but did not enhance the formation of active, intracellular D-PhgAT. These results indicate that the chaperones have the potential to enhance production of active enzymes when present in the same trafficking pathway. This is the first report on the improvement of extracellular bacterial protein production through co-expression with ER residing bacterial chaperones in the Pichia system. The modified P. pastoris expression system may be beneficial for extracellular expression of other prokaryotic proteins.

Functional characterization of two members of histidine phosphatase superfamily in Mycobacterium tuberculosis.

BACKGROUND: Functional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is 'RHA' unlike 'RHG' found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal. RESULTS: Characterization of the purified recombinant proteins revealed that Rv0489 possesses phosphoglycerate mutase activity while Rv2135c does not. However Rv2135c has an acid phosphatase activity with optimal pH of 5.8. Kinetic parameters of Rv2135c and Rv0489 are studied, confirming that Rv0489 is a cofactor dependent phosphoglycerate mutase of M. tuberculosis. Additional characterization showed that Rv2135c exists as a tetramer while Rv0489 as a dimer in solution. CONCLUSION: Most of the proteins orthologous to Rv2135c in other bacteria are annotated as phosphoglycerate mutases or hypothetical proteins. It is possible that they are actually phosphatases. Experimental characterization of a sufficiently large number of bacterial histidine phosphatases will increase the accuracy of the automatic annotation systems towards a better understanding of this important group of enzymes.

Comparative analysis of the responses of related pathogenic and environmental bacteria to oxidative stress.

Bacillus anthracis, the causative agent of anthrax, is exposed to host-mediated antibacterial activities, such as reactive oxygen species (ROS), during the early stages of its disease process. The ability to resist these host-mediated stresses is an essential characteristic of a successful pathogen while it is generally assumed that non-pathogenic environmental bacteria succumb to these antimicrobial activities. In order to gain insights into the underlying mechanisms that pathogens use to resist host-mediated oxidative stress, we have compared the oxidative stress responses of B. anthracis and Bacillus subtilis, a well-studied environmental bacterium. Among the four putative catalases encoded by B. anthracis we identified KatB as the main vegetative catalase. Comparative analysis of catalase production in B. anthracis and B. subtilis in response to superoxide and peroxide stress reveals different expression profiles, even though both are regulated by the PerR repressor, which senses and responds to peroxide stress. A B. anthracis perR deletion mutant exhibits enhanced KatB activity and is hyper-resistant to peroxide stress. Superoxide dismutase A1 (SodA1) is the main contributor to the intracellular superoxide dismutase activity in vegetative cells and the gene encoding this enzyme is constitutively expressed. Although aspects of the ROS detoxifying systems of B. anthracis and B. subtilis are similar, their responses to superoxide stress are different. The observed differences are likely to reflect adaptations to specific environmental niches.

A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris.

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K (m) and V (max) for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe(2+), Fe(3+), and Al(3+). When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).

Characterization, gene cloning, and heterologous expression of ß-mannanase from a thermophilic Bacillus subtilis.

Bacillus subtilis BCC41051 producing a thermostable ß-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular ß-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pi value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60°C, respectively. The enzyme was stable over a pH range of 5.0-11.5, and at temperatures of up to 60°C for 30 min, with more than 80% of its activity retained. ManA was strongly inhibited by Hg(2+) (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).

Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme.

A sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in (2)H(2)O with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'-(2)H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The (2)H at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the (2)H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of (2)H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.