Cellulase Yield Research Articles

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

BackgroundIn 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.ResultsThe 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.ConclusionsOptimization 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.

Bimolecular optimization of cellulase production by <i>Richoderma citrinoviride</i> and <i>Aspergillus niger</i> isolates on corn cob, rice bran and sorghum bran as biomass substrates

This work focuses on the assessment of the conditions relevant for the improvement of enzymes hydrolysis of pretreated corn cob, rice bran and sorghum bran by using Trichoderma Citrinoviride and Aspergillus niger. To achieve this, different fermentation conditions were applied to assess their effect in the optimization of cellulase production. Effects of fermentation duration, inoculation size, temperature and pH of fermentation on cellulase production were investigated. At 96 hrs of fermentation, maxim cellusale product was found to be at optimum in both organisms. Hence, 6% substrates concentration with 10 discs of 8mm inoculum size yielded maximum cellulase production in both A. niger and T. citrinoviride after 5 days of incubation. At 35ºC, A niger and T citrinoviride recorded maximum cellulase production 0.50 mg/ml in sorghum bran while 40ºC was optimum for maximum cellulase production for T, citrinoviride on corn cob. Whereas, pH 5.0 T. citrinoviride exhibits maximum cellulase production with sorghum bran 1.30 mg/ml compared to carboxymetyyl cellulose which served as control and sorghum bran 1.2mg/ml. These results highlight the potentials of T. citrinoviride as species of fungus for the industrial production of cellulase using Agricultural wastes as substrates. Cellulase yield was repressed in the presence of glucose and was induced in the presence of corn cob, rice bran and sorghum using T. citrinoviride. Cellulase yield from Corn cob, rice bran and sorghum bran differed significantly at (P<0.05) from glucose.

Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum

BackgroundLow-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain.ResultsWe began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum.ConclusionsOur study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion.

A thermo-tolerant cellulase enzyme produced by Bacillus amyloliquefaciens M7, an insight into synthesis, optimization, characterization, and bio-polishing activity

Abstract In the current study, among 36 isolates, the bacterial strain M7 was selected as the highest cellulase producer and underwent traditional and molecular identification as Bacillus amyloliquefaciens M7. The productivity of the cellulase enzyme was optimized using the one-factor-at-a-time method. The optimization analysis showed that the best pH value for cellulase production was 7, in the presence of 1% bacterial inoculum size, 5 g·L−1 of carboxymethyl cellulose, 5 g·L−1 of peptone as nitrogen source, and incubation period of 24 h at a temperature of 35°C. The highest cellulase activity (64.98 U·mL−1) was obtained after optimizing conditions using BOX-Behnken Design. The maximum cellulase yield (75.53%) was obtained after precipitation by 60% ammonium sulfate, followed by purification by dialysis bag and Sephadex G-100 column chromatography. The purified cellulase enzyme was characterized by 6.38-fold enrichment, with specific activity (60.54 U·mg−1), and molecular weight of approximately 439.0 Da. The constituent of purified cellulase was 18 amino acids with high concentrations of 200 and 160 mg·L−1 for glycine and arginine, respectively. The purified cellulase enzyme was more stable and active at pH 8 and an incubation temperature of 50°C. The metal ions CaCl2, NaCl, and ZnO enhanced the activity of purified cellulase enzyme. Finally, the B. amyloliquefaciens M7-cellulase exhibits high bio-polishing activity of cotton fabrics with low weight loss (4.3%) which was attained at a maximum concentration (1%, v/v) for 90 min.

Bioprocess development for extraction and purification of cellulases from Aspergillus niger 3ASZ using statistical experimental design techniques

The amount of cellulosic materials is large and may lead to environmental pollution, so they can be converted into useful materials for use in food or energy. Statistical design (Plackett–Burman and Box-Behnken) was the main topic of this study and was used to optimize the effect of environmental factors on cellulase production by Aspergillus niger. Cellulase production using Plackett–Burman was 6.86-fold higher than the production of cellulase using the basal medium. B0X-Benken showed an increase in the cellulase production equal to 18 times compared to the basal medium, where the cellulase produced had an activity equal to 79.4 U/mL/min. Ammonium sulfate precipitation was applied to the crude enzyme, followed by sequential fractionation with an Amicon system. The Amicon was used to demonstrate the final volume, total enzyme activity, specific activity, purification fold, and yield of cellulase (partially purified enzyme). Numerous cellulolytic enzymes are abundant in Aspergillus species. All of the data showed that Aspergillus sp. might be a reliable source of industrially and economically useful cellulases. By statistically calculating the relevance of a large number of elements in one experiment using a multifactorial statistical design, time may be saved while still maintaining the validity of each component.

Cellulase production from disposed COVID-19 personal protective equipment (PPE) using cyclic fed-batch strategies

Wild fungal strain-employing, cellulase production bio-processes require 17-fold higher quantities of lignocelluloses than biofuel production. Globally-available, conventional lignocelluloses are 3 × 103-fold lesser than their requirement for bio-alcohol-fossil fuel blending purposes. In an attempt to explore biomass alternatives, demonstrate their potential in cellulase production, and to simultaneously-address process-deterring conditions of solid-state and submerged fermentations, cellulose-rich, COVID-19 personal protective equipment-based substrates were used in time-extended, empirical data-driven, optimal condition-maintaining cyclic fed-batch strategies. While 37% and 43% increases of cumulative cellulase yields were recorded for solid and submerged cyclic operations, respectively, they were 21% and 28% more economical than multiple, individual batches. Furthermore, the operations improved kinetics of fungal growth, oxygen transfer, nutrient utilization, and maintained optimal parameter balance between variables, thereby improving consistency in cellulase titers. Apart from being sustainable, the demonstration suggests a recycle route for managing such health hazard-posing wastes, in turn alleviating environmental pollution and earning carbon credits.

Economic assessment of cellulase production in batch and semi-batch solid-state fermentation processes

AbstractThe vast range of applications for enzymes in the production of biofuels has led to a major increase in market demand in recent years. Cellulase's high cost and poor catalytic efficiency are two key barriers to the industrial usage of lignocellulosic biomass as feedstocks for fuels in biorefineries. In this study, the economic evaluation of cellulase production in solid-state fermentation was done by designing two process routes (batch and semi-batch) to optimize the process. SuperPro Designer Software has been used to create a process model that assessed the economics of fermentation operations while Monte Carlo Software was used to conduct the sensitivity analysis investigation. The impacts of cellulase yield, productivity, aeration rate and specific power input on the unit production cost (UPC) of cellulase enzyme were studied. The batch procedure requires more sterilized air for the aerobic fermentation process than the semi-batch approach, according to the results. In comparison to the batch fermentation process, the semi-batch approach had a payback period of less than 2 years, an internal rate of return of 39.14% and a net present value of $142 089 000. The semi-batch process approach has a higher economic feasibility when compared with the batch fermentation process since it is more certain to attain its base case value of UPC at the maximum. This research was key to developing a process pathway that reduces cellulase production costs in solid-state fermentation while improving market supply for its use in a biorefinery to produce sustainable fuel.

Study on the effects of different pectinase/cellulase ratios and pretreatment times on the preparation of nanocellulose by ultrasound-assisted bio-enzyme heat treatment.

With the development of science and technology, efficient, fast and green methods are increasingly being pursued. The production of nanocellulose by green methods, such as bio-enzymes-assisted ultrasound treatment, has been the focus of many studies. However, the yield of cellulose nanocrystals prepared by this method is very low. In this paper, by pretreatment of microcrystalline cellulose (MCC), nanocellulose was prepared by heating and stirring + pectinase/cellulase + ultrasonic treatment (HSt - P/C - Ultr). The effects of the ratios of pectinase and cellulase and the hydrolysis time on the yield of nanocellulose were studied. FTIR, XRD, SEM, TEM and TG were used to determine the structure, crystallinity, morphology and thermal stability of nanocellulose. The results showed that optimal hydrolysis conditions were determined as a pectinase : cellulase ratio of 1 : 1, 90 min and 50 °C. The yield of nanocellulose was about 32.0%. The yield of pectinase cellulase = 1 : 1 was higher than that of microcrystalline cellulose (MCC) treated by a single bio-enzyme. This indicated that the synergistic effects of pectinase and cellulase have a certain effect on the formation of nanocellulose. During the preparation, the crystalline form of cellulose did not change. It was still cellulose I with a crystallinity of 73.5%, which is 9.50% higher than that of microcrystalline cellulose (MCC), a width of 20-50 nm, a high aspect ratio and a winding network structure. Therefore, nanocellulose prepared by this method is an ideal toughening material for manufacturing composite materials.

Porous inert material as promising carrier enhanced cellulase production from Trichoderma reesei in solid-state fermentation

Developing a promising fermentation strategy with high cellulase yield and stable fermenting performance is an imperative way to overcome the cellulase costs limitation. In this study, a novel solid-state fermentation (SSF) strategy using polyurethane carbon foams (PAF) as inert carriers was developed to improve cellulase production by Trichoderma reesei (T. reesei). The effects of significant process parameters such as pore size and thickness of carrier on cellulase production were evaluated. Repeated cycle fermentation using inert carrier for cellulase production was assessed by measuring cellulase activity. Results indicated that inert carrier was conducive to cellulase production and the higher cellulase activity of 0.368 FPU/mL was achieved in SSF with PAF rather than PPF or PUF. More cellulase components could be obtained from SSF with inert carrier compared with submerged fermentation. In addition, higher cellulase activity (0.393 FPU/mL) could be achieved at 0.6 mm pore and 15 mm thickness of PAF. The repeated cycle fermentation experiments showed that maximum cellulase activity of 0.514 FPU/mL was obtained over 6 cycles and the fermentation performance was still stable in SSF with PAF. Therefore, SSF using inert carrier is simple, low cost, and environmentally benign, making it an attractive method for cellulase production.

Techno-economic analysis of Cellulase Production by Trichoderma reesei in Submerged Fermentation Processes using a Process Simulator

Enzymes are valuable in many biofuels production processes, and their wide application has generated significant market demand in recent years. To develop lignocellulosic fuels in a biorefinery, high cost and low catalytic efficiency of cellulase are a major obstacle in the industrial use of this enzyme. A techno-economic assessment of cellulase production by Trichoderma reesei in submerged fermentation (SmF) processes was carried out in this study. SuperPro Designer Software was used to develop a process model that estimated the economic analysis of the fermentation processes by creating alternative schemes (batch and fed-batch) to improve the process. Sensitivity analysis study was carried out using Monte Carlo Software. Economic profitability indicators were used to assess cellulase yield, productivity, aeration rate, and specific power input on the unit production cost (UPC) of cellulase enzyme. Results show that the batch process had a higher aeration requirement when compared to the fed-batch process. Fed-batch process route had a shorter payback time (1.88 years), higher internal rate of return IRR (36.95%), and a more positive Net present value ($140,328,000) than the batch process. In addition, the Fed-batch process route is more economically viable than the batch process route, since the certainty of achieving its base case value of UPC is at the optimum. This study has been able to design a process route that reduces the production cost of cellulase, while increasing its availability in the market for sustainable fuel production in a biorefinery.

Enzymatic depolymerization of wheat straw polysaccharides

The purpose of this study is to develop a technology for enzymatic processing for depolymerization of polysaccharides in wheat straw to obtain the maximum yield of glucose and sorbitol. Cellulolytic enzymes endo-1,4-β-glucanase (EC 3.2.1.4) and cellobiose (1,3-β-glucosidase) (CF 3.2.1.21) were isolated and studied in local strains Tr. viride 121, which are grown under deep cultivation conditions. A technology has been developed for obtaining a complex preparation “Cellozyme G20x” with a high yield and specific activity of cellulase, xylanase, β-glucanase and pectinase, and a scheme for purification from cellulases by precipitation, ultrafiltration, and freeze drying is not inferior in efficiency to commercial preparations. The physicochemical properties of the preparation “Cellozyme G20x” have been studied, the optimal parameters of the action and stability of the enzyme preparation have been established. The efficiency of Cellozyme G20x for hydrolysis of straw polysaccharides was 35-40% in terms of glucose yield.

Optimization of Cellulase Production by a Novel Endophytic Fungus Penicillium oxalicum R4 Isolated from Taxus cuspidata

Endophytic fungi inside a plant can degrade a portion of plant lignin and cellulose. Endophytic Penicillium is one of the industrial microorganisms with the advantage of producing enzymes with a complete enzyme system that can be secreted into the extracellular space. The natural evolution of ancient tree species from special natural geographic environments to screen out cellulase-producing strains with excellent characteristics provides a promising direction for future industrial enzymes. The present study successfully isolated and screened a novel fungal endophyte, Penicillium oxalicum R4, with higher cellulase activity from Taxus cuspidata. Under the optimized culture conditions obtained by a Box–Behnken design (BBD) and an artificial neural network–genetic algorithm (ANN–GA), yields of Filter Paperase (FPase), Carboxymethyl Cellulase (CMCase) and β-glucosidase (βGLase) produced by P. oxalicum R4 were 1.45, 5.27 and 6.35 U/mL, which were approximately 1.60-fold, 1.59-fold and 2.16-fold higher than those of the non-optimized culture, respectively. The discovery of cellulase-producing strains of endophytic fungi located in special natural geographic environments, such as Taxus cuspidata, which is known as a living plant fossil, provides new research directions for future industrial enzymes.

Bioprocess Optimization for Enhanced Production of Bacterial Cellulase and Hydrolysis of Sugarcane Bagasse

Cellulolytic enzymes have gained considerable importance as potential candidates for the efficient bioconversion of agricultural biomass into second-generation biofuel. This study aimed to improve cellulase production from a bacterial strain isolated from cow dung. The biochemical and molecular identification was performed, and the capacity of the isolated bacterium to hydrolyze sugarcane bagasse was also determined. Response surface methodology (RSM) and one-variable-at-a-time (OVAT) designs were employed to maximize the cellulase production. The bacterium identified as Bacillus subtilis gave the highest cellulase yield at temperature of 45 °C, pH-7.0, and 72 h of incubation time. Optimization by the Plackett–Burman design consisting of 12 experimental runs at two levels of nine independent variables displayed the significant contribution of galactose (22%) and sodium nitrate (16%) in cellulase production. The responses in the form of contour and 3D plots (generated by Box-Behnken design) predicted maximum cellulase production with an activity of 0.8 U/mL/min in a medium containing 3.0 g/L carboxymethyl cellulose, 3.0 g/L galactose, 0.5 g/L sodium nitrate, 0.75 g/L KH2PO4, and 0.375 g/L MgSO4·7H2O. The experimentally deduced activity value was 0.698 U/mL/min, showing 90% validity of the predicted value and gave a threefold increase in cellulase production. The process adequately represented by the quadratic model (p < 0.0343) for all the responses was significant. Results obtained show that optimization of fermentation medium favored the increased production of cellulase by the bacterium. The bacterial isolate B. subtilis CD001 was found to be a potential candidate to hydrolyze the lignocellulosic feedstock, sugarcane bagasse.

Isolation, Identification and In Silico Study of Native Cellulase Producing Bacteria

Aims: The purpose of this study was to screen the bacteria producing cellulase enzymes and their bioinformatics studies. Background: Cellulose is a long-chain polymer of glucose that hydrolyzes by cellulases to glucose molecules. In order to design the new biotechnological applications, some strategies have been used as increasing the efficiency of enzyme production, generating cost-effective enzymes, producing stable enzymes and identification of new strains. Objective: On the other hand, some bacteria special features have made them suitable candidates for the identification of the new source of enzymes. In this regard, some native strains of bacteria were screened. Methods: These bacteria were grown on a culture containing the liquid M9 media containing CMC to ensure the synthesis of cellulase. The formation of a clear area in the culture medium indicated decomposition of cellulose. In the following, the DNA of these bacteria were extracted and their 16S rDNA genes were amplified. Result: The results show that nine samples were able to synthesize cellulase. In following, these strains were identified using 16S rDNA. The results show that these screened bacteria belonged to the Bacillus sp., Alcaligenes sp., Alcaligenes sp., and Enterobacter sp. Conclusion: The enzyme activity analysis shows that the Bacillus toyonensis, Bacillus sp. strain XA15-411 Bacillus cereus have produced the maximum yield of cellulases. However, these amounts of enzyme production in these samples are not proportional to their growth rate. As the bacterial growth chart within 4 consecutive days shows that the Alcaligenes sp. Bacillus cereus, Bacillus toyonensis, Bacillus sp. strain XA15-411 have a maximum growth rate. The study of the phylogenetic tree also shows that Bacillus species are more abundant in the production of cellulase enzyme. These bioinformatics analyses show that the Bacillus species have different evolutionary relationships and evolved in different evolutionary time. However, for maximum cellulase production by this bacteria, some information as optimum temperature, optimum pH, carbon and nitrogen sources are needed for the ideal formulation of media composition. The cellulase production is closely controlled in microorganisms and the cellulase yields appear to depend on a variety of factors. However, the further studies are needed for cloning, purification and application of these new microbial cellulases in the different commercial fields as in food, detergent, and pharmaceutical, paper, textile industries and also various chemical industries. However, these novel enzymes can be further engineered through rational design or using random mutagenesis techniques.

Влияние источников азота в питательной среде на рост и целлюлазную активность стрептомицетов

Biomass accumulation and cellulase activity were determined in two promising soil isolates during liquid phase cultiva-tion depending on organic (peptone, casein hydrolysate) and mineral (NH4Cl, NaNO3, NH4NO3, (NH4)2SO4, KNO3) nitrogen sources. The only source of carbon in the medium was straw (1ob.%). Using two-factor analysis of variance, it was found that the production of cellulase complex enzymes is equally influenced by the nitrogen source (F= 225,92; p&lt;0,0001), the producer strain (F=205,98; p&lt;0,0001), the interaction of these factors (F=225,06; p&lt; 0,0001). Inorganic nitrogen sources were the most preferred for cellulase production: NH4NO3 provided enzymatic activity in Streptomyces sp. Mb 4-2 is 912,82±60,40 units/10 min /g, and NH4Cl in Streptomyces sp. 1.3 NH4Cl is 531,37±27,25 units/10 min /g, which is 58–481 % and 34,5–409% more, respectively, than on other tested nitrogen sources. The accumulation of biomass during growth was more dependent on the nitrogen source (F= 65,47; p&lt;0,0001) than on the strain of streptomycetes (F=18,65; p&lt;0,0001). The interaction of these factors did not have a significant effect on the accumulation of biomass (F=1,11; p=0,38). Peptone and casein hyd-rolysate became the best nitrogen source for the growth of streptomycetes, providing an average of 19 % more biomass than inorganic nitrogen sources. It is shown that the optimization of the nutrient medium by the nitrogen source makes it possible to regulate the growth rates of streptomycetes-producing cultures and achieve maximum cellulase yield. The results obtained are of interest for the development of new environmentally friendly technological processes using streptomycetes in the production of cellulase complex enzymes and in the destruction of cellulose-containing agricultural waste.

Cost‐effective cellulase production, improvement strategies, and future challenges

AbstractThe most abundant biopolymer accessible on earth is lignocellulose. The hydrolysis of lignocelluloses is completed through enzyme groups such as ligninases, hemicelluloses, and cellulases as independently and together called lignocellulolytic enzymes. This review is focused on dealing with enhanced production of cellulose using modern techniques like strain improvement, consolidated bioprocessing, metabolic engineering, and recombinant technology, and defines the technology to boost the enzyme activity and its yield. Furthermore, the restrains linked with the production of cellulolytic enzyme and the direction of upcoming research to offer a complete strategy to improve cellulase yield with novel properties for industrial application at cost‐effective levels are mentioned.Practical applicationsCellulase is a complex enzyme consisting of an endoglucanase, exoglucanase, and β‐D‐glucosidase which acts in combination to release a small unit of glucose. Cellulase has vast potential in the industrial sector especially in the food and beverage as well as paper and pulp industries. The parameters such as strain improvement and heterologous gene expression are involved to enhance the cellulase production. This review discusses the cost‐effective production of cellulolytic enzyme and also emphasizes the limitations and future prospects of this enzyme production on a large scale.

Optimised cellulase production from fungal co-culture of Trichoderma reesei NCIM 1186 and Penicillium citrinum NCIM 768 under solid state fermentation

Microbial cellulases find applications in many industries and constitute a significant share of the world’s industrial enzyme market. In order to improve the cost function of the cellulose producing processes with enhanced yield and novel activities, superior bioprocesses are formulated these days. Intending to produce higher titre of cellulase enzyme, present study deals with fungal co-culture and to investigate its feasibility of producing higher cellulase titre via solid state fermentation (SSF). Various process parameters were optimized for induced cellulase production from the co-culture of Trichoderma reesei NCIM 1186 and Penicillium citrinum NCIM 768 in SSF. Among different low cost and easily available agricultural lignocellulosic waste, wheat bran was found to be the most suitable substrate. The highest cellulase yield of 6.71 FPU/gds is obtained on 6th day of fermentation using steam pre-treated wheat bran with 70 % moisture content when inoculated with 106 spores of mixed suspension at 30 °C and pH 5. A further possibility to use whole fermented matter for saccharification instead of extracted cellulase was also investigated with the aim of abolishing an extraction step. Akin sugar yield of 6.45 mg/mL and 6.58 mg/mL was obtained at 72 h of hydrolysis of steam pre-treated wheat bran, using cellulase extract and whole fermented matter, respectively. Fungal co-culture of T. reesei and P. citrinum in SSF results in the highest cellulase titre using low cost agricultural waste as a substrate, which can be further used for successful saccharification in the form of whole fermented matter or as extracted enzyme.

Optimization of cellulase production by a novel endophytic fungus Pestalotiopsis microspora TKBRR isolated from Thalakona forest

The present work focused on cellulase production in solid-state fermentation by a fungal isolate of Thalakona forest in Andhra Pradesh identified as Pestalotiopsis microspora TKBRR. Under normal conditions, yields of Filter Paperase (FPase), Carboxymethyl Cellulase (CMCase) and β-glucosidase (BGL) produced by P. microspora TKBRR in SSF were 13, 12 and 11 U/g DS whereas the corresponding yields of FPase, CMCase and BGL after optimization of process conditions and medium components through one stage approach—Central Composite Design in RSM were 15, 16 and 13 U/g DS, respectively in the present study. There was a marginal improvement in yields of cellulolytic enzymes by 2–4 units with the least interactions between factors. Of the four factors tested in the present study, only temperature and pH influenced production of the cellulolytic enzymes and P. microspora TKBRR in SSF exhibited a higher ratio of BGL to FPase in comparison to other cellulolytic organisms reported.

Chitosan modified Fe3O4/graphene oxide nanocomposite as a support for high yield and stable immobilization of cellulase: its application in the saccharification of microcrystalline cellulose

In the present study, specialized methodology of utilizing novel nanobiocatalyst, chitosan coated magnetic graphene (Fe3O4/GO/CS), for efficacious immobilization of Trichoderma reesei cellulase, an important industrial enzyme was revealed. The cellulase was covalently immobilized onto the nanocomposite (NC) using covalent-glutaraldehyde coupling methodology. Successful immobilization of the cellulase with Fe3O4/GO/CS NC was affirmed by transmission electron microscopy, Fourier transform infrared spectroscopy and scanning electron microscopy. The nanobiocatalyst preparations exhibited significantly improved activity, retaining 78% of the initial activity as compared to its soluble counterpart. Immobilization of cellulase also highlighted significant broadening in pH, thermal and storage stability profiles. The kinetic properties of cellulase bound Fe3O4/GO/CS NC showed lower Km indicating increased affinity (1.87 times) of nanobioconjugate toward the substrate. Cellulase bound Fe3O4/GO/CS NC was able to maintain higher percentages of its primary activity after 8 repeated uses. This nanobioconjugate preparation proves to be industrially robust biocatalyst with enhanced nano-biocatalytic activity, stability and reusability attributes, which can be exploited for hydrolysis of microcrystalline cellulose providing increased amount of sugar compared to free cellulase.

Cellulase production by Aspergillus fumigatus MS13.1 mutant generated by heavy ion mutagenesis and its efficient saccharification of pretreated sweet sorghum straw

Abstract Developing a potential strain with high cellulase yield and stable fermenting performance is an imperative way to overcome the enzyme costs limitation. A cellulose degrading fungus was isolated from the agricultural wastes and identified as A. fumigatus MS13.1. For further improving the production capacity of enzyme, the original A. fumigatus MS13.1 was subjected to mutagenesis with heavy-ion beams and mutant MS160.53 was selected. The MS160.53 exhibited higher filter paper (1.81 IU/ml) and β-glucosidase (1.65 IU/ml) activities than parent strain MS13.1, which increased by 40.3% and 15.1%, respectively. Soluble protein concentrations in supernatants from strain MS160.53 were reached 1.497 mg/ml at 240 h which were higher than that in the reported literature of A. fumigatus. In addition, the SDS-PAGE profiles revealed that more enzymes of mutant were secreted during fermentation compared with parent strain MS131.1. The crude enzyme were further applied for saccharification of pretreated sweet sorghum straw, where a dosage of 5 IU/g FPase produced high reducing sugar titers of 789.75 mg/g. SEM images revealed the obviously microstructural changes of straw after the pretreatment and enzymatic hydrolysis. Thus, the enzyme cocktails produced by A. fumigatus MS160.53 has great potential for the saccharification of pretreated biomass and further bioethanol production.