Expression and characterization of a novel ß-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater.

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.

Hydrolysis of ionic liquid-treated substrate with an Iocasia fonsfrigidae strain SP3-1 endoglucanase.

Recently, we reported the discovery of a novel endoglucanase of the glycoside hydrolase family 12 (GH12), designated IfCelS12A, from the haloalkaliphilic anaerobic bacterium Iocasia fonsfrigidae strain SP3-1, which was isolated from a hypersaline pond in the Samut Sakhon province of Thailand (ca. 2017). IfCelS12A exhibits high substrate specificity on carboxymethyl cellulose and amorphous cellulose but low substrate specificity on b-1,3;1,4-glucan. Unlike some endoglucanases of the GH12 family, IfCelS12A does not exhibit hydrolytic activity on crystalline cellulose (i.e., Avicel™). High-Pressure Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) analyses of products resulting from IfCelS12-mediated hydrolysis indicate mode of action for this enzyme. Notably, IfCelS12A preferentially hydrolyzes cellotetraoses, cellopentaoses, and cellohexaoses with negligible activity on cellobiose or cellotriose. Kinetic analysis with cellopentaose and barely b-D-glucan as cellulosic substrates were conducted. On cellopentaose, IfCelS12A demonstrates a 16-fold increase in activity (KM = 0.27 mM; kcat = 0.36 s-1; kcat/KM = 1.34 mM-1 s-1) compared to the enzymatic hydrolysis of barley b-D-glucan (KM: 0.04 mM, kcat: 0.51 s-1, kcat/KM = 0.08 mM-1 s-1). Moreover, IfCelS12A enzymatic efficacy is stable in hypersaline sodium chlorids (NaCl) solutions (up to 10% NaCl). Specifically, IfCel12A retains notable activity after 24 h at 2M NaCl (10% saline solution). IfCelS12A used as a cocktail component with other cellulolytic enzymes and in conjunction with mobile sequestration platform technology offers additional options for deconstruction of ionic liquid-pretreated cellulosic feedstock. KEY POINTS: • IfCelS12A from an anaerobic alkaliphile Iocasia fronsfrigidae shows salt tolerance • IfCelS12A in cocktails with other enzymes efficiently degrades cellulosic biomass • IfCelS12A used with mobile enzyme sequestration platforms enhances hydrolysis.

Acidothermus cellulolyticus E1 endoglucanase expressed in planta undergoes extensive hydroxyproline-O-glycosylation and exhibits enhanced impact on biomass digestibility.

KEY MESSAGE: E1 holoenzyme was extensively Hyp-O-glycosylated at the proline rich linker region in plants, which substantially increased the molecular size and improved the enzymatic digestibility of the biomass of transgenic plants. Thermophilic E1 endo-1,4-ß-glucanase derived from Acidothermus cellulolyticus has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the expressed holoenzyme exhibited a larger molecular size (~ 100 kDa) than the theoretical one (57 kDa), possibly due to posttranslational modifications in the recombinant enzyme within plant cells. This study investigates the glycosylation of the E1 holoenzyme expressed in tobacco plants and determines its impact on enzyme activity and biomass digestibility. The E1 holoenzyme, E1 catalytic domain (E1cd) and E1 linker (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides. Compared with E1cd, the holoenzyme displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. However, the final yield of plant biomass was not significantly affected. Finally, plant biomass engineering with E1 holoenzyme showed 1.7- to 1.8-fold higher saccharification efficiency than the E1cd lines and 2.4- to 2.7-fold higher than the wild-type lines, which was ascribed to the synergetic action of the E1Lk and cellulose binding module in reducing cell wall recalcitrance.

Cultivation of recombinant Aspergillus niger strains on dairy whey as a carbohydrate source.

Agricultural waste valorisation provides a sustainable solution to waste management, and combining waste utilisation with commodity production allows for responsible production processes. Recombinant Aspergillus niger D15 strains expressing fungal endoglucanases (Trichoderma reesei eg1 and eg2 and Aspergillus carneus aceg) were evaluated for their ability to utilise lactose as a carbon source to determine whether dairy waste could be used as a feedstock for enzyme production. The recombinant A. niger D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains produced maximum endoglucanase activities of 34, 54, and 34 U/mL, respectively, on lactose and 23, 27, and 22 U/mL, respectively, on whey. The A. niger D15[eg2]PyrG strain was used to optimise the whey medium. Maximum endoglucanase activity of 46 U/mL was produced on 10% whey medium containing 0.6% NaNO3. The results obtained indicate that dairy whey can be utilised as a feedstock for recombinant enzyme production. However, variations in enzyme activities were observed and require further investigation.

A critical process variable-regulated, parameter-balancing auxostat, performed using disposed COVID-19 personal protective equipment-based substrate mixture, yields sustained and improved endoglucanase titers.

Fifty percent of the overall operational expenses of biorefineries are incurred during enzymatic-saccharification processes. Cellulases have a global-market value of $1621 USD. Dearth of conventional lignocelluloses have led to the exploration of their waste stream-based, unconventional sources. Native fungus-employing cellulase-production batches fail to yield sustained enzyme titers. It could be attributed to variations in the enzyme-production broth's quasi-dilatant behavior, its fluid and flow properties; heat and oxygen transfer regimes; kinetics of fungal growth; and nutrient utilization. The current investigation presents one of the first-time usages of a substrate mixture, majorly comprising disposed COVID-19 personal protective-equipment (PPE). To devise a sustainable and scalable cellulase-production process, various variable-regulated, continuous-culture auxostats were performed. The glucose concentration-maintaining auxostat recorded consistent endoglucanase titers throughout its feeding-cum-harvest cycles; furthermore, it enhanced oxygen transfer, heat transfer co-efficient, and mass transfer co-efficient by 91.5, 36, and 77%, respectively. Substrate-characterization revealed that an unintended, autoclave-based organsolv pretreatment caused unanticipated increases in endoglucanase titers. The cumulative lab-scale cellulase-production cost was found to be $16.3. The proposed approach is economical, and it offers a pollution-free waste management process, thereby generating carbon credits.

Potential of camel rumen derived Bacillus subtilis and Bacillus velezensis strains for application in plant biomass hydrolysis.

Rumen inhabiting Bacillus species possesses a high genetic potential for plant biomass hydrolysis and conversion to value-added products. In view of the same, five camel rumen-derived Bacillus strains, namely B. subtilis CRN 1, B. velezensis CRN 2, B. subtilis CRN 7, B. subtilis CRN 11, and B. velezensis CRN 23 were initially assayed for diverse hydrolytic activities, followed by genome mining to unravel the potential applications. CRN 1 and CRN 7 showed the highest endoglucanase activity with 0.4 U/ml, while CRN 23 showed high ß-xylosidase activity of 0.36 U/ml. The comprehensive genomic insights of strains resolve taxonomic identity, clusters of an orthologous gene, pan-genome dynamics, and metabolic features. Annotation of Carbohydrate active enzymes (CAZymes) reveals the presence of diverse glycoside hydrolases (GH) GH1, GH5, GH43, and GH30, which are solely responsible for the effective breakdown of complex bonds in plant polysaccharides. Further, protein modeling and ligand docking of annotated endoglucanases showed an affinity for cellotrioside, cellobioside, and ß-glucoside. The finding indicates the flexibility of Bacillus-derived endoglucanase activity on diverse cellulosic substrates. The presence of the butyrate synthesis gene in the CRN 1 strain depicts its key role in the production of important short-chain fatty acids essential for healthy rumen development. Similarly, antimicrobial peptides such as bacilysin and non-ribosomal peptides (NRPS) synthesized by the Bacillus strains were also annotated in the genome. The findings clearly define the role of Bacillus sp. inside the camel rumen and its potential application in various plant biomass utilizing industry and animal health research sectors.

Purification and enzymatic properties of a new thermostable endoglucanase from Aspergillus oryzae HML366.

Aspergillus oryzae HML366 is a newly screened cellulase-producing strain. The endoglucanase HML ED1 from A. oryzae HML366 was quickly purified by a two-step method that combines ammonium sulfate precipitation and strong anion exchange column. SDS-PAGE electrophoresis indicated that the molecular weight of the enzyme was 68 kDa. The optimum temperature of the purified endoglucanase was 60 ℃ and the enzyme activity was stable below 70 ℃. The optimum pH was 6.5, and the enzyme activity was stable at pH between 4.5 and 9.0. The analysis indicated that additional Na+, K+, Ca2+, and Zn2+ reduced the catalytic ability of enzyme to the substrate, but Mn2+ enhanced its catalytic ability to the substrate.The Km and Vmax of the purified endoglucanase were 8.75 mg/mL and 60.24 µmol/min·mg, respectively. In this study, we report for the first time that A. oryzae HML366 can produce a heat-resistant and wide pH tolerant endoglucanase HML ED1, which has potential industrial application value in bioethanol, paper, food, textile, detergent, and pharmaceutical industries.

Novel hyperthermoacidic archaeal enzymes for removal of thermophilic biofilms from stainless steel.

AIMS: To test the efficacy of novel hot/acid hyperthermoacidic enzyme treatments on the removal of thermophilic spore-forming biofilms from stainless steel surfaces. METHODS AND RESULTS: The present study measured the efficacy of hyperthermoacidic enzymes (protease, amylase, and endoglucanase) that are optimally active at low pH (≈3.0) and high temperatures (≈80°C) at removing thermophilic bacilli biofilms from stainless steel (SS) surfaces. Plate counts, spore counts, impedance microbiology, as well as epifluorescence microscopy, and scanning electron microscopy (SEM) were used to evaluate the cleaning and sanitation of biofilms grown in a continuous flow biofilm reactor. Previously unavailable hyperthermoacidic amylase, protease, and the combination of amylase and protease were tested on Anoxybacillus flavithermus and Bacillus licheniformis, and endoglucanase was tested on Geobacillus stearothermophilus. In all cases, the heated acidic enzymatic treatments significantly reduced biofilm cells and their sheltering extracellular polymeric substances (EPS). CONCLUSIONS: Hyperthermoacidic enzymes and the associated heated acid conditions are effective at removing biofilms of thermophilic bacteria from SS surfaces that contaminate dairy plants.

Discovery and characterization of a novel PKD-Fn3 domains containing GH44 endoglucanase from a Tibetan metagenomic library.

AIMS: To explore novel microbial endoglucanases with unique properties derived from extreme environments by using metagenomics approach. METHODS AND RESULTS: A Tibetan soil metagenomic library was applied for screening cellulase-active clones by function-based metagenomics. The candidate genes in the active clones were identified through bioinformatic analyses and heterologously expressed using an Escherichia coli system. The recombinant endoglucanases were purified and characterized using enzyme assays to determine their bioactivities, stabilities, substrate specificities, and other enzymatic properties. A novel endoglucanase gene Zfeg1907 was identified, which consisted of a glycoside hydrolase family 44 (GH44) catalytic domain along with a polycystic kidney disease (PKD) domain and a fibronectin type Ⅲ (Fn3) domain at the C terminal. Recombinant enzyme ZFEG1907 and its truncated mutant ZFEG1907t (ΔPKDΔFn3) were successfully expressed and purified. The two recombinants exhibited catalytic activities toward carboxymethyl cellulose, konjac glucomannan (KGM), and lichenan. Both enzymes had an optimal temperature of 50°C and an optimal pH value of 5.0. The catalytic activities of both recombinant enzymes were promoted by adding Zn2+ and Ca2+ at the final concentration of 10 mM. The Km value of ZFEG1907 was lower, while the kcat/Km value of ZFEG1907 was higher than those of of ZFEG1907t when using carboxymethyl cellulose, KGM, and lichenan as substrates. Structure prediction of two recombinants revealed that PKD-Fn3 domains consisted of a flexible linker and formed a ß-sandwich structure. CONCLUSIONS: A novel endoglucanase ZFEG1907 contained a GH44 catalytic domain and a PKD-Fn3 domain was characterized. The PKD-Fn3 domains were not indispensable for the activity but contributed to the enzyme binding of the polysaccharide substrates as a carbohydrate-binding module (CBM).

Predominant contribution of an endogenous cellulase (OlCel) to the cellulolysis in the digestive system of larvae of banana pseudostem weevil, Odoiporus longicollis (Coleoptera: Curculionidae).

Insects have evolved with effective strategies to utilize cellulose as an energy source by possessing cellulolytic enzymes which can be used as an optimal resource in the bioenergy sector. The study was aimed at evaluating the cellulolytic enzyme in the larval gut of the banana pseudostem weevil, Odoiporus longicollis Olivier (Coleoptera: Curculionidae). Primarily, cellulase activity was localized along the gut, in which the midgut showed the highest activity (2858 U/mg). The thermo-tolerance of cellulase activity was found to be up to 80°C (highest at 60°C), and the enzyme was stable at a pH between 5 and 6. Various concentrations of divalent cations (CaCl2 , MgCl2 , and CuCl2 ) have differential enhancing and inhibitory effects on cellulase activity. The cellulase (OlCel) was purified using anion exchange chromatography. The molecular weight of the cellulase was determined to be 47 kDa. The physicochemical parameters of the purified enzyme were similar to that of enzyme activity of whole gut extract. Mass spectrometry results identified sequence similarities of purified cellulase to the glycosyl hydrolase family 5 (GHF5) family. The gut microbial cellulase activity as exogenous source showed no competence compared with the endogenous activity.

Structure and enzymatic characterization of CelD endoglucanase from the anaerobic fungus Piromyces finnis.

Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (CelD) from Piromyces finnis, a modular, cellulosome-incorporated endoglucanase that possesses three GH5 domains followed by two C-terminal fungal dockerin domains (double dockerin). We present the crystal structures of an apo wild-type CelD GH5 catalytic domain and its inactive E154A mutant in complex with cellotriose at 2.5 and 1.8 Å resolution, respectively, finding the CelD GH5 catalytic domain adopts the (ß/α)8-barrel fold common to many GH5 enzymes. Structural superimposition of the apo wild-type structure with the E154A mutant-cellotriose complex supports a catalytic mechanism in which the E154 carboxylate side chain acts as an acid/base and E278 acts as a complementary nucleophile. Further analysis of the cellotriose binding pocket highlights a binding groove lined with conserved aromatic amino acids that when docked with larger cellulose oligomers is capable of binding seven glucose units and accommodating branched glucan substrates. Activity analyses confirm P. finnis CelD can hydrolyze mixed linkage glucan and xyloglucan, as well as carboxymethylcellulose (CMC). Measured kinetic parameters show the P. finnis CelD GH5 catalytic domain has CMC endoglucanase activity comparable to other fungal endoglucanases with kcat = 6.0 ± 0.6 s-1 and Km = 7.6 ± 2.1 g/L CMC. Enzyme kinetics were unperturbed by the addition or removal of the native C-terminal dockerin domains as well as the addition of a non-native N-terminal dockerin, suggesting strict modularity among the domains of CelD. KEY POINTS: • Anaerobic fungi host a wealth of industrially useful enzymes but are understudied. • P. finnis CelD has endoglucanase activity and structure common to GH5_4 enzymes. • CelD's kinetics do not change with domain fusion, exhibiting high modularity.

A multi-plex protein expression system for production of complex enzyme formulations in Trichoderma reesei.

Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have applied the 2A-peptide multi-gene expression system to co-express four proteins, which include three cellulases: a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a ß-D-glucosidase (BGL1), as well as the enhanced green fluorescent protein (eGFP) marker protein. We designed a new chassis vector, pTrEno-4X-2A, for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each cellulase was assessed using chromogenic substrates, which confirmed the functionality of the enzymes. Expression and activity of these enzymes were proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. An 18-fold differencein protein expression was observed between the first and third genes within the 2A-peptide construct. The availability of this new multi-gene expression and screening tool is expected to greatly impact multi-enzyme applications, such as the production of complex commercial enzyme formulations and metabolic pathway enzymes, especially those destined for cell-free applications.

A unique self-truncation of bacterial GH5 endoglucanases leads to enhanced activity and thermostability.

BACKGROUND: ß-1,4-endoglucanase (EG) is one of the three types of cellulases used in cellulose saccharification during lignocellulosic biofuel/biomaterial production. GsCelA is an EG secreted by the thermophilic bacterium Geobacillus sp. 70PC53 isolated from rice straw compost in southern Taiwan. This enzyme belongs to glycoside hydrolase family 5 (GH5) with a TIM-barrel structure common among all members of this family. GsCelA exhibits excellent lignocellulolytic activity and thermostability. In the course of investigating the regulation of this enzyme, it was fortuitously discovered that GsCelA undergoes a novel self-truncation/activation process that appears to be common among GH5 enzymes. RESULTS: Three diverse Gram-positive bacterial GH5 EGs, but not a GH12 EG, undergo an unexpected self-truncation process by removing a part of their C-terminal region. This unique process has been studied in detail with GsCelA. The purified recombinant GsCelA was capable of removing a 53-amino-acid peptide from the C-terminus. Natural or engineered GsCelA truncated variants, with up to 60-amino-acid deletion from the C-terminus, exhibited higher specific activity and thermostability than the full-length enzyme. Interestingly, the C-terminal part that is removed in this self-truncation process is capable of binding to cellulosic substrates of EGs. The protein truncation, which is pH and temperature dependent, occurred between amino acids 315 and 316, but removal of these two amino acids did not stop the process. Furthermore, mutations of E142A and E231A, which are essential for EG activity, did not affect the protein self-truncation process. Conversely, two single amino acid substitution mutations affected the self-truncation activity without much impact on EG activities. In Geobacillus sp. 70PC53, the full-length GsCelA was first synthesized in the cell but progressively transformed into the truncated form and eventually secreted. The GsCelA self-truncation was not affected by standard protease inhibitors, but could be suppressed by EDTA and EGTA and enhanced by certain divalent ions, such as Ca2+, Mg2+, and Cu2+. CONCLUSIONS: This study reveals novel insights into the strategy of Gram-positive bacteria for directing their GH5 EGs to the substrate, and then releasing the catalytic part for enhanced activity via a spontaneous self-truncation process.

A Large-Scale Hydroponic Evaluation of Rice Mutants for Pythium Resistance.

Rice is a major staple crop worldwide. However, the occurrence of rice diseases during cultivation poses a significant challenge to achieving optimal yields. Among the major pathogens, Pythium species, which cause seedling blight, are of particular concern. Pythium infects rice roots through zoospores, mycelia, or oospores, leading to root rot, stunting, yellowing, and ultimately seedling damping-off. While many disease resistance-related genes have been reported in rice, only very limited research has been associated with resistance to Pythium infection. In this study, we aimed to establish a rapid screening system to identify rice lines that are resistant or susceptible to Pythium pathogen in rice nurseries. We conducted evaluations on important factors, including virulence, inoculation method, seed soaking period, and the measurement of disease severity. As a result, we successfully developed a screening system that allows for high-throughput and rapid screening of the Taiwan Rice Insertional Mutant (TRIM) library for mutant lines exhibiting resistance to P. arrhenomanes. Furthermore, we identified a slightly resistant TRIM line and explored potential genes encoding endglucanase-1 precursor and malonyl-CoA decarboxylase that may be involved in conferring resistance to P. arrhenomanes.

Biological pretreatment of sugarcane bagasse for the production of fungal laccase and bacterial cellulase.

Sugarcane bagasse (SB) is a promising source of appreciable quantities of fermentable sugars. However, the presence of lignin hinders utilization of these carbohydrates and hence pretreatment to remove lignin is necessarily carried out. Here, a biological pretreatment method was synchronized with the production of a thermostable cellulase using SB as a raw material. Initially, bagasse was fermented by a laccase producing fungus, Trametes pubescens MB 89 under solid state fermentation (SSF) and a titer of 1758 IU mL-1 of laccase was obtained. Investigations of nine factors affecting laccase production through Plackett Burman design improved the titers to 6539 IU mL-1 . Five factors (incubation period, concentration of CuSO4 , temperature, moisture content, and particle size) were found significant which were optimized through Central Composite design leading to an improvement in the titers by ~5 folds (8841 IU mL-1 ). Biologically pretreated SB was fermented by a thermophilic bacterium, Neobacillus sedimentimangrovi UE25, that yielded 8.64 IU mL-1 of cellulase. Delignification and cellulose utilization were affirmed by structural analysis through FTIR and SEM. The synchronized process yielded higher titers of laccase and cellulase under SSF of SB with the minimum use of corrosive chemicals.

Multipurpose cellulases of Promicromonospora sp. VP111, with broad substrate specificity and tolerance properties.

Cellulolytic actinobacterium, Promicromonospora sp. VP111 concomitantly produced cellulases (CELs), xylanase and pectinase when grown on commercial cellulose and untreated agricultural lignocellulosic residues (wheat straw and sugarcane bagasse). Secreted CELs hydrolyzed (enhanced with Co2+ ion) multiple cellulosic substrates, including sodium carboxymethyl cellulose (Na-CMC), Whatman filter paper no. 1, microcrystalline cellulose (avicel), p-nitrophenyl-ß-D-glucopyranoside (pNPG), laminarin, and cellulose powder. The CELs showed stabilities in the presence of various chemicals, including glucose (0.2 M), detergents (1%, w/v or v/v), denaturants (1%, w/v or v/v), and sodium chloride (NaCl, 30%, w/v). The CELs were fractionated using ammonium sulfate precipitation and dialysis. Activities (%) of fractionated CELs were retained at 60°C for endoglucanase/carboxymethyl cellulase (CMCase) (88.38), filter paper cellulase (FPase) (77.55), and ß-glucosidase (90.52), which indicated of thermo-stability. Similarly, the activities (%) for CMCase (85.79), FPase (82.48), and ß-glucosidase (85.92) at pH 8.5 indicated of alkaline-stability. Kinetic factors, Km and Vmax for endoglucanase component of fractionated CELs were 0.014 g/l and 158.23 µM glucose/min/mL, respectively. Fractionated CELs yielded activation energies (kJ/mol) of 17.933, 6.294, and 4.207 for CMCase, FPase, and ß-glucosidase activities, respectively in linear thermostable Arrhenius plots. Thus, this study reports on the multipurpose CELs from an untreated agricultural residue utilizing Promicromonospora in relation to broad substrate specificity, halo-tolerance, alkaline-tolerance, detergent-tolerance, thermo-tolerance, organic solvent-tolerance, and end product-tolerance.

Optimisation of ß-Glucosidase Production in a Crude Aspergillus japonicus VIT-SB1 Cellulase Cocktail Using One Variable at a Time and Statistical Methods and its Application in Cellulose Hydrolysis.

Pulp and paper mill sludge (PPMS) is currently disposed of into landfills which are reaching their maximum capacity. Valorisation of PPMS by enzymatic hydrolysis using cellulases is an alternative strategy. Existing commercial cellulases are expensive and contain low titres of ß-glucosidases. In this study, ß-glucosidase production was optimised by Aspergillus japonicus VIT-SB1 to obtain higher ß-glucosidase titres using the One Variable at a Time (OVAT), Plackett Burman (PBD), and Box Behnken design (BBD)of experiments and the efficiency of the optimised cellulase cocktail to hydrolyse cellulose was tested. ß-Glucosidase production was enhanced from 0.4 to 10.13 U/mL, representing a 25.3-fold increase in production levels after optimisation. The optimal BBD production conditions were 6 days of fermentation at 20 °C, 125 rpm, 1.75% soy peptone, and 1.25% wheat bran in (pH 6.0) buffer. The optimal pH for ß-glucosidase activity in the crude cellulase cocktail was (pH 5.0) at 50 °C. Optimal cellulose hydrolysis using the crude cellulase cocktail occurred at longer incubation times, and higher substrate loads and enzyme doses. Cellulose hydrolysis with the A. japonicus VIT-SB1 cellulase cocktail and commercial cellulase cocktails resulted in glucose yields of 15.12 and 12.33 µmol/mL glucose, respectively. Supplementation of the commercial cellulase cocktail with 0.25 U/mg of ß-glucosidase resulted in a 19.8% increase in glucose yield.

Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies.

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process.

Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis.

Zymomonas mobilis (Z. mobilis) is a potential candidate strain for consolidated bioprocessing (CBP) in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion that is affected by cell wall peptidoglycan is also not well understood. Here, we constructed several penicillin-binding protein (PBP)-deficient strains derived from Z. mobilis S192 to perturb the cell wall peptidoglycan network and then investigated the effects of peptidoglycan on the endoglucanase secretion. The results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBP mutant strains, notably, Δ1089/0959 (4.09-fold) and Δ0959 (5.76-fold) in comparison to parent strains. For PBP-deficient strains, the growth performance was not significantly inhibited, but cell morphology was altered. In addition, enhanced antibiotic sensitivity and reduced inhibitor tolerance were also detected in our study. The concentration of intracellular soluble peptidoglycan was increased, especially for single-gene deletion. Outer membrane permeability of PBP-deficient strains was also improved, notably, Δ1089/0959 (1.14-fold) and Δ0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our findings indicated that PBP-deficient Z. mobilis was capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance, and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness and acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacteria. Herein, we perturbed the peptidoglycan synthesis network via knocking out PBPs (ZMO0197, ZMO0959, ZMO1089) to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study could lay the foundation for understanding the regulatory network of cell wall synthesis and guide the construction of CBP strains in Z. mobilis.

Coevolutionary analysis reveals a distal amino acid residue pair affecting the catalytic activity of GH5 processive endoglucanase from Bacillus subtilis BS-5.

EG5C-1, processive endoglucanase from Bacillus subtilis, is a typical bifunctional cellulase with endoglucanase and exoglucanase activities. The engineering of processive endoglucanase focuses on the catalytic pocket or carbohydrate-binding module tailoring based on sequence/structure information. Herein, a computational strategy was applied to identify the desired mutants in the enzyme molecule by evolutionary-coupling analysis; subsequently, four residue pairs were selected as evolutionary mutational hotspots. Based on iterative-saturation mutagenesis and subsequent enzymatic activity analysis, a superior mutant K51T/L93T has been identified away from the active center. This variant had increased specific activity from 4170 U/µmol of wild-type (WT) to 5678 U/µmol towards carboxymethyl cellulose-Na and an increase towards the substrate Avicel from 320 U/µmol in WT to 521 U/µmol. In addition, kinetic measurements suggested that superior mutant K51T/L93T had a high substrate affinity (Km ) and a remarkable improvement in catalytic efficiency (kcat /Km ). Furthermore, molecular dynamics simulations revealed that the K51T/L93T mutation altered the spatial conformation at the active site cleft, enhancing the interaction frequency between active site residues and substrate, and improving catalytic efficiency and substrate affinity. The current studies provided some perspectives on the effects of distal residue substitution, which might assist in the engineering of processive endoglucanase or other glycoside hydrolases.