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Xylan is one of the major structural components of the plant cell wall. Xylan present in the human diet reaches the large intestine undigested and becomes a substrate to species of the gut microbiota. Here, we characterised the capacity of Limosilactobacillus reuteri and Blautia producta strains to utilise xylan derivatives. We showed that L. reuteri ATCC 53608 and B. producta ATCC 27340 produced β-D-xylosidases, enabling growth on xylooligosaccharide (XOS). The recombinant enzymes were highly active on artificial (p-nitrophenyl β-D-xylopyranoside) and natural (xylobiose, xylotriose, and xylotetraose) substrates, and showed transxylosylation activity and tolerance to xylose inhibition. The enzymes belong to glycoside hydrolase family 120 with Asp as nucleophile and Glu as proton donor, as shown by homology modelling and confirmed by site-directed mutagenesis. In silico analysis revealed that these enzymes were part of a gene cluster in L. reuteri but not in Blautia strains, and quantitative proteomics identified other enzymes and transporters involved in B. producta XOS utilisation. Based on these findings, we proposed a model for an XOS metabolism pathway in L. reuteri and B. producta strains. Together with phylogenetic analyses, the data also revealed the extended xylanolytic potential of the gut microbiota.

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The bacterial flora associated with Gracilaria dura, the sole producer of natural agarose was obtained. Screening for the presence of various enzymes and bioactive compounds resulted in discerning different clinically and industrially important enzymes. Three isolates were identified to be producing L-asparaginase (LA), a potent anticancer drug. The strain 4T1-C26E registered highest LA activity (25.2 IU ml− 1) in the enzyme assay, was selected for yield improvement by optimizing process parameters through Response Surface Methodology (RSM). Various parameters like glucose concentration, L-asparagine concentration, pH of the media, and incubation period were optimized through Central Composite Design (CCD) for enhanced yield of LA by 4T1-C26E, identified as Bacillus licheniformis. The CCD table was exploited for application in artificial neural networking (ANN) for the development of optimum method of cultivation. In this study, we obtained 2.5-fold (88.4 IU ml− 1) increase in LA production by this strain through RSM optimization. Further, Artificial Neural Networking Particle Swarm Optimization (ANN-PSO) predicted the input combinations. The experimental validation of LA production was found to be 4-fold (126.4 IU ml− 1) higher with respect to the unoptimized conditions. This illustrates the effectiveness of ANN-PSO based optimization technique, the error percent between the predicted and experimental output was 4.03%. The most important benefit of LA from 4T1-C26E is that it possesses no L-glutaminase activity. Further, it is extracellularly produced which is suitable for easier downstream processing thereby reducing chances of immunogenic responses by endotoxins. However, characterization of kinetic parameters and cytotoxic studies on cell cultures are mandatory to confirm its pharmaceutical application.

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