Rice straw hydrolysis using in-situ produced enzymes: Feedstock influences fungal enzyme composition and hydrolytic efficiency.

Trichoderma reesei RUT-C30 was cultivated on differentially pretreated rice straw and pure cellulose as a carbon source/inducer for cellulase production, and the enzymes were evaluated for hydrolysis of sequential acid and alkali pretreated rice straw. Growth on pretreated rice straw enhanced protein secretion and cellulase activities compared to pure cellulose as a carbon source. The yield of cellulolytic enzymes was higher for alkali pretreated rice straw (ALP-RS), while H2O2-treated (HP-RS) could not induce cellulases to a larger level compared to pure cellulose. Protein concentration was 3.5-fold higher on ALP-RS as compared to pure cellulose, with a maximum filter-paper cellulase (FPase) activity of 1.76 IU/ml and carboxy-methyl cellulase (CMCase) activity of 40.16 IU/ml (2.18 fold higher). Beta-glucosidase (BGL) activity was more or less the same with the different substrates and supplementation of heterologous BGL could result in a quantum jump in hydrolytic efficiencies, which in the case of ALP-RS induced enzymes was 34% (increased from 69.26% to 92.51%). The use of lignocellulosic biomass (LCB) itself as a substrate for the production of cellulase is advantageous not only in terms of raw material costs but also for obtaining a more suitable enzyme profile for biomass hydrolysis.

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.

Comparison of the solid-state and submerged fermentation derived secretomes of hyper-cellulolytic Penicillium janthinellum NCIM 1366 reveals the changes responsible for differences in hydrolytic performance.

Solid-state fermentation (SSF) and submerged fermentation (SmF) have often been compared for production of biomass hydrolyzing enzymes highlighting the superiority of the SSF produced enzymes, but the reasons for the performance differences are under-explored. Penicillium janthinellum NCIM 1366 culture extracts from SSF had better hydrolytic performance along with a higher initial rate of reaction. Secretome analyses of the SSF and SmF enzymes using LC/MS-MS, indicated that while the type of proteins secreted were similar in both modes, the abundance of specific beta glucosidases, lytic polysaccharide monooxygenases and hemicellulolytic enzymes were very high in SSF resulting in efficient initiation, low accumulation of cellobiose and high initial reaction rates. Key enzymes that catalyse lignocellulose breakdown under SSF and SmF are therefore different and the fungus may be speculated to have regulation mechanisms that aid differential expression under different cultivation modes.

Sequential mild acid and alkali pretreatment of rice straw to improve enzymatic saccharification for bioethanol production.

Sequential pretreatment using different NaOH concentrations (0.5%, 1.0%, 1.5%, w/w) and 1% H2SO4 (w/w) was evaluated as a strategy for effective hydrolysis of rice straw. The efficiency of sequential NaOH and H2SO4 (SNA) pretreatment against sequential H2SO4 and NaOH (SH) was assessed. SH pretreated biomass attained more sugar yield compared to SNA pretreated biomass. The sugar yields from pretreated biomass improved with increasing NaOH concentration in both SH and SNA treatments. The maximum sugar release of 40.6 mg/ml (83.2% efficiency) was obtained from SH pretreated biomass when the stage 2 alkali treatment was performed at 1.5% w/w NaOH. The non-detoxified hydrolysate from this biomass was fermented with 96.8% efficiency.

Lignocellulose degradation by Penicillium janthinellum enzymes is influenced by its variable secretome and a unique set of feedstock characteristics.

Substrate characteristics and proteins that affect lignocellulose-hydrolysis by the hypercellulolytic fungus Penicillium janthinellum NCIM 1366 (PJ-1366) were investigated. The hydrolysis rate of PJ-1366 enzymes was very high, with upto 75 % of the reaction being completed in initial 4 h. Comparison of the hydrolytic efficiencies on differently pretreated biomass indicated that the greatest (negative) effect was imparted by lignin, suggesting that improving ligninase activity of the PJ-1366 enzymes may help to improve hydrolysis. Larger pore sizes and higher crystallinity of substrates, which favor enzyme penetration and processive hydrolysis, positively influenced hydrolysis efficiency. For alkali-pretreated substrates, 16 FPU/g of PJ-1366 cellulases released the sugar-equivalent of using 10 FPU/g of a commercial biomass hydrolyzing enzyme. By correlation analysis, 41 proteins, including 20 CAZymes were identified, whose abundance in the secretome positively correlated with the cellulase activities of the culture filtrate. These proteins may be considered as the primary drivers of FPase/CMCase/pNPGase/xylanase activity in PJ-1366.

High-solids loading processing for an integrated lignocellulosic biorefinery: Effects of transport phenomena and rheology - A review.

This review aims to present an analysis and discussion on the processing of lignocellulosic biomass in terms of biorefinery concept and circular bioeconomy operating at high solids lignocellulosic (above 15% [w/w]) at the pretreatment, enzymatic hydrolysis stage, and fermentation strategy for an integrated lignocellulosic bioprocessing. Studies suggest high solids concentration enzymatic hydrolysis for improved sugars yields and methods to overcome mass transport constraints. Rheological and computational fluid dynamics models of high solids operation through evaluation of mass and momentum transfer limitations are presented. Also, the review paper explores operational feeding strategies to obtain high ethanol concentration and conversion yield, from the hydrothermal pretreatment and investigates the impact of mass load over the operational techniques. Finally, this review contains a brief overview of some of the operations that have successfully scaled up and implemented high-solids enzymatic hydrolysis in terms of the biorefinery concept.

A highly efficient stratagem for protoplast isolation and genetic transformation in filamentous fungus Colletotrichum falcatum.

Red rot of sugarcane caused by the hemi-biotrophic fungal pathogen, Colletotrichum falcatum, is a major threat to sugarcane cultivation in many tropical countries such as India, Bangladesh, and Pakistan. With the accumulating information on pathogenicity determinants, namely, effectors and pathogen-associated molecular patterns (PAMPs) of C. falcatum, it is of paramount importance to decipher the functional role of these molecular players that may ultimately decide upon the outcome of sugarcane-C. falcatum interaction. Since C. falcatum is a multinucleated filamentous fungus, the conventional Agrobacterium-mediated transformation method could not be effectively utilized for targeted manipulation of genomic DNA. Hence, we developed a highly efficient protoplast-based transformation method for the virulent pathotype of C. falcatum - Cf671, which involves isolation of protoplast, polyethylene glycol (PEG)-mediated transformation, and regeneration of transformed protoplasts into hyphal colonies. In this study, germinating conidiospores of Cf671 were treated with different enzyme-osmoticum combinations, out of which 20 mg/mL lysing enzyme with 5 mg/mL ß-glucanase in an osmoticum of 1.2 mol/L MgSO4 yielded maximum number of viable protoplasts. The resultant protoplasts were transformed with pAsp shuttle vector. Transformed protoplasts were regenerated into hyphal colonies under hygromycin selection and observed for GFP fluorescence. This protocol resulted in a transformation efficiency of > 130 transformants per µg of plasmid DNA. This method of transformation is rapid, simple, and more efficient for gene knockout, site-directed mutagenesis, ectopic expression, and other genetic functional characterization experiments in C. falcatum, even with large vectors (> 10 kb) and can also be applied for other filamentous fungi.

Integrated bioprocess for structured lipids, emulsifiers and biodiesel production using crude acidic olive pomace oils.

Olive pomace oil (OPO), a by-product of olive oil industry, is directly consumed after refining. The novelty of this study consists of the direct use of crude high acidic OPO (3.4-20% acidity) to produce added-value compounds, using sn-1,3-regioselective lipases: (i) low-calorie dietetic structured lipids (SL) containing caprylic (C8:0) or capric (C10:0) acids by acidolysis or interesterification with their ethyl esters, (ii) fatty acid methyl esters (FAME) for biodiesel, and (iii) sn-2 monoacylglycerols (emulsifiers), as by-product of FAME production by methanolysis. Immobilized Rhizomucor miehei lipase showed similar activity in acidolysis and interesterification for SL production (yields: 47.8-53.4%, 7 h, 50℃) and was not affected by OPO acidity. Batch operational stability decreased with OPO acidity, but it was at least three-fold in interesterification that in acidolysis. Complete conversion of OPO into FAME and sn-2 monoacylglycerols was observed after 3 h-transesterification (glycerol stepwise addition) and lipase deactivation was negligeable after 11 cycles.

Repurposing proteases: An in-silico analysis of the binding potential of extracellular fungal proteases with selected viral proteins.

Proteases have long been the target of many drugs, but their potential as therapeutic agents is a well-known, but under-explored area. Due to the heightened threat from new and emerging infectious agents, it is worthwhile to tap into the vast microbial protease resource to identify potential therapeutics. By docking proteases of the fungus Penicillium janthinellum NCIM 1366 with the proteins encoded by the SARS-CoV-2 virus, the enzymes that have the potential to bind with, and thereby degrade viral proteins were identified. In-silico docking analysis revealed that both fungal and commercially available proteases belonging to the A1A, M20A, S10, S8A and T1A families were able to bind the viral spike, envelope, ORF-7a and Nsp2 proteins (binding energy < -50 kJ/mol), thereby opening up the possibility of developing additional therapeutic applications for these enzymes.

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.

Thermophilic Chitinases: Structural, Functional and Engineering Attributes for Industrial Applications.

Chitin is the second most widely found natural polymer next to cellulose. Chitinases degrade the insoluble chitin to bioactive chitooligomers and monomers for various industrial applications. Based on their function, these enzymes act as biocontrol agents against pathogenic fungi and invasive pests compared with conventional chemical fungicides and insecticides. They have other functional roles in shellfish waste management, fungal protoplast generation, and Single-Cell Protein production. Among the chitinases, thermophilic and thermostable chitinases are gaining popularity in recent years, as they can withstand high temperatures and maintain the enzyme stability for longer periods. Not all chitinases are thermostable; hence, tailor-made thermophilic chitinases are designed to enhance their thermostability by direct evolution, genetic engineering involving mutagenesis, and proteomics approach. Although research has been done extensively on cloning and expression of thermophilic chitinase genes, there are only few papers discussing on the mechanism of chitin degradation using thermophiles. The current review discusses the sources of thermophilic chitinases, improvement of protein stability by gene manipulation, metagenomics approaches, chitin degradation mechanism in thermophiles, and their prospective applications for industrial, agricultural, and pharmaceutical purposes.

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".

Mild alkaline pretreatment can achieve high hydrolytic and fermentation efficiencies for rice straw conversion to bioethanol.

Mild alkaline pretreatment was evaluated as a strategy for effective lignin removal and hydrolysis of rice straw. The pretreatment efficiency of different NaOH concentrations (0.5, 1.0, 1.5 or 2.0% w/w) was assessed. Rice straw (RS) pretreated with 1.5% NaOH achieved better sugar yield compared to other concentrations used. A cellulose conversion efficiency of 91% (45.84 mg/ml glucose release) was attained from 1.5% NaOH pretreated rice straw (PRS), whereas 1% NaOH pretreated rice straw yielded 35.10 mg/ml of glucose corresponding to a cellulose conversion efficiency of 73.81%. The ethanol production from 1% and 1.5% NaOH pretreated RS hydrolysates was similar at ∼3.3% (w/v), corresponding to a fermentation efficiency of 86%. The non-detoxified hydrolysate was fermented using the novel yeast strain Saccharomyces cerevisiae RPP-03O without any additional supplementation of nutrients.

Production of low-calorie structured lipids from spent coffee grounds or olive pomace crude oils catalyzed by immobilized lipase in magnetic nanoparticles.

In this study, crude oils extracted from spent coffee grounds (SCG) and olive pomace (OP) were used as raw-material to synthesize low-calorie triacylglycerols, either by acidolysis with capric acid, or by interesterification with ethyl caprate, in solvent-free media, catalyzed by sn-1,3 regioselective lipases. The Rhizopus oryzae lipase (ROL) was immobilized in magnetite nanoparticles (MNP-ROL) and tested as novel biocatalyst. MNP-ROL performance was compared with that of the commercial immobilized Thermomyces lanuginosus lipase (Lipozyme TL IM). For both oils, Lipozyme TL IM preferred interesterification over acidolysis. MNP-ROL catalyzed reactions were faster and acidolysis was preferred with yields of c.a. 50% new triacylglycerols after 3 h acidolysis of OP or SCG oils. MNP-ROL was very stable following the Sadana deactivation model with half-lives of 163 h and 220 h when reused in batch acidolysis and interesterification of OP oil, respectively.

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.

Valorization of lignocellulosic residues from the olive oil industry by production of lignin, glucose and functional sugars.

Spent olive pomace from the two-phase extraction system of virgin olive oil and olive pomace oil, is a major agro-industrial residue. Present study aimed at the valorization of residual olive pomace and stones (seeds) by hydrothermal treatment and enzymatic hydrolysis of glucans. Both residues contain lignin (31.2% and 42.1%), glucans (13.8% and 15.3%) and xylans (18.9% and 20.3%). After hydrothermal pretreatment (130 °C, 30 min; severity factor log R0 = 2.99), 65% and 75% of hemicelluloses (65% of xylan) were hydrolysed into xylo-oligosaccharides in pomace and stones, respectively. Cellulose and lignin were not substantially affected. Three commercial enzyme preparations, Saczyme Yield, Ultimase BWL 40 and Celluclast 1.5 L, were evaluated for saccharification of pomace or stones at three biomass loads (10, 20 and 30%, w/v). Saczyme and Ultimase were active with high solid loads (30%), reaching 80 and 90% of glucan conversion in pomace, and 40 and 55% in stones, respectively, after 5 h.