Maximum Endoglucanase Activity Research Articles

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 highly thermostable crude endoglucanase produced by a newly isolated Thermobifida fusca strain UPMC 901

A thermophilic Thermobifida fusca strain UPMC 901, harboring highly thermostable cellulolytic activity, was successfully isolated from oil palm empty fruit bunch compost. Its endoglucanase had the highest activity at 24 hours of incubation in carboxymethyl-cellulose (CMC) and filter paper. A maximum endoglucanase activity of 0.9 U/mL was achieved at pH 5 and 60 °C using CMC as a carbon source. The endoglucanase properties were further characterized using crude enzyme preparations from the culture supernatant. Thermal stability indicated that the endoglucanase activity was highly stable at 70 °C for 24 hours. Furthermore, the activity was found to be completely maintained without any loss at 50 °C and 60 °C for 144 hours, making it the most stable than other endoglucanases reported in the literature. The high stability of the endoglucanase at an elevated temperature for a prolonged period of time makes it a suitable candidate for the biorefinery application.

Genomically Defined Paenibacillus polymyxa ND24 for Efficient Cellulase Production Utilizing Sugarcane Bagasse as a Substrate.

Cellulolytic bacteria from cattle rumen with ability to hydrolyze cellulose rich biomass were explored. The study selected Paenibacillus polymyxa ND24 from 847 isolates as the most potent strain, which can efficiently produce cellulase by utilizing sugarcane bagasse, rice straw, corn starch, CMC, and avicel as a sole carbon source. On annotation of P. polymyxa ND24 genome, 116 members of glycoside hydrolase (GH) family from CAZy clusters were identified and the presence of 10 potential cellulases was validated using protein folding information. Cellulase production was further demonstrated at lab-scale 5-L bioreactor exhibiting maximum endoglucanase activity up to 0.72U/mL when cultivated in the medium containing bagasse (2% w/v) after 72h. The bagasse hydrolysate so produced was further utilized for efficient biogas production. The presence of diverse hydrolytic enzymes and formidable cellulase activity supports the use of P. polymyxa ND24 for cost-effective bioprocessing of cellulosic biomass.

Isolation of endo-1,4-b-D-glucanase producing Bacillus subtilis sp. from fermented foods and enhanced enzyme production bydeveloping the mutant strain

Cellulolytic bacteria living in food can be applied to microbial feed additives to improve fiber digestion in animal feeds. In this study, a cellulase-producing bacteria was isolated from salted clam and treated with physical or chemical agents to enhance their enzyme production. The bacteria was identified as a strain of Bacillus subtilis on the basis of 16S rRNA analysis. Endo-1,4-b-D-glucanase (endoglucanase) was produced by the wild type using 0.4% carboxy-methyl-cellulose as a carbon source with maximal activity (0.04 U/mL) after 24 h incubation. Insoluble cellulose and oat spelt xylan were also used as carbon sources for investigation of exoglucanase and xylanase, however, these enzymes were not found in the culture supernatant. Maximum endoglucanase activity of Bacillus subtilis sp. was measured at 50°C and pH 5, respectively. Then, the strain was subjected to classical mutagenesis (UV-irradiation and chemical treatment) to improve endoglucanase production. A mutant strain, P11 treated with ethyl methyl sulfonate was finally selected. Mutant P11 was sub-cultured and tested for endoglucanase production, which was 0.05 U/mL after 24 h growth. The significant difference of endoglucanase production between wild type and mutant P11 was prolonged to 10th generation. Thus, the mutant strain was found to have enhanced endoglucanase production.

Enhanced Endoglucanase Production by Bacillus aerius on Mixed Lignocellulosic Substrates

Selected carbon sources including soluble carboxymethyl cellulose (CMC), insoluble microcrystalline cellulose (MCC), and single (SS)/mixed lignocellulosic substrates (MS), were evaluated for endoglucanase production by B. aerius S5.2. The lignocellulosic substrates of oil palm empty fruit bunch (EFB), oil palm frond (OPF), rice husk (RH), and their mixture (MS) supported growth of the strain better than CMC and MCC. The maximum endoglucanase activity on MS was 7.3-, 2.6-, 1.7-, and 1.2-fold higher than those recorded on MCC, CMC, EFB/OPF, and RH, respectively. While the reducing sugar concentration of the CMC medium was comparable to that of the EFB and MS media, wide variability was observed in the reducing sugar concentrations among the lignocellulosic substrates. Extremely low levels of sugar were detected in the MCC medium, reflecting its poor digestibility and hence unsuitability for growth and endoglucanase production. Endoglucanase production was predominantly extracellular when the strain was grown on CMC and MS. After seven days of fermentation, there was an approximately 25% reduction in MS dry weight. These findings show that the use of mixed lignocellulosics could potentially reduce the cost of cellulase production. Certain novel aspects of the cellulase system of B. aerius are reported in this study.

Potential use of soybean hulls and waste paper as supports in SSF for cellulase production by Aspergillus niger

Abstract Cellulase has by vast applications in the biofuel, pulp and paper, detergent and textile industries. The three components of the enzyme complex (endoglucanase, exoglucanase and β-glucosidase) can effectively depolymerize the cellulose chains in lignocellulosic substrate. Solid-state fermentation (SSF) by fungi is a preferable production route for cellulase because of its low cost, among other advantages. This work describes the cellulase production by Aspergillus niger NRRL3 grown on SSF. SSF was carried out on soybean hulls and waste paper as supports. The effect of the support on cellulase production was assessed under a completely randomized factorial design. The support-time interaction was significant for all the variables studied. Both materials were characterized in terms of water absorption index and critical humidity point. Samples of culture were analyzed with scanning electron microscopy (SEM) to study spores and fungal growth. Maximum endoglucanase activity was found at 96 h using soybean hulls as support (5914.29 U L−1), being four times higher than that obtained using waste paper at the same fermentation time. The exoglucanase activity in soybean hulls was maximal at 96 h (4551.19 U L−1), being 9.6 times higher than that obtained in waste paper at the same time. The maximum β-glucosidase activity in soybean hulls (984.01 U L−1) was reached at 96 h, being 1.7 times greater than that obtained in waste paper. Besides, the use of soybean hulls provided high volumetric productivities at shorter times, which may decrease production costs considering a scaled process.

Determination of endoglucanase activity of paper decaying fungi from an old library at the ancient Medina of Fez

Paper from an ancient library of the cultural city of Fez (Morocco) is exposed to rapid deterioration by variety of microorganisms, especially cellulolytic fungi. For this, ten isolates fungi previously isolated from historical biodeteriorated paper were screened for their ability to produce endoglucanase (CMCase), amylase, polygalacturonase and ligninase enzymes. The CMCase activity of cellulolytic strains was essayed in liquid media at 25°C for 10 days. Influence of temperature and pH were assessed for the production of CMCase by all the fungus isolated from decaying paper. The research findings from the present study demonstrate that all the tested isolates had cellulase, amylase, pectinase and ligninase activities. It was found that Mucor racemosus PF15, Aspergillus niger, and Aspergillus oryzae exhibited the maximum endoglucanase activity in liquid medium (0.256, 0.236, and 0.216 UI/mL in descending order) for six days. Temperature profiling revealed optimum endoglucanase activity at 25 and 30°C. Maximum activity was observed at pH 5 and pH 6.

Optimization of Endoglucanase Production from a Novel Bacterial Isolate, Arthrobacter sp. HPG166 and Characterization of Its Properties

In this study, a potential novel cellulolytic bacteriumArthrobacter sp. HPG166 was isolated from the hindgut of root-feeding larvaeHolotrichia parallela. Optimization of fermentation factors for endoglucanase production byArthrobacter sp. HPG166 was carried out via response surface methodology. Sodium carboxymethylcellulose 1.19% (w/v) and beef extract 0.35% (w/v) were the ideal combination of carbon and nitrogen sources for enzyme production; the optimum temperature and pH for cellulase production were 34°C and pH 8.0 respectively. Under the optimized fermentation conditions, the maximum endoglucanase activity of 1.411 U mL-1 was obtained. The crude endoglucanase was thermotolerant as it retained 50.31% of its activity after incubation at 70°C for an hour. Metal profile of the enzyme indicated that Mg2+ and Na+ were strong stimulators while Mn2+ and Co+ drastically inhibited its activity. Due to its particular characteristics, this enzyme could have potential for industrial applications.

Isolation, Screening, and Optimization of the Fermentation Conditions of Highly Cellulolytic Bacteria from the Hindgut of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae)

From the hindgut contents of Holotrichia parallela, 93 cellulolytic bacterial isolates were isolated after enrichment in carboxymethyl cellulose medium. Among these isolates, a novel bacterium, designated HP207, with the highest endoglucanase productivity was selected for further study. This bacterium was identified as Pseudomonas sp. based on the results of the 16S ribosomal DNA analysis, morphological characteristics, and biochemical properties. The production of the endoglucanase was optimized by varying various physical culture conditions using a submerged fermentation method. Under the optimized fermentation conditions, the maximum endoglucanase activity of 1.432 U mL(-1) in bacterial cultures was obtained, higher than those of the most widely studied bacteria and fungi, which are the attractive candidates for the commercial producer of cellulase. And the crude endoglucanase enzyme was also highly thermostable; approximately 55% of the original activity was maintained after pretreatment at 70 °C for 1 h. Thus, from the present study, the bacterium can be added up to the database of cellulolytic bacteria.

Bioconversion and saccharification of some lignocellulosic wastes by Aspergillus oryzae ITCC-4857.01 for fermentable sugar production

The recent interest in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensive studies on cellulolytic enzymes produced by microorganisms. In the present study three lignocellulosic substrates viz. sugarcane bagasse, sawdust and water hyacinth were pre-treated with alkali and enzyme and their effect on bioconversion has been investigated. The ability of selected substrates for induction of cellulase enzyme by A. oryzae ITCC 4857.01 and for the potentiality of the induced enzyme to saccharify the substrates were also assessed. The maximum degree of conversion of substrate (0.415%) and improved specific substrate consumption (0.99 g substrate/g dry biomass) was exhibited in sugarcane bagasse after alkali treatment at 96 hrs. Both alkali-treatment and enzyme-treatment, water hyacinth was the best for cellulase induction and showed maximum endoglucanase activity of 11.42 U/ml. Reducing sugar yield ranged from 1.12 mg/ml for enzyme treated sawdust at 48 hrs to 7.53 mg/ml for alkali treated sugarcane bagasse at 96 hrs. Alkali-treated sugarcane bagasse gave the highest saccharification rate of 9.03% after 96 hrs. The most resistant substrate was sawdust which produced 5.92% saccharification by alkaline treatment. The saccharification of lignocellulosic substrates by enzyme produced by A. oryzae ITCC 4857.01 indicates the enzymes specificity towards the substrates. The use of such enzyme in lingo-cellulose hydrolysis will lead to efficient conversion of cellulose materials to other important products.

Development and Application of a PCR-Targeted Gene Disruption Method for Studying CelR Function in Thermobifida fusca

Thermobifida fusca is a high-G+C-content, thermophilic, Gram-positive soil actinobacterium with high cellulolytic activity. In T. fusca, CelR is thought to act as the primary regulator of cellulase gene expression by binding to a 14-bp inverted repeat [5'-(T)GGGAGCGCTCCC(A)] that is upstream of many known cellulase genes. Previously, the ability to study the roles and regulation of cellulase genes in T. fusca has been limited largely by a lack of established genetic engineering methods for T. fusca. In this study, we developed an efficient procedure for creating precise chromosomal gene disruptions and demonstrated this procedure by generating a celR deletion strain. The celR deletion strain was then characterized using measurements for growth behavior, cellulase activity, and gene expression. The celR deletion strain of T. fusca exhibited a severely crippled growth phenotype with a prolonged lag phase and decreased cell yields for growth on both glucose and cellobiose. While the maximum endoglucanase activity and cellulase activity were not significantly changed, the endoglucanase activity and cellulase activity per cell were highly elevated. Measurements of mRNA transcript levels in both the celR deletion strain and the wild-type strain indicated that the CelR protein potentially acts as a repressor for some genes and as an activator for other genes. Overall, we established and demonstrated a method for manipulating chromosomal DNA in T. fusca that can be used to study the cellulolytic capabilities of this organism. Components of this method may be useful in developing genetic engineering methods for other currently intractable organisms.

Cloning and expression of cellulase gene EG1 from Rhizopus stolonifer var. reflexus TP-02 in Escherichia coli

A novel gene, EG encoding enzymes involved in carboxymethyl cellulose (CMC) degradation was isolated, sequenced from the filamentous fungus Rhizopus stolonifer var. reflexus TP-02, and expressed in Escherichia coli BL21. The results showed that the gene amplified from the cDNA of the strain could be classified as the family of endoglucanase. During the fermentation process, the maximum endoglucanase activity (i.e. 0.715 IU/ml) of the recombinant bacteria was obtained at 36 h. The SDS–PAGE analysis on purified samples showed that a band with apparent molecular weight of about 40 kDa was detected after staining with Coomassie brilliant blue.

Production of endoglucanase by the native strains of Streptomyces isolates in submerged fermentation

Cellulase is a complex enzyme system, commercially produced by filamentous fungi under solid-state and submerged cultivation. It has wide applicability in textile, food and beverage industry for effective saccharification process. In this study, cellulolytic enzyme activity, particularly endoglucanase of 26 Streptomyces strains isolated from garden soil was examined, including two isolates selected on the basis of potential cellulolytic activity on Bennett's agar medium. To enhance the endoglucanase formation in broth culture, different conditions including carbon and nitrogen sources, and growth conditions were tested. The maximum endoglucanase activity (11.25-11.90 U/mL) was achieved within 72-88 h in fermentation medium containing Tween-80, followed by phosphate sources. Both cellulolytic Streptomyces isolates gave almost equal quantity of enzyme in all trials. However the effect of medium ingredients on endoglucanase induction diverged with strains in some extent.

Production of endoglucanase by the native strains of Streptomyces isolates in submerged fermentation.

Cellulase is a complex enzyme system, commercially produced by filamentous fungi under solid-state and submerged cultivation. It has wide applicability in textile, food and beverage industry for effective saccharification process. In this study, cellulolytic enzyme activity, particularly endoglucanase of 26 Streptomyces strains isolated from garden soil was examined, including two isolates selected on the basis of potential cellulolytic activity on Bennett’s agar medium. To enhance the endoglucanase formation in broth culture, different conditions including carbon and nitrogen sources, and growth conditions were tested. The maximum endoglucanase activity (11.25-11.90 U/mL) was achieved within 72-88 h in fermentation medium containing Tween-80, followed by phosphate sources. Both cellulolytic Streptomyces isolates gave almost equal quantity of enzyme in all trials. However the effect of medium ingredients on endoglucanase induction diverged with strains in some extent.

Constitutive expression of the Trichoderma reesei beta-1,4-xylanase gene (xyn2) and the beta-1,4-endoglucanase gene (egl) in Aspergillus niger in molasses and defined glucose media.

The xylanase II (xyn2)- and endoglucanase I (egl)-encoding regions of Trichoderma reesei QM6a were successfully expressed in Aspergillus niger D15 under the transcriptional control of the glyceraldehyde-6-phosphate dehydrogenase (gpd) promoter from A. niger and the glaA terminator of Aspergillus awamori. A stable xyn2 transformant produced beta-xylanase activity of 8,000 nkat/ml and 5,000 nkat/ml in shake-flask cultures containing defined or 20% (v/v) molasses medium, respectively. The recombinant Xyn2 enzyme expressed highest activity at pH 5-6 and 50-60 degrees C and retained more than 75% of its activity after 3 h of incubation at 50 degrees C. A stable egl transformant produced endo-P-1,4-glucanase activity of 2,300 nkat/ml in shake-flask cultures containing defined media and about half the activity in 20% molasses medium. Maximum endoglucanase activity was obtained at pH 5 and 60 degrees C. Both Xyn2 and EgI retained >80% activity after incubation at 50 degrees C for 3 h. The heterologous Xyn2 and EgI represent a significant portion of the total extracellular proteins produced.

Production of endo-1,4-β-glucanase and xylanase with nylon-web immobilized and free Trichoderma reesei

On all media studied, the endo-1,4-β-glucanase and xylanase activities obtained with the nylon-web immobilized Trichoderma reesei were higher than the activities with the free mycelium. The nitrogen source had a marked effect on endoglucanase and xylanase production on lactose, cellulose, and l-sorbose-based medium with immobilized T. reesei. The highest enzyme activities with this medium, 880 nkat ml −1 for endoglucanase and 8,020 nkat ml −1 for xylanase, were obtained in the presence of proteose peptone and yeast extract. Urea alone was an insufficient nitrogen source for enzyme production. A lactose-containing industrial by-product-based medium offered a good alternative for endoglucanase and xylanase production. In this case, the maximum endoglucanase activity was 940 nkat ml −1, xylanase activity was 4,410 nkat ml −1, and filter paper activity was 1.9 FPU ml −1. On synthetic cellulose-based medium, the activities were 840 nkat ml −1, 4.790 nkat ml −1, and 3.0 FPU ml −1, respectively. The nylon-web immobilized biocatalyst could be used for two successive batch cultures with only a little decrease in enzyme production observed.

Production of extracellular enzymes by immobilizedTrichoderma reesei in shake flask cultures

Polyurethane foam and nylon-web carriers were compared for simultaneous production of endo-1, 4-β-glucanase and xylanase by immobilizedTrichoderma reesei on a medium based on lactose (27 g/l), cellulose (3 g/l) and sorbose (0.3 g/l). Nylon sheet with 1.2 cm2 carrier surface/ml medium was superior to the others, and it was selected for further studies. The carbon source had a marked effect on enzyme production by the immobilized fungus. With pure cellulose (10 g/l) as substrate, the maximum endoglucanase activity was 690 nkat/ml and xylanase activity 4800 nkat/ml. Supplementation with 0.5 g/l of sorbose resulted in an increase in both endoglucanase and xylanase activities in all media studied. A more detailed study on the effect of sorbose on a lactose(7 g/l)-and cellulose(3 g/l)-based medium revealed a clear optimum sorbose concentration of 1.5 g/l, with a maximum endoglucanase activity of 660 nkat/ml, xylanase activity of 3670 nkat/ml, and filter-paper activity (overall cellulolytic activity) of 2.0 filter-paper units/ml. However, the addition of 1.5 g/l sorbose to the pure-cellulose(10 g/l)-based medium resulted in a slight decrease in the enzyme production.

Purification and properties of the Endo-1,4-β-glucanase from Ruminococcus albus and its gene product in Escherichia coli

Endoglucanases, EGI and EgI, were produced from the same Ruminococcus albus gene in R. albus and recombinant Escherichia coli, respectively. EGI was purified from R. albus culture supernatant and EgI was extracted from the transformant E. coli (JM101/pURA1) and purified. The purified enzymes EGI and EgI revealed maximum endoglucanase activity at a same pH of 6.8 and a temperature of 37°C. Both enzymes were stable at temperatures below 30°C. In addition, about 10% of their original activities were conserved even after boiling for 10 min. Amino acid sequences of both enzymes at the N-terminal (Ala-Ala-Asp-Glu-Ser-Glu-Thr-Glu-Asn-Val-Pro-Val-Ser-Gln-Thr-His--) were consistent with each other. The antiserum against EgI reacted with both EgI and EGI, indicating that both their protein moieties were the same immunologically. However, the molecular size of EGI (43,000) was larger than that of EgI (39,000) due to the presence of sugar moiety. The specific activity (54 units/mg) of EGI was almost double that (27 units/mg) of EgI. EGI was immunologically different from the endoglucanase purified in the previous paper [Ohmiya et al.: Carbohydrate Res., 166, 145–155 (1987)].

Purification and properties of an endo-1,4-beta-glucanase translated from a Clostridium josui gene in Escherichia coli

An endoglucanase encoded by a gene of Clostridium josui was expressed in Escherichia coli and purified. The homogeneous enzyme, with a molecular weight of 39,000, revealed maximum endoglucanase activity at pH 7.2 to 7.5 and a temperature of 65 to 70 degrees C. The enzyme was stable at a temperature lower than 45 degrees C (the growth temperature of the bacterium) in the range of pH 4.5 to 9.0. The amino acid sequence of the enzyme at the N terminus was Val-Glu-Glu-Asp-Ser-Ser-His-Leu-Ile-Thr-Asn-Gln-Ala-Lys-Lys----. The enzyme hydrolyzed cellotetraose to cellobiose and then transferred cellobiose to the residual cellotetraose. The resulting cellohexaose was cleaved to cellotriose.