Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Structural insights and functional characterization of a novel ß-glucosidase derived from Thermotoga profunda.

ß-Glucosidase is a crucial cellulase, as its activity determines the efficiency of cellulose hydrolysis into glucose. This study addresses the functional and structural characteristics of Thermotoga profunda ß-glucosidase (Tp-BGL). Tp-BGL exhibited a Km of 0.3798 mM for p-nitrophenyl-ß-d-glucopyranoside (pNPGlc) and 4.44 mM for cellobiose, with kcat/Km of 1211.16 and 4.18 s-1 mM-1, respectively. In addition, Tp-BGL showed significant pH adaptability and thermal stability, with a Tm of 85.7 °C and retaining >90 % of its activity after incubation at 80 °C for 90 min. The crystal structure of Tp-BGL was resolved at 1.95 Å resolution, and reveals a typical TIM barrel structure. Comparative structural analysis highlighted that the major distinction between Tp-BGL and the other glucosidases lies in their loop regions.

Glycosylation of cellulase: a novel strategy for improving cellulase.

Protein glycosylation is the most complex posttranslational modification process. Most cellulases from filamentous fungi contain N-glycosylation and O-glycosylation. Here, we discuss the potential roles of glycosylation on the characteristics and function of cellulases. The use of certain cultivation, inducer, and alteration of engineering glycosylation pathway can enable the rational control of cellulase glycosylation. Glycosylation does not occur arbitrarily and may tend to modify the 3D structure of cellulases by using specially distributed glycans. Therefore, glycoengineering should be considered comprehensively along with the spatial structure of cellulases. Cellulase glycosylation may be an evolution phenomenon, which has been considered as an economical way for providing different functions from identical proteins. In addition to gene and transcription regulations, glycosylation may be another regulation on the protein expression level. Enhanced understanding of the potential regulatory role of cellulase glycosylation will enable synthetic biology approaches for the development of commercial cellulase.

Specific activity and utility of recombinant cellobiohydrolase II (Cel6A) produced in maize endosperm.

Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed. The transgenic line recovered from tissue culture was bred into male and female elite Stine inbred corn lines, stiff stalk 16083-025 (female) and Lancaster MSO411 (male), for future production in their hybrid. The enzyme increases its accumulation throughout its 6 generations of back crosses, 27-266-fold between T1 and T2, and 2-10-fold between T2 and T3 generations with lesser increases in T4-T6. The germplasm of the inbred lines replaces the tissue culture corn variety germplasm with each generation, with the ultimate goal of producing a high-yielding hybrid with the transgene. The CBH II enzyme was purified from T5 inbred male grain 10-fold to homogeneity with 47.5% recovery. The specific activity was determined to be 1.544 units per µg protein. The corn-derived CBH II works in biopolishing of cotton by removing surface fibers to improve dyeability and increasing glucose from corn flour for increasing ethanol yield from starch-based first-generation processes.

Effect of cellulose reducing ends and primary hydroxyl groups modifications on cellulose-cellulase interactions and cellulose hydrolysis.

Cellulose reducing ends are believed to play a vital role in the cellulose recalcitrance to enzymatic conversion. However, their role in insoluble cellulose accessibility and hydrolysis is not clear. Thus, in this study, reducing ends of insoluble cellulose derived from various sources were modified by applying reducing and/or oxidizing agents. The effects of cellulose reducing ends modification on cellulose reducing ends, cellulose structure, and cellulose accessibility to cellulase were evaluated along with the impact on cellulose hydrolysis with complete as well purified cellulase components. Sodium borohydride (NaBH4) reduction and sodium chlorite-acetic acid (SC/AA) oxidation were able to modify more than 90% and 60% of the reducing ends, respectively, while the bicinchoninic acid (BCA) reagent applied for various cycles oxidized cellulose reducing ends to various extents. X-ray diffractograms of the treated solids showed that these treatments did not change the cellulose crystalline structure and the change in crystallinity index was insignificant. Surprisingly, it was found that the cellulose reducing ends modification, either through selective NaBH4 reduction or BCA oxidation, had a negligible impact on cellulose accessibility as well on cellulose hydrolysis rates or final conversions with complete cellulase at loadings as low as 0.5 mg protein/g cellulose. In fact, in contrast to what is traditionally believed, modifications of cellulose reducing ends by these two methods had no apparent impact on cellulose conversion with purified cellulase components and their synergy. However, SC/AA oxidation resulted in significant drop in cellulose conversion (10%-50%) with complete as well purified cellulase components. Nonetheless, further research revealed that the cause for drop in cellulose conversion for the SC/AA oxidation case was due to primary hydroxyl groups (PHGs) oxidation and not the oxidation of reducing ends. Furthermore, it was found that the PHGs modification affects cellulose accessibility and slows the cellulase uptake as well resulting in significant drop in cellulose conversions.

An extracellular glucose sensor for substrate-dependent secretion and display of cellulose-degrading enzymes.

The efficient hydrolysis of lignocellulosic biomass into fermentable sugars is key for viable economic production of biofuels and biorenewable chemicals from second-generation feedstocks. Consolidated bioprocessing (CBP) combines lignocellulose saccharification and chemical production in a single step. To avoid wasting valuable resources during CBP, the selective secretion of enzymes (independent or attached to the surface) based on the carbon source available is advantageous. To enable enzyme expression and secretion based on extracellular glucose levels, we implemented a G-protein-coupled receptor (GPCR)-based extracellular glucose sensor; this allows the secretion and display of cellulases in the presence of the cellulosic fraction of lignocellulose by leveraging cellobiose-dependent signal amplification. We focused on the glucose-responsiveness of the HXT1 promoter and engineered PHXT1 by changing its core to that of the strong promoter PTHD3 , increasing extracellular enzyme activity by 81%. We then demonstrated glucose-mediated expression and cell-surface display of the ß-glucosidase BglI on the surface of Saccharomyces cerevisiae. The display system was further optimized by re-directing fatty acid pools from lipid droplet synthesis toward formation of membrane precursors via knock-out of PAH1. This resulted in an up to 4.2-fold improvement with respect to the baseline strain. Finally, we observed cellobiose-dependent signal amplification of the system with an increase in enzymatic activity of up to 3.1-fold when cellobiose was added.

Caatinga, Amazon and Atlantic Forest as natural sources for microbial lignocellulolytic enzymes.

Brazilian biomes are important sources for environmental microorganisms, including efficient metabolic machineries, like actinomycetes. These bacteria are known for their abilities to produce many bioactive compounds, including enzymes with multiple industrial applications. The present work aimed to evaluate lignocellulolytic abilities of actinomycetes isolated from soil and rhizosphere samples collected at Caatinga, Atlantic and Amazon Forest. Laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and cellulase were evaluated for their efficiency. These enzymes have an essential role in lignin decomposition, through oxidation of phenolic and non-phenolic compounds, as well as enzymatic hydrolysis of vegetal biomass. In this sense, a total of 173 actinomycetes were investigated. Eleven (11) of them were selected by their enzymatic performance. The actinomycete AC166 displayed some activity in all analysed scenarios in terms of Lac, MnP and LiP activity, while AC171 was selected as the most promising strain, showing the following activities: 29.7 U.L-1 for Lac; 2.5 U.L-1 for LiP and 23 U.L-1 for MnP. Cellulolytic activities were evaluated at two pH conditions, 4.8 and 7.4, obtaining the following results: 25 U.L-1 and 71 U.L-1, respectively. Thermostability (4, 30 and 60 o C) and salinity concentrations (0 to 4 M) and pH variation (2.0 to 9.0) stabilities of the obtained LiP and Lac enzymatic extracts were also verified. The actinomycete strain AC171 displayed an adaptable response in distinct pH and salt profiles, indicating that bacterial LiP was some halophilic type. Additionally, the strain AC149 produced an alkali and extreme halophilic lignin peroxidase, which are promising profiles for their future application under lignocellulosic biomass at bioethanol biorefineries.

Metabolomics-based study of chemical compositions in cellulase additives derived from a tobacco-origin Bacillus subtilis and their impact on tobacco sensory attributes.

To enhance the quality of tobacco leaves and optimize the smoking experience, diverse strains of functional bacteria and their associated metabolites have been used in tobacco aging. Exogenous cellulase additives are frequently employed to facilitate the degradation of cellulose and other macromolecular matrices and enhance the quality of the tobacco product. However, little is known about how microbial metabolites present in exogenous enzyme additives affect tobacco quality. In this study, crude cellulase solutions, produced by a tobacco-originating bacterium Bacillus subtilis FX-1 were employed on flue-cured tobacco. The incorporation of cellulase solutions resulted in the reduction of cellulose crystallinity in tobacco and the enhancement of the overall sensory quality of tobacco. Notably, tobacco treated with cellulase obtained from laboratory flask fermentation demonstrated superior scent and flavor attributes in comparison to tobacco treated with enzymes derived from industrial bioreactor fermentation. The targeted and untargeted metabolomic analysis revealed the presence of diverse flavor-related precursors and components in the cellulase additives, encompassing sugars, alcohols, amino acids, organic acids, and others. The majority of these metabolites exhibited significantly higher levels in the flask group compared to the bioreactor group, probably contributing to a pronounced enhancement in the sensory quality of tobacco. Our findings suggest that the utilization of metabolic products derived from B. subtilis FX-1 as additives in flue-cured tobacco holds promise as a viable approach for enhancing sensory attributes, establishing a solid theoretical foundation for the potential development of innovative tobacco aging additives.

Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

Cloning and expression of Neurospora crassa cellobiohydrolase II in Pichia pastoris.

A major cellobiohydrolase of Neurospora crassa CBH2 was successfully expressed in Pichia pastoris. The maximum Avicelase activity in shake flask among seven transformants which selected on 4.0 g/L G418 plates was 0.61 U/mL. The optimal pH and temperature for Avicelase activity of the recombinant CBH2 were determined to be 4.8 and 60 °C, respectively. The new CBH2 maintained 63.5 % Avicelase activity in the range of pH 4.0-10.4, and 60.2 % Avicelase activity in the range of 30-90 °C. After incubation at 70-90 °C for 1 h, the Avicelase activity retained 60.5 % of its initial activity. The presence of Zn2+, Ca2+ or Cd2+ enhanced the Avicelase activity of the CBH2, of which Cd2+ at 10 mM causing the highest increase. The recombinant CBH2 was used to enhance the Avicel hydrolysis by improving the exo-exo-synergism between CBH2 and CBH1 in N.crassa cellulase. The enzymatic hydrolysis yield was increased by 38.1 % by adding recombinant CBH2 and CBH1, and the yield was increased by 215.4 % when the temperature is raised to 70 °C. This work provided a CBH2 with broader pH range and better heat resistance, which is a potential enzyme candidate in food, textile, pulp and paper industries, and other industrial fields.

Mass production and characterization of an endoglucanase from Coleoptera insect (Monochamus saltuarius) in yeast Kluyveromyceslactis.

To harness the diverse industrial applications of cellulase, including its use in the food, pulp, textile, agriculture, and biofuel sectors, this study focused on the high-yield production of a bioactive insect-derived endoglucanase, Monochamus saltuarius glycoside hydrolase family 5 (MsGHF5). MsGHF5 was introduced into the genome of Kluyveromyces lactis to maintain expression stability, and mass production of the enzyme was induced using fed-batch fermentation. After 40 h of cultivation, recombinant MsGHF5 was successfully produced in the culture broth, with a yield of 29,000 U/L, upon galactose induction. The optimal conditions for the activity of purified MsGHF5 were determined to be a pH of 5 and a temperature of 35 °C, with the presence of ferrous ions enhancing the enzymatic activity by up to 1.5-fold. Notably, the activity of MsGHF5 produced in K. lactis was significantly higher than that produced in Escherichia coli, suggesting that glycosylation is crucial for the functional performance of the enzyme. This study highlights the potential use of K. lactis as a host for the production of bioactive MsGHF5, thus paving the way for its application in various industrial sectors.

Influence of coloured lights on growth and enzyme production of beneficial endophytic fungi.

The influence of light regulation on fungal growth and enzyme production was tested on endophytic isolates of Fusarium proliferatum (CCH), Colletotrichum boninense (PL1, PL9, OL2), Colletotrichum gloeosporiodes (OL3) and Colletotrichum siamense (PL3). The isolates were treated with blue, red, green, and yellow light, while white fluorescent light (12 h light/12 h dark photoperiod) and 24 h dark conditions were applied as control. Results revealed that coloured light treatments induced formation of circadian rings, while exposure to white light and dark conditions showed less pronounced circadian rings. Growth and sporulation of endophytes were not significantly influenced by light. By contrast, enzyme production was affected by coloured light treatments, notably with red (amylase), blue (cellulase) and yellow (cellulase, xylanase, L-asparaginase) light, resulting in lower enzyme levels for certain isolates. Under control conditions, enzyme production was relatively higher for amylase, cellulase, xylanase (for cultures incubated in the dark), and for L-asparaginase (for cultures incubated in white fluorescent light). Among the endophytic isolates, F. proliferatum (CCH) showed better response to coloured light treatment as higher sporulation and enzyme production was detected, although growth was significantly suppressed. On the contrary, C. gloeosporiodes (OL3) showed better growth but significantly lower enzyme production and sporulation when treated with the various coloured light. This study revealed that coloured light may have the potential to manipulate growth, sporulation and enzyme production in certain fungal species as strategies for fungal control or for harnessing of valuable enzymes.

C-terminus of serine-arginine protein kinase-like protein, SrpkF, is involved in conidiophore formation and hyphal growth under salt stress in Aspergillus aculeatus.

The serine-arginine protein kinase-like protein, SrpkF, was identified as a regulator for the cellulose-responsive induction of cellulase genes in Aspergillus aculeatus. To analyze various aspects of SrpkF function, we examined the growth of the control strain (MR12); C-terminus deletion mutant, which produced SrpkF1-327 (ΔCsrpkF); whole gene-deletion mutant of srpkF (ΔsrpkF), srpkF overexpressing strain (OEsprkF); and the complemented strain (srpkF+) under various stress conditions. All test strains grew normally on minimal medium under control, high salt (1.5 M KCl), and high osmolality (2.0 M sorbitol and 1.0 M sucrose). However, only ΔCsrpkF showed reduced conidiation on 1.0 M NaCl media. Conidiation of ΔCsrpkF on 1.0 M NaCl media was reduced to 12% compared with that of srpkF+. Further, when OEsprkF and ΔCsrpkF were pre-cultured under salt stress conditions, germination under salt stress conditions was enhanced in both strains. By contrast, deletion of srpkF did not affect hyphal growth and conidiation under the same conditions. We then quantified the transcripts of the regulators involved in the central asexual conidiation pathway in A. aculeatus. The findings revealed that the expression of brlA, abaA, wetA, and vosA was reduced in ΔCsrpkF under salt stress. These data suggest that in A. aculeatus, SrpkF regulates conidiophore development. The C-terminus of SrpkF seems to be important for regulating SrpkF function in response to culture conditions such as salt stress.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity.

BACKGROUND: Lignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process. RESULTS: The current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities. CONCLUSION: The results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Green ecofriendly enhancement of cellulase productivity using agricultural wastes by Aspergillus terreus MN901491: statistical designs and detergent ability on cotton fabrics.

BACKGROUND: Cellulase is considered a group member of the hydrolytic enzymes, responsible for catalyzing the hydrolysis of cellulose and has various industrial applications. Agricultural wastes are used as an inexpensive source for several utilizable products throughout the world. So, searching for cellulase enzymes from fungal strains capable of utilizing agricultural wastes to increase productivity, reduce costs and overcome waste accumulation in the environment is very important to evaluate its potency as a bio-additive to detergent agents. RESULTS: In the current study, the previously identified fungal strain Aspergillus terreus MN901491 was screened and selected for cellulase production. Medium parameters were optimized using one-factor-at-a-time (OFAT) and multi-factorial (Plackett-Burman and Box-Behnken) design methods. OFAT showed the ability of the fungal strain to utilize agricultural wastes (corn cob and rice straw) as a substrate. Also, yeast extract was the best nitrogen source for enhancing cellulase productivity. The most significant variables were determined by Plackett-Burman Design (PBD) and their concentrations were optimized by Response Surface Methodology (RSM) using Box-Behnken Design (BBD). Among eleven independent variables screened by PBD, malt extract, (NH4)2SO4, and KCl were the most significant ones followed by rice straw which affected cellulase production positively. The ANOVA results particularly the R2-value of PBD (0.9879) and BBD (0.9883) confirmed the model efficiency and provided a good interpretation of the experiments. PBD and BBD improved cellulase productivity by 6.1-fold greater than that obtained from OFAT. Medium optimization using OFAT and statistical models increased cellulase production from A. terreus MN901491 by 9.3-fold compared to the non-optimized medium. Moreover, the efficiency of cellulase activity on cotton fabrics as a bio-additive detergent was evaluated and estimated using whiteness and scanning electron microscope (SEM) that affirmed its potential effect and remarkable detergent ability to improve whiteness by 200% in comparison with non-washed fabric and by 190% in comparison with fabric washed by water. CONCLUSION: The presented work was stabilized as a multi-efficiency in which wastes were used to produce cellulase enzyme from the fungal strain, Aspergillus terreus MN901491 as a bio-additive to detergent applications that involved ecofriendly and green processes.

Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Aspergillus awamori MK788209 cellulase: production, statistical optimization, pea peels saccharification and textile applications.

BACKGROUND: The demand for low-cost cellulolytic enzyme synthesis is rising in the enzyme market. This work aims to produce cellulase by utilizing various agricultural wastes and investigating the use of enzyme in saccharification and textile industries. RESULTS: Solid state fermentation (SSF) was applied to produce industrial enzymes, particularly cellulase, through utilizing Molokhia (Corchorus olitorius) stems by Aspergillus awamori MK788209 isolate. Two stages of statistical factorial designs Plackett-Burman (PB) and Central Composite Design (CCD) were applied to enhance the A. awamori MK788209 cellulase production from Molokhia stems (MS). The fold increase of enzyme production by PB followed by CCD was 2.51 and 4.86, respectively. Additionally, the A. awamori MK788209 culture filtrate was highly effective in saccharifying various agricultural wastes, particularly pea peels (PP) (yielding 98.33 mg reducing sugar/ml), due to its richness in cellulase, laccase, xylanase, pectinase, and amylase. By optimizing the three main variables; pea peel weight, culture filtrate volume added, and saccharification time by CCD, the sugar recovery from PP was enhanced, leading to a 3.44-fold increase in reducing sugar recovery (338 mg reducing sugar /ml). Furthermore, the A. awamori MK788209 culture filtrate showed high efficacy in textile applications, enhancing the roughness, weight loss, white index, and printing capability of treated cotton fabrics. CONCLUSIONS: A. Awamori MK788209 produced cellulase which was effective in PP saccharification. The enzyme was also capable of enhancing cotton fabric properties.

Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations.

BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.

An overview on the current status and future prospects in Aspergillus cellulase production.

Cellulase is a new research point besides glucoamylase, amylase, and protease in the enzyme industry. Cellulase can decompose lignocellulosic biomass into small-molecule sugars, which facilitates microbial utilization; thus, it has a vast market potential in the field of feed, food, energy, and chemistry. The Aspergillus was the first strain used in cellulase preparation because of its safety and non-toxicity, strong growth ability, and high enzyme yield. This review provides the latest research and advances on preparing cellulase from Aspergillus. The metabolic mechanisms of cellulase secretion by Aspergillus, the selection of fermentation substrates, the comparison of the fermentation modes, and the effect of fermentation conditions have been discussed in this review. Also, the subsequent separation and purification techniques of Aspergillus cellulase, including salting out, organic solvent precipitation, ultrafiltration, and chromatography, have been declared. Further, bottlenecks in Aspergillus cellulase preparation and corresponding feasible approaches, such as genetic engineering, mixed culture, and cellulase immobilization, have also been proposed in this review. This paper provides theoretical support for the efficient production and application of Aspergillus cellulase.

Production of cello-oligosaccharides from corncob residue by degradation-synthesis reactions.

The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3 g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. KEY POINTS: • Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. • Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. • Spraying cello-oligosaccharides promoted tomato growth.