Geobacillus thermoleovorans MTCC 13131: An Amide-Hydrolyzing Thermophilic Bacterium Isolated from a Hot Spring of Manikaran.

Geobacillus thermoleovorans MTCC 13131, an amide hydrolyzing bacteria was isolated from a hot spring in Himachal Pradesh and identified through 16S rRNA gene sequence analysis. The amidase derived from this bacterium exhibited hydrolyzing catalytic ability against aliphatic and aromatic amides. The isolate was characterized for morphological and biochemical properties. Further, the production of amidase enzyme from this isolate was evaluated using approach of one-variable-at-a-time and response surface method. The Response Surface Methodology based study indicated the importance of nitrogen sources and growth period for amidase production. Optimal production was achieved at a temperature 55 °C, and production pH 7.5 in the production medium comprising diammonium hydrogen phosphate (0.4%), peptone (0.45%) and yeast extract (0.3%). The wide substrate affinity of the strain suggests its potential role in biotransformation of amides to corresponding acids of industrial significance along with its strong capacity to degrade the toxic amide in polluted environmental samples. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01042-9.

Gut-microbiota derived bioactive metabolites and their functions in host physiology.

Every individual harbours a complex, diverse and mutualistic microbial flora in their intestine and over the time it became an integral part of the body, affecting a plethora of activities of the host. Interaction between host and gut-microbiota affects several aspects of host physiology. Gut-microbiota affects host metabolism by fermenting unabsorbed/undigested carbohydrates in the large intestine. Not only the metabolic functions, any disturbances in the composition of the gut-microbiota during first 2-3 years of life may impact on the brain development and later affects cognition and behaviour. Thus, gut-dysbiosis causes certain serious pathological conditions in the host including metabolic disorders, inflammatory bowel disease and mood alterations, etc. Microbial-metabolites in recent times have emerged as key mediators and are responsible for microbiota induced beneficial effects on host. This review provides an overview of the mechanism of microbial-metabolite production, their respective physiological functions and the impact of gut-microbiome in health and diseases. Metabolites from dietary fibres, aromatic amino acids such as tryptophan, primary bile acids and others are the potential substances and link microbiota to host physiology. Many of these metabolites act as signalling molecules to a number of cells types and also help in the secretion of hormones. Moreover, interaction of microbiota derived metabolites with their host, immunity boosting mechanisms, protection against pathogens and modulation of metabolism is also highlighted here. Understanding all these functional attributes of metabolites produced from gut-microbiota may lead to the opening of a new avenue for preventing and developing potent therapies against several diseases.

Microbial metabolites in nutrition, healthcare and agriculture.

Microorganisms are a promising source of an enormous number of natural products, which have made significant contribution to almost each sphere of human, plant and veterinary life. Natural compounds obtained from microorganisms have proved their value in nutrition, agriculture and healthcare. Primary metabolites, such as amino acids, enzymes, vitamins, organic acids and alcohol are used as nutritional supplements as well as in the production of industrial commodities through biotransformation. Whereas, secondary metabolites are organic compounds that are largely obtained by extraction from plants or tissues. They are primarily used in the biopharmaceutical industry due to their capability to reduce infectious diseases in human beings and animals and thus increase the life expectancy. Additionally, microorganisms and their products inevitably play a significant role in sustainable agriculture development.

Bio-statistical enhancement of acyl transfer activity of amidase for biotransformation of N-substituted aromatic amides.

Acyl transfer activity (ATA) of amidase transfers an acyl group of different amides to hydroxylamine to form the corresponding hydroxamic acid. Bacterial isolate BR-1 was isolated from cyanogenic plant Cirsium vulgare rhizosphere and identified as Pseudomonas putida BR-1 by 16S rDNA sequencing. This organism exhibited high ATA for the biotransformation of N-substituted aromatic amide to the corresponding hydroxamic acid. Optimization of media, tryptone (0.6%), inducer, pH 8.5, and a growth temperature 25°C for 56 h, resulted in a 7-fold increase in ATA. Further, Response Surface Methodology (RSM) and multiple feeding approach (20 mM after 14 h) of inducer led to a 29% enhancement of ATA from this organism. The half life (t1/2) of this enzyme at 50°C and 60°C was 3 h and 1 h, respectively. The ATA of amidase of Pseudomonas putida BR-1 makes it a potential candidate for the production of a variety of N-substituted aromatic hydroxamic acid.

Enhanced production of thermostable amidase from Geobacillus subterraneus RL-2a MTCC 11502 via optimization of physicochemical parameters using Taguchi DOE methodology.

The specific effect of chemical and physical factors on amidase production from Geobacillus subterraneus RL-2a was investigated using design of experiments (DOE) methodology. The one-factor-at-a-time (OFAT) method was used to study the effects of carbon and nitrogen sources on amidase production. Subsequently, optimal levels of physical parameters and key media components, namely temperature, pH, sucrose, K2HPO4, NaCl, yeast, CaCl2·2H2O and MgSO4·7H2O, were determined using the Taguchi orthogonal array (OA) experimental design (DOE) methodology. Taguchi method based on three levels with a OA layout of L18 (21 × 37) with eight most influential factors on amidase synthesis for the proposed experimental design. Analysis of variance was performed on the obtained results and optimum condition suggested by statistical calculations was tested in a verification test. An increase of 169.56 % in amidase production compared to the unoptimized conditions was observed and the conversion of isonicotinamide was significantly improved after performing optimization techniques, including OFAT and Taguchi method. The result indicated that Taguchi method was effective in optimizing the culture conditions of amidase production.

Microbial enzymes: industrial progress in 21st century.

Biocatalytic potential of microorganisms have been employed for centuries to produce bread, wine, vinegar and other common products without understanding the biochemical basis of their ingredients. Microbial enzymes have gained interest for their widespread uses in industries and medicine owing to their stability, catalytic activity, and ease of production and optimization than plant and animal enzymes. The use of enzymes in various industries (e.g., food, agriculture, chemicals, and pharmaceuticals) is increasing rapidly due to reduced processing time, low energy input, cost effectiveness, nontoxic and eco-friendly characteristics. Microbial enzymes are capable of degrading toxic chemical compounds of industrial and domestic wastes (phenolic compounds, nitriles, amines etc.) either via degradation or conversion. Here in this review, we highlight and discuss current technical and scientific involvement of microorganisms in enzyme production and their present status in worldwide enzyme market.

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a.

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg(-1). The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5-11.5) and temperature (40-90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co(2+), Hg(2+), Cu(2+), Ni(2+), and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K m and V max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 µmol min(-1) mg(-1) protein by analyzing Michaelis-Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.

Bench scale production of benzohydroxamic acid using acyl transfer activity of amidase from Alcaligenes sp. MTCC 10674.

The acyl transfer activity of the amidase of Alcaligenes sp. MTCC 10674 has been applied to the conversion of benzamide and hydroxylamine to benzohydroxamic acid. The unique features of the acyl transfer activity of this organism include its optimal activity at 50 °C and very high substrate (100 mM benzamide) and product (90 mM benzohydroxamic acid) tolerance among the hitherto reported enzymes. The bench scale production of benzohydroxamic acid was carried out in a fed-batch reaction (final volume 1 l) by adding 50 mM benzamide and 250 mM of hydroxylamine after every 20 min for 80 min in 0.1 M potassium phosphate buffer (pH 7.0) at 50 °C, using resting cells equal to 4.0 mg dcm/ml of reaction mixture. From 1 l of reaction mixture 33 g of benzohydroxamic acid was recovered with 24.6 g l(-1) h(-1) productivity. The acyl transfer activity of the amidase of Alcaligenes sp. MTCC 10674 and the process developed in the present study are of industrial significance for the enzyme-mediated production of benzohydroxamic acid.