Glycoside Hydrolase Enzymes Research Articles

Species-level verification of Phascolarctobacterium association with colorectal cancer.

We have previously demonstrated an association between increased abundance of Phascolarctobacterium and colorectal cancer (CRC) and adenomas in two independent Norwegian cohorts. Here we seek to verify our previous findings using new cohorts and methods. In addition, we characterize lifestyle and sex specificity, the functional potential of the Phascolarctobacterium species, and their interaction with other microbial species. We analyze Phascolarctobacterium with 16S rRNA sequencing, shotgun metagenome sequencing, and species-specific qPCR, using 2350 samples from three Norwegian cohorts-CRCAhus, NORCCAP, and CRCbiome-and a large publicly available data set, curatedMetagenomicData. Using metagenome-assembled genomes from the CRCbiome study, we explore the genomic characteristics and functional potential of the Phascolarctobacterium pangenome. Three species of Phascolarctobacterium associated with adenoma/CRC were consistently detected by qPCR and sequencing. Positive associations with adenomas/CRC were verified for Phascolarctobacterium succinatutens and negative associations were shown for Phascolarctobacterium faecium and adenoma in curatedMetagenomicData. Men show a higher prevalence of P. succinatutens across cohorts. Co-occurrence among Phascolarctobacterium species was low (<6%). Each of the three species shows distinct microbial composition and forms distinct correlation networks with other bacterial taxa, although Dialister invisus was negatively correlated to all investigated Phascolarctobacterium species. Pangenome analyses showed P. succinatutens to be enriched for genes related to porphyrin metabolism and degradation of complex carbohydrates, whereas glycoside hydrolase enzyme 3 was specific to P. faecium.IMPORTANCEUntil now Phascolarctobacterium has been going under the radar as a CRC-associated genus despite having been noted, but overseen, as such for over a decade. We found not just one, but two species of Phascolarctobacterium to be associated with CRC-Phascolarctobacterium succinatutens was more abundant in adenoma/CRC, while Phascolarctobacterium faecium was less abundant in adenoma. Each of them represents distinct communities, constituted by specific microbial partners and metabolic capacities-and they rarely occur together in the same patients. We have verified that P. succinatutens is increased in adenoma and CRC and this species should be recognized among the most important CRC-associated bacteria.

Draft genome sequence of Vibrio sp. PBL-C16, isolated from Batu Laut Beach in Selangor, Malaysia.

Vibrio sp. PBL-C16 is a bacterium that was isolated from Batu Laut Beach in Selangor, Malaysia. Here, we present a high-quality annotated draft genome of strain PBL-C16 and suggest its potential glycoside hydrolase enzymes for polysaccharide degradation.

Simple Genomic Traits Predict Rates of Polysaccharide Biodegradation.

Natural and chemically modified polysaccharides are extensively employed across a wide array of industries, leading to their prevalence in the waste streams of industrialized societies. With projected increasing demand, a pressing challenge is to swiftly assess and predict their biodegradability to inform the development of new sustainable materials. In this study, we developed a scalable method to evaluate polysaccharide breakdown by measuring microbial growth and analyzing microbial genomes. Our approach, applied to polysaccharides with various structures, correlates strongly with well-established regulatory methods based on oxygen demand. We show that modifications to the polysaccharide structure decreased degradability and favored the growth of microbes adapted to break down chemically modified sugars. More broadly, we discovered two main types of microbial communities associated with different polysaccharide structures─one dominated by fast-growing microbes and another by specialized degraders. Surprisingly, we were able to predict biodegradation rates based only on two genomic features that define these communities: the abundance of genes related to rRNA (indicating fast growth) and the abundance of glycoside hydrolases (enzymes that break down polysaccharides), which together predict nearly 70% of the variation in polysaccharide breakdown. This suggests a trade-off, whereby microbes are either adapted for fast growth or for degrading complex polysaccharide chains, but not both. Finally, we observe that viral elements (prophages) encoded in the genomes of degrading microbes are induced in easily degradable polysaccharides, leading to complex dynamics in biomass accumulation during degradation. In summary, our work provides a practical approach for efficiently assessing polymer degradability and offers genomic insights into how microbes break down polysaccharides.

Unravel the Supremacy of Klebsiella variicola over Native Microbial Strains for Aroma-Enhancing Compound Production in Reconstituted Tobacco Concentrate through Metagenomic Analysis.

Sensory attributes strongly influence consumers' preferences for products. The inoculation of the Klebsiella variicola H8 strain in a reconstituted tobacco leaf concentrate (RTLC) solution increased neutral aroma-enhancing compound (NAEC) production by 45%, decreased the nicotine level by 25%, decreased the water-soluble total sugar content by ~36%, and improved the sensory quality by 5.71%. The production of NAECs such as dihydrokiwi lactone (DHKL: 192.86%), 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene (THTMN: 177.77%), 2,4-di-tert-butylphenol (DTBP: 25%), 4-oxoisofolkone (OIFK: 116.66%,) 1,9-heptadecadiene-4,6-diyn-3-ol (HDD: 116.67%), β-damastrone (BDS: 116.67), and megastigmatrienone A (MSTA: 116.67%) was increased. A metagenomics analysis of the microbial community in the fermented RTLC (FRTLC) was performed to elucidate the mechanism by which NAECs were produced. As a result, 24 groups of functional genes were identified, and among them, five families of carbohydrate-active enzymes, (i) glycoside hydrolase (GH), (ii) glycosyltransferase (GT), (iii) polysaccharide lyase (PL), (iv) carbohydrate esterase (CE), and (v) auxiliary active enzyme (AA), were found to be positively correlated with the production of NAECs. However, among the GHs, the GHs annotated from the H8 strain chromosome displayed the highest relative abundance and a positive correlation with the production of NAECs. Specifically, the GH13-14, GH13-20, GH13-38, GH13-25, GH13-10, GH42, and GH28 genes of the H8 strain were relatively more abundant and were key contributors to the production of NAECs. The correlation analyses revealed that the H8 strain plays a leading role among all the microorganisms in FRTLC in the production of NAECs. Our findings support the application of Klebsiella variicola in NAEC production and a reduction in nicotine content in tobacco products.

Simulated digestions of free oligosaccharides and mucin-type O-glycans reveal a potential role for Clostridium perfringens.

The development of a stable human gut microbiota occurs within the first year of life. Many open questions remain about how microfloral species are influenced by the composition of milk, in particular its content of human milk oligosaccharides (HMOs). The objective is to investigate the effect of the human HMO glycome on bacterial symbiosis and competition, based on the glycoside hydrolase (GH) enzyme activities known to be present in microbial species. We extracted from UniProt a list of all bacterial species catalysing glycoside hydrolase activities (EC 3.2.1.-), cross-referencing with the BRENDA database, and obtained a set of taxonomic lineages and CAZy family data. A set of 13 documented enzyme activities was selected and modelled within an enzyme simulator according to a method described previously in the context of biosynthesis. A diverse population of experimentally observed HMOs was fed to the simulator, and the enzymes matching specific bacterial species were recorded, based on their appearance of individual enzymes in the UniProt dataset. Pairs of bacterial species were identified that possessed complementary enzyme profiles enabling the digestion of the HMO glycome, from which potential symbioses could be inferred. Conversely, bacterial species having similar GH enzyme profiles were considered likely to be in competition for the same set of dietary HMOs within the gut of the newborn. We generated a set of putative biodegradative networks from the simulator output, which provides a visualisation of the ability of organisms to digest HMO and mucin-type O-glycans. B. bifidum, B. longum and C. perfringens species were predicted to have the most diverse GH activity and therefore to excel in their ability to digest these substrates. The expected cooperative role of Bifidobacteriales contrasts with the surprising capacities of the pathogen. These findings indicate that potential pathogens may associate in human gut based on their shared glycoside hydrolase digestive apparatus, and which, in the event of colonisation, might result in dysbiosis. The methods described can readily be adapted to other enzyme categories and species as well as being easily fine-tuneable if new degrading enzymes are identified and require inclusion in the model.

Probiotic consortium modulating the gut microbiota composition and function of sterile Mediterranean fruit flies

The sterile insect technique (SIT) remains a successful approach in managing pest insects. However, the long-term mass rearing and sterilizing radiation associated with SIT have been observed to induce physiological and ecological fitness decline in target insects. This decline may be attributed to various factors, including commensal microbiota dysbiosis, selection procedures, loss of heterozygosity, and other complex interactions.. There is evidence that the bacterial symbiont of insects may play critical roles in digestion, development, reproduction, and behavior. Probiotics are an increasingly common approach for restoring the intestinal microbiota structure and fitness parameters of sterile insects, particularly in the Vienna 8 genetic sexing strain (V8-GSS) of the Mediterranean fruit fly (medfly), Ceratitis capitata. Here, we explore the influence of the previously isolated bacterial strain, Lactococcus lactis, Enterobacter sp., and Klebsiella oxytoca, administration as probiotic consortia (LEK-PC) to the larvae and/or adult diet over the course of 20 rearing generations on fitness parameters. The experiment was carried out in four colonies: a control colony (C), one to which probiotics were not added, one to which probiotics were added to the larval medium (L+), one to which probiotics were added to the adult medium (A+), and one to which probiotics were added to both the larval and adult mediums (AL+). Emergence, flight ability, survival under stress conditions, and mating competitiveness, were all significantly improved by the LEK-PC treatment independently of the administration stage. The intestinal microbiota structure of various medfly V8-GSS colonies also underwent a significant shift, despite the fact that the core microbial community was unaffected by the LEK-PC administration stage, according to 16S metagenomics sequencing. Comparison of the metabolic function prediction and associated carbohydrate enzymes among colonies treated with “LEK-PC” showed an enrichment of metabolic functions related to carbohydrates, amino acids, cofactors, and vitamins metabolism, as well as, glycoside hydrolase enzymes in the AL+ colony compared to the control. This study enriches the knowledge regarding the benefits of probiotic treatment to modulate and restore the intestinal microbiota of C. capitata sterile males for a better effectiveness of the SIT.

Self-driving laboratories to autonomously navigate the protein fitness landscape

Protein engineering has nearly limitless applications across chemistry, energy and medicine, but creating new proteins with improved or novel functions remains slow, labor-intensive and inefficient. Here we present the Self-driving Autonomous Machines for Protein Landscape Exploration (SAMPLE) platform for fully autonomous protein engineering. SAMPLE is driven by an intelligent agent that learns protein sequence–function relationships, designs new proteins and sends designs to a fully automated robotic system that experimentally tests the designed proteins and provides feedback to improve the agent’s understanding of the system. We deploy four SAMPLE agents with the goal of engineering glycoside hydrolase enzymes with enhanced thermal tolerance. Despite showing individual differences in their search behavior, all four agents quickly converge on thermostable enzymes. Self-driving laboratories automate and accelerate the scientific discovery process and hold great potential for the fields of protein engineering and synthetic biology.

A Multiplexing Activity-Based Protein-Profiling Platform for Dissection of a Native Bacterial Xyloglucan-Degrading System.

Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a "low-tech", easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.

Recent advances in enzymatic properties, preparation methods, and functions of glycoside hydrolase from Bifidobacterium: a review.

Bifidobacterium is a major probiotic of intestinal gut flora and exerts many physiological activities, and it is widely applied in the fields of food and medicine. As an important part of Bifidobacterium, glycoside hydrolase plays a role in its physiological activity. With the continuous development and improvement of genetic engineering technology, research on this type of enzyme will play a crucial role in promoting the further development of Bifidobacterium in the field of probiotics. In this review, the preparation methods, enzymatic properties, and functions of glycoside hydrolase extracted from Bifidobacterium are described and summarized. The common method for preparing glycoside hydrolase derived from Bifidobacterium is heterologous expression in Escherichia coli BL21. The optimal pH range for these glycoside hydrolase enzymes is between 4.5 and 7.5; the optimal temperature is between 30 and 50 °C, which is close to the optimal growth condition of Bifidobacterium. Based on substrate specificity, these glycoside hydrolase could hydrolyze synthetic substrates and natural oligosaccharides, including a series of pNP artificial substrates, disaccharide, and trisaccharides, while they have little ability to hydrolyze polysaccharide substrates. This review will be expected to provide a basis for the development of Bifidobacterium as a probiotic element.

Influence of pH on Inulin Conversion to 2,3-Butanediol by Bacillus licheniformis 24: A Gene Expression Assay.

2,3-Butanediol (2,3-BD) is an alcohol highly demanded in the chemical, pharmaceutical, and food industries. Its microbial production, safe non-pathogenic producer strains, and suitable substrates have been avidly sought in recent years. The present study investigated 2,3-BD synthesis by the GRAS Bacillus licheniformis 24 using chicory inulin as a cheap and renewable substrate. The process appears to be pH-dependent. At pH 5.25, the synthesis of 2,3-BD was barely detectable due to the lack of inulin hydrolysis. At pH 6.25, 2,3-BD concentration reached 67.5 g/L with rapid hydrolysis of the substrate but was accompanied by exopolysaccharide (EPS) synthesis. Since inulin conversion by bacteria is a complex process and begins with its hydrolysis, the question of the acting enzymes arose. Genome mining revealed that several glycoside hydrolase (GH) enzymes from different CAZy families are involved. Five genes encoding such enzymes in B. licheniformis 24 were amplified and sequenced: sacA, sacB, sacC, levB, and fruA. Real-time RT-PCR experiments showed that the process of inulin hydrolysis is regulated at the level of gene expression, as four genes were significantly overexpressed at pH 6.25. In contrast, the expression of levB remained at the same level at the different pH values at all-time points. It was concluded that the sacC and sacA/fruA genes are crucial for inulin hydrolysis. They encode exoinulinase (EC 3.2.1.80) and sucrases (EC 3.2.1.26), respectively. The striking overexpression of sacB under these conditions led to increased synthesis of EPS; therefore, the simultaneous production of 2,3-BD and EPS cannot be avoided.

Evaluating the Activity of Neuraminidase in Bacterial Vaginosis Microflora and Imaging Sialic Acid on the Cell Membrane by Boron and Nitrogen Codoped Fluorescent Carbon Dots.

Sialic acids (SAs) are commonly located on the cell surface as terminal ends of glycoproteins and glycolipids. Neuraminidase (NEU) is a class of glycoside hydrolase enzymes that can cleave SAs from receptors. Both SA and NEU play important roles in human physiological and pathological processes of cell-cell interaction, communication, and signaling. Additionally, bacterial vaginosis (BV), a form of gynecological inflammation caused by dysbiosis of the vaginal microbiome, results in the abnormal activity of NEU in vaginal fluid. Here, we developed a novel probe for rapidly and selectively sensing SA and NEU based on a one-step prepared boron and nitrogen codoped fluorescent carbon dots (BN-CDs). The selective recognition reaction between SA and the phenylboronic acid groups on the surface of BN-CDs inhibits fluorescence emission from BN-CDs, while the NEU-catalyzed hydrolysis of SA bound on BN-CDs leads to fluorescence recovery. The probe was applied in diagnosing BV and showed consistent results to Amsel criteria. Moreover, the low cytotoxicity of BN-CDs facilitates its application in fluorescence imaging of SA on the membrane of red blood cells (RBCs) and leukemia cell lines (U937, KAS-1). The excellent sensitivity, accuracy, and applicability of the developed probe support its broad potential applications in future clinical diagnosis and treatment.

Unveiling the genomic potential of a novel thermostable glycoside hydrolases producing Neobacillus sedimentimangrovi UE25.

Genetic and enzymatic potential of Neobacillus sedimentimangrovi has not been assembled to date. Here, we report a high-quality genome assembly of thermophilic bacterium Neobacillus sedimentimangrovi UE25 using Illumina Hi-seq 2500. The strain was isolated from a crocodile pond Manghopir, Karachi, Pakistan. QUAST quality parameters showed 37.75% GC content and exhibited the genome into 110 contigs, with a total size of 3,230,777 bases. Genome of N. sedimentimangrovi UE25 harbors phage mediated DNA through horizontal gene exchange from the phages, symbiotic and pathogenic bacteria. Most of the phage genome encodes for hypothetical proteins, protease, and phage assembly proteins. Gene clusters encoding the intrinsic resistance to glycopeptides, isoniazid, rifamycin, elfamycin, macrolide, aminoglycosides, tetracycline and fluoroquinolone were identified into the genome. Since, the strain has been reported for the production of many industrially important thermostable enzymes, therefore, the genomic data of thermostable enzymes might be helpful to employ this species in commercial sectors. Probing genes of multiple thermostable glycoside hydrolase enzymes especially xylanases of N. sedimentimangrovi UE25 showed genetic diversity among the genes and confer the industrial importance of this microorganism. Furthermore, the genome of N. sedimentimangrovi will greatly improve our understanding of its genetics and evolution.

A Review on the Various Sources of β-Galactosidase and Its Lactose Hydrolysis Property.

β-Galactosidase is a glycoside hydrolase enzyme that possesses both hydrolytic and transgalactosylation properties and has several benefits and advantages in the food and dairy industries. The catalytic process of β-galactosidase involves the transfer of a sugar residue from a glycosyl donor to an acceptor via a double-displacement mechanism. Hydrolysis prevails when water acts as an acceptor, resulting in the production of lactose-free products. Transgalactosylation prevails when lactose acts as an acceptor, resulting in the production of prebiotic oligosaccharides. β-Galactosidase is also obtained from many sources including bacteria, yeast, fungi, plants, and animals. However, depending on the origin of the β-galactosidase, the monomer composition and their bonds may differ, thereby influencing their properties and prebiotic efficacy. Thus, the increasing demand for prebiotics in the food industry and the search for new oligosaccharides have compelled researchers to search for novel sources of β-galactosidase with diverse properties. In this review, we discuss the properties, catalytic mechanisms, various sources and lactose hydrolysis properties of β-galactosidase.

Compost-derived thermophilic microorganisms producing glycoside hydrolase activities as new potential biocatalysts for sustainable processes

BackgroundThe management of the organic waste recycling process determines the interest in the thermophiles microorganisms involved in composting. Although many microbial enzymes have been isolated and studied for their industrial and commercial uses, there is still a continuous search for microorganisms which could synthesize industrially feasible enzymes, especially when the microbial diversity of cow dung itself makes a potential source of biotechnological enzymes.ResultsThe composting process studied at the Experimental Station of the University of Naples Federico II (Castel Volturno, Caserta, Italy) was characterized by fresh saw dust 40%, bovine manure 58%, and 2% mature compost as raw organic substrates, and its thermophilic phase exceeded a temperature of 55 °C for at least 5 days, thus achieving sanitation. Six microbial strains were isolated and designated as follow: CV1-1, CV1-2, CV2-1, CV2-2, CV2-3 and CV2-4. Based on 16S rRNA gene sequence, HRMAS–NMR spectroscopy, and biochemical investigations, they were ascribed to the genera Geobacillus and Bacillus. All the microbial isolates were qualitatively screened on plates for the presence of hydrolytic activities, and they were quantitatively screened in liquid for glycoside hydrolase enzymes in the extracellular, cell-bound, and cytosolic fractions. Based on these results, strains CV2-1 and CV2-3 were also quantitatively screened for the presence of cellulase and pectinase activities, and pH and temperature optimum plus thermostability of cellulase from CV2-1 were analyzed.ConclusionsThe isolation and the identification of these thermophilic microorganisms such as Geobacillus toebii, Geobacillus galactosidasius, Bacillus composti, Bacillus thermophilus and Aeribacillus composti have allowed the study of the biodiversity of compost, with emphasis on their primary metabolome through an innovative and underutilized technique, that is HRMAS–NMR, also highlighting it as a novel approach to bacterial cell analysis. Subsequently, this study has permitted the identification of enzymatic activities able to degrade cellulose and other polymeric substrates, such as the one investigated from strain CV2-1, which could be interesting from an industrial and a biotechnological point of view, furthermore, increasing the knowledge for potential applicability in different industrial fields as an efficient and environmentally friendly technique.Graphical

A combinatorial droplet microfluidic device integrated with mass spectrometry for enzyme screening.

Mass spectrometry (MS) enables detection of different chemical species with a very high specificity; however, it can be limited by its throughput. Integrating MS with microfluidics has a tremendous potential to improve throughput and accelerate biochemical research. In this work, we introduce Drop-NIMS, a combination of a passive droplet loading microfluidic device and a matrix-free MS laser desorption ionization technique called nanostructure-initiator mass spectrometry (NIMS). This platform combines different droplets at random to generate a combinatorial library of enzymatic reactions that are deposited directly on the NIMS surface without requiring additional sample handling. The enzyme reaction products are then detected with MS. Drop-NIMS was used to rapidly screen enzymatic reactions containing low (on the order of nL) volumes of glycoside reactants and glycoside hydrolase enzymes per reaction. MS "barcodes" (small compounds with unique masses) were added to the droplets to identify different combinations of substrates and enzymes created by the device. We assigned xylanase activities to several putative glycoside hydrolases, making them relevant to food and biofuel industrial applications. Overall, Drop-NIMS is simple to fabricate, assemble, and operate and it has potential to be used with many other small molecule metabolites.

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling

Activity-based protein profiling (ABPP) has emerged as a versatile biochemical method for studying enzyme activity under various physiological conditions, with applications so far mainly in biomedicine. Here, we show the potential of ABPP in the discovery of biocatalysts from the thermophilic and lignocellulose-degrading white rot fungus Phanerochaete chrysosporium. By employing a comparative ABPP-based functional screen, including a direct profiling of wood substrate-bound enzymes, we identify those lignocellulose-degrading carbohydrate esterase (CE1 and CE15) and glycoside hydrolase (GH3, GH5, GH16, GH17, GH18, GH25, GH30, GH74 and GH79) enzymes specifically active in presence of the substrate. As expression of fungal enzymes remains challenging, our ABPP-mediated approach represents a preselection procedure for focusing experimental efforts on the most promising biocatalysts. Furthermore, this approach may also allow the functional annotation of domains-of-unknown functions (DUFs). The ABPP-based biocatalyst screening described here may thus allow the identification of active enzymes in a process of interest and the elucidation of novel biocatalysts that share no sequence similarity to known counterparts.

Carbohydrate active enzyme system in rumen fungi: a review

Hydrolysis and dehydration reactions of carbohydrates, which are used as energy raw materials by all living things in nature, are controlled by Carbohydrate Active Enzyme (CAZy) systems. These enzymes are also used in different industrial areas today. There are different types of microorganisms that have the CAZy system and are used in the industrial sector. Apart from current organisms, there are also rumen fungi within the group of candidate microorganisms with the CAZy system. It has been reported that xylanase (EC3.2.1.8 and EC3.2.1.37) enzyme, a member of the glycoside hydrolase enzyme family obtained from Trichoderma sp. and used especially in areas such as bread, paper, and feed industry, is more synthesized in rumen fungi such as Orpinomyces sp. and Neocallimastix sp. Therefore, this study reviews Neocallimastixsp., Orpinomyces sp., Caecomyces sp., Piromyces sp., and Anaeromyces sp., registered in the CAZy and Mycocosm database for rumen fungi to have both CAZy enzyme activity and to be an alternative microorganism in the industry. Furthermore the CAZy enzyme activities of the strains are investigated. The review shows thatNeocallimax sp. and Orpinomyces sp. areconsidered as candidate microorganisms.

Effect of Incubation Time on Amylase Enzyme Activity in Cassava Tape

Cassava tape is a product made by a fermentation process that requires the help of microorganisms. The tape fermentation process involves the enzyme amylase. Amylase is a glycoside hydrolase enzyme that catalyzes the breakdown of starch into simple sugars. Enzyme activity can be calculated by measuring the amount of product formed, or by calculating the amount of substrate reduction in a given unit of time. This study aims to determine the effect of incubation time on amylase enzyme activity in cassava tape. The research method used was the descriptive experimental method. The results of the experiment showed a reduction in the substrate due to amylase enzyme activity. The highest percentage of substrate reduction occurred at 48 hours incubation time and the lowest percentage at 144 hours incubation time. An organoleptic test was conducted with color, aroma, texture, and taste parameters conducted on 30 panelists. Incubation time affected the percentage of substrate reduction and organoleptic parameters

Bacterial species metabolic interaction network for deciphering the lignocellulolytic system in fungal cultivating termite gut microbiota

Fungus-cultivating termite Odontotermes badius developed a mutualistic association with Termitomyces fungi for the plant material decomposition and providing a food source for the host survival. The mutualistic relationship sifted the microbiome composition of the termite gut and Termitomyces fungal comb. Symbiotic bacterial communities in the O. badius gut and fungal comb have been studied extensively to identify abundant bacteria and their lignocellulose degradation capabilities. Despite several metagenomic studies, the species-wide metabolic interaction patterns of bacterial communities in termite gut and fungal comb remains unclear. The bacterial species metabolic interaction network (BSMIN) has been constructed with 230 bacteria identified from the O. badius gut and fungal comb microbiota. The network portrayed the metabolic map of the entire microbiota and highlighted several inter-species biochemical interactions like cross-feeding, metabolic interdependency, and competition. Further, the reconstruction and analysis of the bacterial influence network (BIN) quantified the positive and negative pairwise influences in the termite gut and fungal comb microbial communities. Several key macromolecule degraders and fermentative microbial entities have been identified by analyzing the BIN. The mechanistic interplay between these influential microbial groups and the crucial glycoside hydrolases (GH) enzymes produced by the macromolecule degraders execute the community-wide functionality of lignocellulose degradation and subsequent fermentation. The metabolic interaction pattern between the nine influential microbial species has been determined by considering them growing in a synthetic microbial community. Competition (30%), parasitism (47%), and mutualism (17%) were predicted to be the major mode of metabolic interaction in this synthetic microbial community. Further, the antagonistic metabolic effect was found to be very high in the metabolic-deprived condition, which may disrupt the community functionality. Thus, metabolic interactions of the crucial bacterial species and their GH enzyme cocktail identified from the O. badius gut and fungal comb microbiota may provide essential knowledge for developing a synthetic microcosm with efficient lignocellulolytic machinery.

A processive GH9 family endoglucanase of Bacillus licheniformis and the role of its carbohydrate-binding domain.

One of the critical steps in lignocellulosic deconstruction is the hydrolysis of crystalline cellulose by cellulases. Endoglucanases initially facilitate the breakdown of cellulose in lignocellulosic biomass and are further aided by other cellulases to produce fermentable sugars. Furthermore, if the endoglucanase is processive, it can adsorb to the smooth surface of crystalline cellulose and release soluble sugars during repeated cycles of catalysis before dissociating. Most glycoside hydrolase family 9 (GH9) endoglucanases have catalytic domains linked to a CBM (carbohydrate-binding module) (mostly CBM3) and present the second-largest cellulase family after GH5. GH9 endoglucanases are relatively less characterized. Bacillus licheniformis is a mesophilic soil bacterium containing many glycoside hydrolase (GH) enzymes. We identified an endoglucanase gene, gh9A, encoding the GH9 family enzyme H1AD14 in B. licheniformis and cloned and overexpressed H1AD14 in Escherichia coli. The purified H1AD14 exhibited very high enzymatic activity on endoglucanase substrates, such as β-glucan, lichenan, Avicel, CMC-Na (sodium carboxymethyl cellulose) and PASC (phosphoric acid swollen cellulose), across a wide pH range. The enzyme is tolerant to 2 M sodium chloride and retains 74% specific activity on CMC after 10 days, the highest amongst the reported GH9 endoglucanases. The full-length H1AD14 is a processive endoglucanase and efficiently saccharified sugarcane bagasse. The deletion of the CBM reduces the catalytic activity and processivity. The results add to the sparse knowledge of GH9 endoglucanases and offer the possibility of characterizing and engineering additional enzymes from B. licheniformis toward developing a cellulase cocktail for improved biomass deconstruction. KEY POINTS: • H1AD14 is a highly active and processive GH9 endoglucanase from B. licheniformis. • H1AD14 is thermostable and has a very long half-life. • H1AD14 showed higher saccharification efficiency than commercial endoglucanase.