Early cellular events and potential regulators of cellulase induction in Penicillium janthinellum NCIM 1366.

Cellulase production by fungi is tightly regulated in response to environmental cues, and understanding this mechanism is a key pre-requisite in the efforts to improve cellulase secretion. Based on UniProt descriptions of secreted Carbohydrate Active enZymes (CAZymes), 13 proteins of the cellulase hyper-producer Penicillium janthinellum NCIM 1366 (PJ-1366) were annotated as cellulases- 4 cellobiohydrolases (CBH), 7 endoglucanases (EG) and 2 beta glucosidases (BGL). Cellulase, xylanase, BGL and peroxidase activities were higher for cultures grown on a combination of cellulose and wheat bran, while EG was stimulated by disaccharides. Docking studies indicated that the most abundant BGL- Bgl2- has different binding sites for the substrate cellobiose and the product glucose, which helps to alleviate feedback inhibition, probably accounting for the low level of glucose tolerance exhibited. Out of the 758 transcription factors (TFs) differentially expressed on cellulose induction, 13 TFs were identified whose binding site frequencies on the promoter regions of the cellulases positively correlated with their abundance in the secretome. Further, correlation analysis of the transcriptional response of these regulators and TF-binding sites on their promoters indicated that cellulase expression is possibly preceded by up-regulation of 12 TFs and down-regulation of 16 TFs, which cumulatively regulate transcription, translation, nutrient metabolism and stress response.

Anisotropic Alignment of Bacterial Nanocellulose Ionogels for Unconventionally High Combination of Stiffness and Damping.

Ionogels are emerging materials for advanced electrochemical devices; however, their mechanical instability to external stresses has raised concerns about their safety. This study reports aligned bacterial nanocellulose (BC) ionogel films swelled with the model ionic liquid (IL) of 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF4) for an unprecedented combination of high stiffness and high energy dissipation without significant loss of ionic conductivity. The aligned BC ionogel films are prepared through wet-state stretching methods, followed by drying and swelling by ILs. The aligned ionogel films exhibit significantly improved dynamic mechanical properties, overcoming the mechanical conventional limit of traditional materials by 2.0 times at 25 °C and by a maximum of 4.0 times at 0 °C. Additionally, the same samples exhibit relatively high ionic conductivities of 0.16 mS cm-1 at 20 °C and 0.45 mS cm-1 at 60 °C with storage moduli over 10 GPa. The synergistic effect of the mechanical reinforcements by alignment of the BC nanofibers and the plasticizing effects by ILs could be attributed to the significant enhancement of dynamic mechanical properties and the retention of ionic conductivities. These results will lead to a deeper understanding of the material design for mechanically superior ionogel systems with increasing demands for advanced electronic and electrochemical devices.

Characterisation of bacterial nanocellulose and nanostructured carbon produced from crude glycerol by Komagataeibacter sucrofermentans.

Bacterial nanocellulose (BNC), which has tunable properties, is a precursor of nanostructured energy storage materials; however, the cost of BNC production is challenging. This study uses crude glycerol from the biodiesel industry as a carbon nutrient and first-time carbonised BNC from K. sucrofermentans that is applied in energy storage. From crude glycerol in static cultivation, 6.4 g L-1 BNC was produced with a high crystallinity index (85%) and tensile properties in comparison to conventionally used pure carbon substrates. Carbon materials were derived from the BNC retained fibrous and crystalline features with disordered porous structures. The electrochemical properties of the carbon materials have a specific capacitance of 140 F g-1. This study highlights the valorisation of waste glycerol from the biodiesel industry as a substrate for efficient BNC production and the energy storage potential of carbon derived from BNC as renewable energy materials.

Draft genome of the glucose tolerant ß-glucosidase producing rare Aspergillus unguis reveals complete cellulolytic machinery with multiple beta-glucosidase genes.

Draft genome sequence of the glucose tolerant beta glucosidase (GT-BGL) producing rare fungus Aspergillus unguis NII 08,123 was generated through Next Generation Sequencing (NGS). The genome size of the fungus was estimated to be 37.1 Mb. A total of 3116 contigs were assembled using SPades, and 15,161 proteins were predicted using AUGUSTUS 3.1. Among them, 13,850 proteins were annotated using UniProt. Distribution of CAZyme genes specifically those encoding lignocellulose degrading enzymes were analyzed and compared with those from the industrial cellulase producer Trichoderma reesei in view of the huge differences in detectable enzyme activities between the fungi, despite the ability of A. unguis to grow on lignocellulose as sole carbon source. Full length gene sequence of the inducible GT-BGL could be identified through tracing back from peptide mass fingerprint. A total of 403 CAZymes were predicted from the genome, which includes 232 glycoside hydrolases (GHs), 12 carbohydrate esterases (CEs), 109 glycosyl transferases (GTs), 15 polysaccharide lyases (PLs), and 35 genes with auxiliary activities (AAs). The high level of zinc finger motif containing transcription factors could possibly hint a tight regulation of the cellulolytic machinery, which may also explain the low cellulase activities even when a complete repertoire of cellulase degrading enzyme genes are present in the fungus.

Addressing challenges in production of cellulases for biomass hydrolysis: Targeted interventions into the genetics of cellulase producing fungi.

Lignocellulosic materials are the favoured feedstock for biorefineries due to their abundant availability and non-completion with food. Biobased technologies for refining these materials are limited mainly by the cost of biomass hydrolyzing enzymes, typically sourced from filamentous fungi. Therefore, considerable efforts have been directed at improving the quantity and quality of secreted lignocellulose degrading enzymes from fungi in order to attain overall economic viability. Process improvements and media engineering probably have reached their thresholds and further production enhancements require modifying the fungal metabolism to improve production and secretion of these enzymes. This review focusses on the types and mechanisms of action of known fungal biomass degrading enzymes, our current understanding of the genetic control exerted on their expression, and possible routes for intervention, especially on modulating catabolite repression, transcriptional regulators, signal transduction, secretion pathways etc., in order to improve enzyme productivity, activity and stability.

Anaerobic co-digestion of bioplastics as a sustainable mode of waste management with improved energy production - A review.

The world of bioplastics has expanded rapidly in recent decades, and the new waste stream generated is creating major barriers to waste processing. Anaerobic co-digestion is to be considered one of the best options for the efficient processing of bioplastic waste due to its minimal space requirements, lower degrees of environmental pollution, and renewable energy generation. The higher carbon to nitrogen (C/N) ratio of bioplastics poses a challenge to anaerobic digestion, but co-digestion with lower C/N ratio biowastes can efficiently degrade bioplastics and improve biogas production in the system. In the future, the collection of organic waste in biodegradable plastic bags makes the waste management process easier for anaerobic digestion plants. The present review paper discusses current trends of bioplastic usage, degradation strategies, and the potential of anaerobic co-digestion for waste management with improved energy production in anaerobic digesters.

Penicillium janthinellum NCIM1366 shows improved biomass hydrolysis and a larger number of CAZymes with higher induction levels over Trichoderma reesei RUT-C30.

BACKGROUND: Major cost of bioethanol is attributed to enzymes employed in biomass hydrolysis. Biomass hydrolyzing enzymes are predominantly produced from the hyper-cellulolytic mutant filamentous fungus Trichoderma reesei RUT-C30. Several decades of research have failed to provide an industrial grade organism other than T. reesei, capable of producing higher titers of an effective synergistic biomass hydrolyzing enzyme cocktail. Penicillium janthinellum NCIM1366 was reported as a cellulase hyper producer and a potential alternative to T. reesei, but a comparison of their hydrolytic performance was seldom attempted. RESULTS: Hydrolysis of acid or alkali-pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei indicated 37 and 43% higher glucose release, respectively, with P. janthinellum enzymes. A comparison of these fungi with respect to their secreted enzymes indicated that the crude enzyme preparation from P. janthinellum showed 28% higher overall cellulase activity. It also had an exceptional tenfold higher beta-glucosidase activity compared to that of T. reesei, leading to a lower cellobiose accumulation and thus alleviating the feedback inhibition. P. janthinellum secreted more number of proteins to the extracellular medium whose total concentration was 1.8-fold higher than T. reesei. Secretome analyses of the two fungi revealed higher number of CAZymes and a higher relative abundance of cellulases upon cellulose induction in the fungus. CONCLUSIONS: The results revealed the ability of P. janthinellum for efficient biomass degradation through hyper cellulase production, and it outperformed the established industrial cellulase producer T. reesei in the hydrolysis experiments. A higher level of induction, larger number of secreted CAZymes and a high relative proportion of BGL to cellulases indicate the possible reasons for its performance advantage in biomass hydrolysis.

Correction to: Characterization of a glucose tolerant ß-glucosidase from Aspergillus unguis with high potential as a blend-in for biomass hydrolyzing enzyme cocktails.

In the original publication of the article, the affiliation of two co-authors Prajeesh Kooloth-Valappil and Meera Christopher was published incompletely. The correct affiliation of the authors should read " Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India".

Pretreatment strategies for enhanced biogas production from lignocellulosic biomass.

The inclusion of a pretreatment step in anaerobic digestion processes increases the digestibility of lignocellulosic biomass and enhances biogas yields by promoting lignin removal and the destruction of complex biomass structures. The increase in surface area enables the efficient interaction of microbes or enzymes, and a reduction in cellulose crystallinity improves the digestion process under anaerobic conditions. The pretreatment methods may vary based on the type of the lignocellulosic biomass, the nature of the subsequent process and the overall economics of the process. An improved biogas production by 1200% had been reported when ionic liquid used as pretreatment strategy for anaerobic digestion. The different pretreatment techniques used for lignocellulosic biomasses are generally grouped into physical, chemical, physicochemical, and biological methods. These four modes of pretreatment on lignocellulosic biomass and their impact on biogas production process is the major focus of this review article.

Characterization of a glucose tolerant ß-glucosidase from Aspergillus unguis with high potential as a blend-in for biomass hydrolyzing enzyme cocktails.

OBJECTIVES: Characterization of glucose tolerant beta glucosidase (GT-BGL) secreted by Aspergillus unguis NII 08123, determination of the gene and protein sequences of the enzyme and establishing its performance in blends for lignocellulose hydrolysis. RESULTS: Supplementation of A. unguis beta glucosidase (BGL) to cellulase released 1.6 times more sugar within 12 h during the hydrolysis of lignocellulosic biomass. The enzyme was determined to be similar to BGL-F from Emericella nidulans by MALDI-TOF analysis, and was found to be a GH3 family protein. Molecular Docking simulation studies showed that the enzyme has lesser affinity for glucose (- 5.7 kcal/mol) compared to its substrate cellobiose (- 7.5 kcal/mol). The residues present in the N-terminal domain are mostly involved in bond formation with both the substrate and the product, while the C-terminal domain contains the catalytic region. In-silico studies showed that its predicted structure is unlike that of previously reported BGLs, which might provide a clue to its exceptional catalytic activity. CONCLUSION: The GT-BGL from A. unguis NII 08123 was proven effective as a blend in for biomass hydrolyzing enzyme cocktails and the possible reasons for its glucose tolerance was determined through studies on its modeled structure.

Non-conventional yeast cell factories for sustainable bioprocesses.

The non-conventional yeasts Kluyveromyces lactis, Yarrowia lipolytica, Ogataea polymorpha and Pichia pastoris have been developed as eukaryotic expression hosts because of their desirable growth characteristics, including inhibitor and thermo-tolerance, utilisation of diverse carbon substrates and high amount of extracellular protein secretion. These yeasts already have established in the heterologous production of vaccines, therapeutic proteins, food additives and bio-renewable chemicals, but recent advances in the genetic tool box have the potential to greatly expand and diversify their impact on biotechnology. The diversity of these yeasts includes many strains possessing highly useful, and in some cases even uncommon, metabolic capabilities potentially helpful for the bioprocess industry. This review outlines the recent updates of non-conventional yeast in sustainable bioprocesses.

Applications of Microbial Enzymes in Food Industry.

The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed.

Production of Pectinase from Bacillus sonorensis MPTD1.

Seven isolates from spoiled fruits and vegetables were screened for pectinase production using pectin agar plates and the most efficient bacterial strain, MPTD1, was identified as Bacillus sonorensis. Optimisation of various process parameters was done using Plackett-Burman and Box-Behnken designs and it was found that parameters like yeast extract, K2HPO4, incubation time, NaNO3 and KCl have a negative impact on pectinase production. Parameters like pH and MgSO4 and pectin mass fractions have a positive impact on pectinase production. The maximum obtained enzyme activity was 2.43 (µM/mL)/min. This is the first report on pectinase production by Bacillus sonorensis.

An effective surfactant-assisted hydrothermal pretreatment strategy for bioethanol production from chili post-harvest residue by separate hydrolysis and fermentation.

Surfactants play major role in the delignification of lignocellulosic biomass. Surfactant-assisted hydrothermal pretreatment was evaluated for chili post-harvest residue. Maximum reducing sugar yield of 0.445 g per g of dry biomass (g/g) was obtained when surfactant PEG 6000 was used. Compositional analysis revealed an efficient removal of lignin and hemicelluloses from the pretreated biomass. Fermentation inhibitors such as furfural, 5-hydroxymethylfurfural and organic acids were absent in the hydrolyzate. After pretreatment, the biomass can be directly hydrolyzed without any neutralization, washing and drying, and the hydrolyzate is devoid of major fermentation inhibitors. Fermentation with Saccharomyces cerevisiae yielded 1.84% of ethanol with a fermentation efficiency of 63.88%.

Recent developments in l-glutaminase production and applications - An overview.

l-glutaminases is an important industrial enzyme which finds potential applications in different sectors ranging from therapeutic to food industry. It is widely distributed in bacteria, actinomycetes, yeast and fungi. l-Glutaminases are mostly produced by Bacillus and Pseudomonas sp. and few reports were available with fungal, actinomycete and yeast system. Modern biotechnological tools help in the improved production as well as with tailor made properties for specific applications. Most of the genetic engineering studies were carried out for the production of l-glutaminase with improved thermo-tolerance and salt tolerance. Considering the potential of in vitro applications of l-glutaminase, extracellular enzymes are important and most microbes produce this enzyme intracellularly. Several research and developmental activities are going on for the extracellular production of l-glutaminase. This review discusses recent trends and developments and applications of l-glutaminases.

Molecular improvements in microbial α-amylases for enhanced stability and catalytic efficiency.

α-Amylases is one of the most important industrial enzyme which contributes to 25% of the industrial enzyme market. Though it is produced by plant, animals and microbial source, those from microbial source seems to have potential applications due to their stability and economic viability. However a large number of α-amylases from different sources have been detailed in the literature, only few numbers of them could withstand the harsh industrial conditions. Thermo-stability, pH tolerance, calcium independency and oxidant stability and starch hydrolyzing efficiency are the crucial qualities for α-amylase in starch based industries. Microbes can be genetically modified and fine tuning can be done for the production of enzymes with desired characteristics for specific applications. This review focuses on the native and recombinant α-amylases from microorganisms, their heterologous production and the recent molecular strategies which help to improve the properties of this industrial enzyme.

Development of a novel ultrasound-assisted alkali pretreatment strategy for the production of bioethanol and xylanases from chili post harvest residue.

A novel ultrasound-assisted alkali pretreatment strategy was developed which could effectively remove lignin and hemicelluloses and improve the sugar yield from chili post harvest residue. Operational parameters that affect the pretreatment efficiency were studied and optimized. Inhibitor analysis of the hydrolyzate revealed that major fermentation inhibitors like furfural, 5-hydroxymethyl furfural as well as organic acids like citric acid, succinic acid and propionic acid were absent. Hence fermentation can be carried out without detoxification of the hydrolyzate. Changes in structural properties of the biomass were studied in relation to the pretreatment process using Scanning Electron Microscopy (SEM) and the changes in chemical composition were also monitored. The biomass pretreated with the optimized novel method could yield 0.428g/g of reducing sugars upon enzymatic hydrolysis. The hydrolyzate obtained by this novel pretreatment strategy was found to be suitable for bioethanol and xylanase production.

Potential of rice straw for bio-refining: An overview.

The biorefinery approach for the production of fuels and chemicals is gaining more and more attraction in recent years. The major advantages of biorefineries are the generation of multiple products with complete utilization of biomass with zero waste generation. Moreover the process will be economically viable when it targets low volume high value products in addition to high volume low value products like bioethanol. The present review discuss about the potential of rice straw based biorefinery. Since rice is a major staple food for many Asian countries, the utilization of the rice straw residue for fuel and chemicals would be very economical. The review focuses the availability and the potential of this residue for the production of fuel and other high value chemicals.

Phenazine-1-carboxylic acid mediated anti-oomycete activity of the endophytic Alcaligenes sp. EIL-2 against Phytophthora meadii.

The oomycete pathogen, Phytophthora meadii, causes various diseases in Hevea brasiliensis at different stages of its life cycle. The study reports the structural characterization of the active principle from the culture filtrate of Alcaligenes sp. EIL-2 (GenBank ID: HQ641257) offering antagonistic activity against P. meadii. Gas Chromatography Mass Spectroscopy (GC-MS) analysis showed the similarity of the compound with phenazine derivatives. The specific representations of FT-IR spectrum such as 3200 cm(-1) (OH stretching, NH stretching and presence of aromatic ring), 1737 cm(-1) (carboxylic acid), 2200-2400 cm(-1) (conjugated dienes) and 1467 cm(-1), and 1422 cm(-1) (CN bonds) were an indicative of phenazine-1-carboxylic acid (PCA). The structure of the compound was further confirmed by (1)H NMR/(13)C NMR spectroscopy, DEPT experiments, and two-dimensional NMR spectral studies, including (1)H-(1)H COSY and (1)H-(13)C HSQC as PCA with the molecular formula of C13H8N2O2. P. meadii was sensitive to purified PCA extract from the endophyte and a concentration of 5 µg/ml completely inhibited the mycelia growth. PCA also showed zoosporicidal activity against P. meadii zoospores. This is the first study of this kind where PCA from an endophyte of H. brasiliensis is being confirmed to carry antagonistic activity against P. meadii.

In silico characterization of a novel ß-1,3-glucanase gene from Bacillus amyloliquefaciens--a bacterial endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii.

We report the molecular characterization of ß-1,3-glucanase-producing Bacillus amyloliquefaciens-an endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii. After cloning and sequencing, the ß-1,3-glucanase gene was found to be 747 bp in length. A homology model of the ß-1,3-glucanase protein was built from the amino acid sequence obtained upon translation of the gene. The target ß-1,3-glucanase protein and the template protein, endo ß-1,3-1,4-glucanase protein (PDB ID: 3o5s), were found to share 94% sequence identity and to have similar secondary and tertiary structures. In the modeled structure, three residues in the active site region of the template-Asn52, Ile157 and Val158-were substituted with Asp, Leu and Ala, respectively. Computer-aided docking studies of the substrate disaccharide (ß-1, 3-glucan) with the target as well as with the template proteins showed that the two protein-substrate complexes were stabilized by three hydrogen bonds and by many van der Waals interactions. Although the binding energies and the number of hydrogen bonds were the same in both complexes, the orientations of the substrate in the active sites of the two proteins were different. These variations might be due to the change in the three amino acids in the active site region of the two proteins. The difference in substrate orientation in the active site could also affect the catalytic potential of the ß-1,3 glucanase enzyme.