Properties and applications of α-galactosidase in agricultural waste processing and secondary agricultural process industries.

Agriculture products form the foundation building blocks of our daily lives. Although they have been claimed to be renewable resources with a low carbon footprint, the agricultural community is constantly challenged to overcome two post-harvest bottlenecks: first, farm bio-waste, a substantial economic and environmental burden to the farming sector, and second, an inefficient agricultural processing sector, plagued by the need for significant energy input to generate the products. Both these sectors require extensive processing technologies that are demanding in their energy requirements and expensive. To address these issues, an enzyme(s)-based green chemistry is available to break down complex structures into bio-degradable compounds that source alternate energy with valuable by-products and co-products. α-Galactosidase is a widespread class of glycoside hydroxylases that hydrolyzes α-galactosyl moieties in simple and complex oligo and polysaccharides, glycolipids, and glycoproteins. As a result of its growing importance, in this review we discuss the source of the enzyme, production and purification systems, and enzyme properties. We also elaborate on the enzyme's potential in agricultural bio-waste management, secondary agricultural industries like sugar refining, soymilk derivatives, food and confectionery, and animal feed processing. Insight into this vital enzyme will provide new avenues for less expensive green chemistry-based secondary agricultural processing and agricultural sustainability. © 2023 Society of Chemical Industry.

Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum.

Syphilis is a sexually transmitted disease caused by the spirochaete bacterium Treponema pallidum. This study has developed a multiplex High-Resolution Melt-curve Loop-mediated isothermal amplification (multiplex HRM-LAMP) assay targeting the marker genes polA and tprL to detect T. pallidum. The multiplex HRM-LAMP assay conditions were optimized at 65 °C for 45 min. Real-time melt-curve analysis of multiplex HRM-LAMP shows two melt-curve peaks corresponding to polA and tprL with a Tm value of 80 ± 0.5 °C and 87 ± 0.5 °C, respectively. The detection limit of multiplex HRM-LAMP was found to be 6.4 × 10-4 ng/µL (3.79 copies/µL) of T. pallidum. The specificity was evaluated using seven different bacterial species, and the developed method was 100% specific in detecting T. pallidum. A total of 64 blood samples of T. pallidum suspected cases were used to validate the assay method. The clinical validation showed that the assay was 96.43% sensitive and 100% specific in detecting syphilis. Thus, the developed method was more rapid and sensitive than other available methods and provides a multigene-based diagnostic approach to detect T. pallidum.

Delayed hydrolysis of Raffinose Family Oligosaccharides (RFO) affects critical germination of chickpeas.

Seed raffinose family oligosaccharides (RFOs) are converted into sucrose and galactose by α-galactosidase during germination. Seed osmopriming with a low concentration of potassium nitrate (KNO3) induces early and synchronized germination by activating hydrolases. Here, we report the effect of osmopriming on the germination indices of chickpea, its effects on α-galactosidase, and the fate of total RFOs. Chickpea seeds primed with 100 µM KNO3 show early and synchronized germination but with reduced vigour after 48 h after imbibition (HAI) due to excess sucrose accumulation. The KNO3 suppressed the activity of α-galactosidase during the imbibition stage that was later derepressed after 24 HAI, hence decreased the RFO levels accumulating high levels of sucrose after 48 HAI. The accumulated sucrose imposed a negative effect on the germination characteristics, particularly on seed vigour. Our results suggested that the sugar release and utilization were highly regulated and crucial during imbibition and germination; the enzyme α-galactosidase regulates sugar release from seed RFO reserve.

The Varied Proportion of Filifactor alocis in Periodontal Health and Disease in the South Indian Subpopulation.

BACKGROUND AND AIM: The periodontal microbiome being complex, this study was aimed to detect and quantify the prevalence of Filifactor alocis in various stages of periodontitis and to evaluate its prospect as a diagnostic marker for periodontal disease. SETTINGS AND DESIGN: Sixty subjects were selected (20 healthy controls, 20 with chronic periodontitis, and 20 with aggressive periodontitis) for the study. MATERIALS AND METHODS: Clinical parameters probing depth and the level of clinical attachment was recorded, subgingival plaque samples were collected. The F. alocis 16srDNA was cloned, sequenced, and used as the standard for real-time quantification of bacterial load using SYBR green chemistry. STATISTICAL ANALYSIS: Clinical, microbiological, and quantitative polymerase chain reaction (PCR) data were analyzed using ANOVA and Pearson's coefficient correlation. RESULTS: (a) Real-time PCR analysis showed the highest average F. alocis count in chronic periodontitis subjects (32,409.85), which was followed by count in healthy controls (3046.15) and the least count in aggressive periodontitis subjects (939.84). The bacterial count was statistically significant at P = 0.005. (b) An intra-group comparison reveals that there was a statistically significant increase in the bacterial count with age and mean probing pocket depth at P = 0.0005. CONCLUSION: F. alocis population in aggressive periodontitis was lower compared to chronic periodontitis and healthy controls. The F. alocis population surge in healthy controls may be due to geographical variations and the ethnicity of the subjects. A higher population of F. alocis in chronic periodontitis proves its high pathogenic potential to invade the host tissues to aid in further periodontal destruction.

Spatial regulation of alpha-galactosidase activity and its influence on raffinose family oligosaccharides during seed maturation and germination in Cicer arietinum.

Alpha-galactosides or Raffinose Family Oligosaccharides (RFOs) are enriched in legumes and are considered as anti-nutritional factors responsible for inducing flatulence. Due to a lack of alpha-galactosidases in the stomachs of humans and other monogastric animals, these RFOs are not metabolized and are passed to the intestines to be processed by gut bacteria leading to distressing flatulence. In plants, alpha(α)-galactosides are involved in desiccation tolerance during seed maturation and act as a source of stored energy utilized by germinating seeds. The hydrolytic enzyme alpha-galactosidase (α-GAL) can break down RFOs into sucrose and galactose releasing the monosaccharide α-galactose back into the system. Through characterization of RFOs, sucrose, reducing sugars, and α-GAL activity in maturing and germinating chickpeas, we show that stored RFOs are likely required to maintain a steady-state level of reducing sugars. These reducing sugars can then be readily converted to generate energy required for the high energy-demanding germination process. Our observations indicate that RFO levels are lowest in imbibed seeds and rapidly increase post-imbibition. Both RFOs and the α-GAL activity are possibly required to maintain a steady-state level of the reducing monosaccharide sugars, starting from dry seeds all the way through post-germination, to provide the energy for increased germination vigor.

Development of sensitive and specific multiplex PCR method for the detection of microcystin producing cyanobacteria in spirulina food supplements.

Spirulina has emerged as the next-generation dietary supplement owing to its health benefits. Despite the advantages, there have been reports of contamination by cyanotoxins such as microcystins that can adversely affect human health. Hence, there is a need to develop a robust, efficient, and cost-effective method to detect microcystin-producing cyanobacteria in these food supplements. In this study, we have demonstrated a multiplex polymerase chain reaction (PCR) method for identification of microcystin-contamination in spirulina dietary supplements. This method involves simultaneous amplification of phycocyanin and microcystin B encoding genes (pcb, mcyB). The sensitivity of the multiplex PCR was assessed, and the limit of detecting mcyB along with pcb was found to be 250 fg/µL. The presence of microcystin was detected in five out of seven fish food supplements indicating poor culture conditions. Hence, rigorous quality control is required for monitoring the spirulina food supplements.

Integration of amplification efficiency in qPCR analysis allows precise and relative quantification of transcript abundance of genes from large gene families using RNA isolated from difficult tissues.

The performance of the quantitative polymerase chain reaction (qPCR) assay in the analysis of gene expression belonging to multigene families in tissues rich in secondary metabolites is technically complicated. Here, we present the qPCR analysis of PMT2 gene, a predominant member of a multigene family from tobacco, expressed in the root tissues and is involved in the biosynthesis of nicotine. Consequently, we provide insight into the effect of polymerase chain reaction (PCR) amplification efficiency (AE) of reference and target genes of calibrator and test samples on the qPCR assay performance. Obviously, we found PCR AE as a critical indicator of qPCR assay performance involving multigene families and secondary metabolite-rich root tissues of tobacco. The integration of consistent and uniform PCR amplification efficiencies of reference and target genes of the samples into the relative quantification analysis is emphasized.

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1.

New Delhi metallo-ß-lactamase-1 gene (bla NDM-1 ) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of ß-lactam ring, and provides resistance against major classes of ß-lactam antibiotics. Dissemination of the plasmid borne bla NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla NDM-1 . Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6-25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla NDM-1 gene.

A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications.

A rapid, cost effective method of metagenomic DNA extraction from soil is a useful tool for environmental microbiology. The present work describes an improved method of DNA extraction namely "powdered glass method" from diverse soils. The method involves the use of sterile glass powder for cell lysis followed by addition of 1% powdered activated charcoal (PAC) as purifying agent to remove humic substances. The method yielded substantial DNA (5.87 ± 0.04 µg/g of soil) with high purity (A260/280: 1.76 ± 0.05) and reduced humic substances (A340: 0.047 ± 0.03). The quality of the extracted DNA was compared against five different methods based on 16S rDNA PCR amplification, BamHI digestion and validated using quantitative PCR. The digested DNA was used for a metagenomic library construction with the transformation efficiency of 4 X 106 CFU mL-1. Besides providing rapid, efficient and economical extraction of metgenomic DNA from diverse soils, this method's applicability is also demonstrated for cultivated organisms (Gram positive B. subtilis NRRL-B-201, Gram negative E. coli MTCC40, and a microalgae C. sorokiniana UTEX#1666).