Production,purific tion,characterization and application of two novel endoglucanases from buffalo rumen metagenome

Background Lignocellulose biomass is the most abundant and renewable material in nature.The objectives of this study were to characterize two endoglucanases Trep Cel3 and Trep Cel4,and determine the effect of the combination of them(1.2 mg Trep Cel3,0.8 mg Trep Cel4)on in vitro rumen fermentation characteristics.In this study,three nature lignocellulosic substrates(rice straw,RS;wheat straw,WS;leymus chinensis,LC)were evaluated for their in vitro digestibility,gas,NH3-N and volatile fatty acid(VFA)production,and microbial protein(MCP)synthesis by adding enzymatic combination.Methods Two endoglucanases’genes were successfully expressed in Escherichia coli(E.coli)BL21(DE3),and enzymatic characteristics were further characterized.The combination of Trep Cel3 and Trep Cel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability.Results The maximum enzymatic activity of Trep Cel3 was determined at p H 5.0 and 40℃,while Trep Cel4 was at p H 6.0 and 50℃.They were stable over the temperature range of 30 to 60℃,and active within the p H range of 4.0 to 9.0.The Trep Cel3 and Trep Cel4 had the highest activity in lichenan 436.9±8.30 and 377.6±6.80 U/mg,respectively.The combination of Trep Cel3 and Trep Cel4 exhibited the highest efficiency at the ratio of 60:40.Compared to maximum hydrolysis of Trep Cel3 or Trep Cel4 separately,this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4%for RS,236.7%for wheat straw WS,222.4%for LC and 131.1%for sugar beet pulp(SBP).Supplemental this combination enhanced the dry matter digestion(DMD),gas,NH3-N and VFA production,and MCP synthesis during in vitro rumen fermentation.Conclusions The Trep Cel3 and Trep Cel4 exhibited the synergistic relationship(60:40)and significantly increased the saccharification yield of lignocellulosic substrates.The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates.This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants.If possible,in the future,experiments in vivo should be carried out to fully evaluate its effect.

A cold adapt and ethanol tolerant endoglucanase from a marine Bacillus subtilis

The catalytic properties and thermodynamic kinetics of the endoglucanase from a marine Bacillus subtilis were analyzed. Optimum pH and temperature of the endoglucanase activity were 5.0 and 35 °C. The endoglucanase activity, melt point temperature was 1.13 folds(247.02 U·ml^(-1)), 2.1 °C higher(39.2 °C) in 6% ethanol solution than that(218.60 U·ml^(-1)),(37.1 °C) in free ethanol. At 40 °C–55 °C, Gibbs free energy, ΔG, and the content ofα-helix was higher in 6% ethanol solution than that in ethanol free solution. The increasing of α-helix content led to higher activity and better thermostability in ethanol solution. The cold adapt ethanol tolerant endoglucanase was valuable for bioethanol product by simultaneous saccharification and fermentation process.

A Rapid Parameter of Enzyme-Treated Cellulosic Material Revealed by Reducing Sugar Release

This study was conducted to evaluate the effectiveness of enzymes in purifying and reducing the degree of polymerization of cellulose for the production of dissolving pulp.Our goal was to determine the contributions of xylanase(X)and endoglucanase(EG)in the treatment of pulp,specifically by quantifying the formation of soluble and insoluble reducing sugars using the dinitrosalycilic acid(DNS)test.Predominantly,the release of soluble reducing sugars(RSSol)was enhanced after xylanase treatment,while endoglucanase(EG)treatment led to changes in insoluble reducing sugars(RSIns).The maximum synergism was observed for RSIns when a high ratio of endoglucanase to xylanase(320EG:5X/g pulp)was used.The relative contribution of endoglucanase to RSins was determined to be 15.6%of the total reducing sugar.The viscosity of pulps treated with xylanase decreased only by 7%,whereas endoglucanase treatment significantly reduced viscosity by 45%.Modifications in the particle size were observed after pulp treatment with the combination of endoglucanase and xylanase.In summary,the DNS test is a rapid and effective method for evaluating the efficiency of enzyme treatments on pulps.The measurement of RSIns correlates with changes in pulp viscosity to different extents,providing valuable insights into the effectiveness of enzyme treatments.

Evaluation of thermostable endoglucanase in Paenibacillus lautus strain BHU3 for yield enhancement

In the present study,Paenibacillus lautus strain BHU3 isolated from landfill soil was evaluated for the presence of potential endoglucanases which are the first candidate of cellulase enzyme system to act on cellulose.In-silico analysis revealed high potential thermostable endoglucanases which can efficiently interact with cellulose.The most potent and thermostable endoglucanase(locus tag id.CPZ30_18280)belonged to glycosyl family-5 and had interaction energy of−12.981 kcal/mol for the best docked cluster containing three out of ten docking conformations,and Tm value of 73.3℃.MD simulation of 100 ns proved highly stable binding interactions of CPZ30_18280 endoglucanase with cellulose with root mean square deviation(RMSD)values ranging from 0.15 to 0.30 nm.Consistent interactions with characteristic active site residues(tyrosine,tryptophan and aspartate)of glycosyl family-5 endoglucanases were found.Further,to enhance the production of endoglucanases,the fermentation conditions were optimized employing approaches like one factor at a time(OFAT)and response surface methodology(RSM).Maximum activity of endoglucanase was determined at 60℃.The optimized condi-tions for enhanced production of endoglucanase(10.15 U/mL)were pH 6.63,yeast extract conc.3.44 g/L,wheat bran 3.59%,and inoculum size 2.65%.Hence,P.lautus strain BHU3 has enormous potential to synthesize highly efficient thermostable endoglucanases under optimized regime using agro-wastes.Thus,it could find immense industrial applications including large scale cellulose conversion to bioethanol.

Carboxymethylcellulase Activity in Lettuce Seeds Prior to Germination

Does the endosperm pose a mechanical resistance on embryonic axis (radicle) growth for lettuce seed (achene) germination? To aid answering this question, the cell wall degrading enzyme, carboxymethylcellulase (CMCase) was extracted and assayed from lettuce seeds imbibed for 0 to 12 h, prior to germination. Measuring the loss of viscosity of carboxymethylcellulose, CMCase activity was high in dry seeds, low after 6 h of imbibition, high after 9 and 10 h, and then reduced again after 12 h. Fractions from Sephadex columns showed CMCase activity in three peaks labeled E1, E2, E3. The greatest change in CMCase activity during imbibition was with E3 (molecular weight of about 40,000 Daltons) and some reduction in E2 (molecular weight about 280,000). The RNA synthesis inhibitor, 6-methyl purine, eliminated CMCase activity when present from 4.5 to 7 h of imbibition and the protein synthesis inhibitor, cycloheximide, eliminated CMCase activity when present between 5.5 and 9 h. Imbibition in darkness lowered CMCase activity while 15 min of light at 3.5 h restored it and 30 min of far-red light at 3 h eliminated it. Increasing the imbibition temperature to 35°C under light reduced activity while under darkness, activity was eliminated under 24°C and 35°C. CMCase activity was localized in the endosperm surrounding the embryonic axis (micropylar end) of 9 h imbibed seeds. These observations showed that CMCase was active in degrading the cell wall in the endosperm surrounding the radicle, weakening it, prior to radicle protrusion so that the radicle remains undamaged.

Carposome productivity of Pleurotus ostreatus and Pleurotus eryngii growing on agro-industrial residues enriched with nitrogen,calcium salts and oils

The suitability of the abundant agro-industrial residues wheat straw(WS;control),barley and oats straw(BOS)and rice husk(RH),supplemented with various sources of oils(sunflower,corn oil),nitrogen(peptone,yeast extract)and calcium salts(CaSO_(4)⋅2H_(2)O,CaCl2),as novel substrates in solid-state fermentation of selected Pleurotus ostreatus and P.eryngii mushrooms was investigated.The possible effect of different additives on mycelial growth rate,biomass production and endoglucanase,laccase and lipase biosynthesis were evaluated.Moreover,their impact on essential cultivation aspects(earliness,total mushroom yield,biological efficiency)and carposome quality parameters(weight,morphological characteristics)was assessed.Both fungi showed their highest growth rates on BOS substrates and the most positive implementation was CaSO_(4)⋅2H_(2)O 6%w/w(Kr=9.58 mm/day;P.ostreatus,Kr=9.42 mm/day;P.eryngii),while different additives led to enhancement of biomass production.Pleurotus species demonstrated minimal levels of endoglucanase activity,with values ranging from 0.01 to 0.42 U/g of dry weight,regardless of the substrate and the stage of colonization.On the contrary,the maximum values of laccase activity were observed at 50%of colonization on BOS and RH,while supplementation with nitrogen and calcium sources positively affected its biosynthesis.P.ostreatus and P.eryngii cultivated on BOS supplemented with peptone at 2 and 5%w/w,synthesized significant laccase amounts,i.e.,12,165.78 and 8,624.55 U/g d.w.,respectively.Satisfactory amounts of lipase were produced,especially in substrates supplemented with sunflower 2%w/w,in quantities up to 1.42 U/g d.w.,whereas the highest lipase activity was achieved by P.eryngii on WS supplemented with corn oil at 2%w/w,with a value of 4.25 U/g d.w.being recorded.Regarding fermentation of Pleurotus species in polypropylene bags,WS and BOS supported faster colonization and shorter earliness period than RH substrates,whereas supplementation did not seem to affect these culture parameters.Furthermore,oils supplementation had a positive effect on BE of both species,with values up to 100%for P.ostreatus and 80%for P.eryngii on WS and BOS,whereas on RH the lowest BE values were detected.Morphological characteristics were not significantly affected by the additives.Results indicate the positive impact that certain additives have on mushroom productivity and production of enzymes with great financial and environmental importance.

Endo-β-1,4-glucanases impact plant cell wall development by influencing cellulose crystallization

Cell walls are vital to the normal growth and development of plants as they protect the protoplast and provide rigidity to the stem. Here, two poplar and Arabidopsis orthologous endoglucanases, which have been proposed to play a role in secondary cell wall development, were examined. The class B endoglucanases, Pt GH9B5 and At GH9B5, are secreted enzymes that have a predicted glycosylphosphatidylinositol anchor, while the class C endoglucanases, Pt GH9C2 and At GH9C2, are also predicted to be secreted but instead contain a carbohydrate-binding module.The poplar endoglucanases were expressed in Arabidopsis using both a 35 S promoter and the Arabidopsis secondary cell wall-specific Ces A8 promoter. Additionally, Arabidopsis t-DNA insertion lines and an RNAiconstruct was created to downregulate At GH9C2 in Arabidopsis. All of the plant lines were examined for changes in cell morphology and patterning, growth and development, cell wall crystallinity, micro fibril angle, and proportion of cell wall carbohydrates. Misregulation of Pt GH9B5/At GH9B5 resulted in changes in xylose content, while misregulation of Pt GH9C2/At GH9C2 resulted in changes in crystallinity, which was inversely correlated with changes in plant height and rosette diameter. Together, these results suggest that these endoglucanases affect secondary cell wall development by contributing to the cell wall crystallization process.

Identification of proteins involved in lignocellulose degradation using in gel zymogram analysis combined with mass spectroscopy-based peptide analysis of gut proteins from larval Asian Ionghorned beetles, Anoplophora glabripennis

Enzyme activities toward lignocellulose substrates were analyzed in the gut of larval Asian longhorned beetle （Anoplophora glabripennis）. Total protein was extracted from gut contents of wild collected larvae from an invasive population in Worchester, MA, USA. From these protein extracts, lignocellulolytic activities were measured （β-1,4- endoglucanase,β-1,4-glucosidase and birch wood xylanase）.β-1,4-glucosidase activity was 0.075 μmol glucose/mg protein per min, endoglucanase activity was measured at 0.41/zmol glucose/mg protein per min and xylanase activity was 0.058μmol xylose/mg protein per min. To identify specific enzymes that may provide these activities, zymogram analysis was performed to detect enzymes active toward carboxymethyl cellulose （CMC）, 4-methylumbelliferyl-β-D-glueopyranoside and birch wood xylan. Three protein bands were found to be active toward CMC, three displayed β-1,4-glucosidase, and one displayed xylanase activity. Proteins from active bands from these zymograms were then identified by in-gel trypsin digestions followed by peptide identification by matrix-assisted laser desorption ionization - time of flight - time of flight mass spectrometry （MS）. A custom A. glabripennis transcriptome database was used for peptide identification, giving highly significant matches in all MS analyses. These matches were then searched against the National Center for Biotechnology Information database to provide annotation to the transcripts and provide possible classification. From these analyses, we were able to detect enzymes active toward cellulose and xylan, and proteins putatively involved in lignocellulose degradation in the gut of this wood-feeding insect. Future research will be focused on characterizing these enzymes through cloning and expression experiments and understanding how the lignocellulose degradation system functions in the gut of this insect.

Cold adaptation of a mesophilic cellulase,EG Ⅲ from Trichoderma reesei,by directed evolution

Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase III (EG III) from Trichoderma reesei, and obtained a cold- adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG III with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG III calculated from Arrhenius equation are 13.3 kJ@mol-1 and 26.2 kJ@mol-1, respectively. Therefore, the increased specific activity of w-3 at lower temperatures could result from increased Kcat value and decreased activation energy.

Cloning and expression of endo-β-glucanase II gene in Humicola insolens H31-3

Endo-β-glucanase II(EG II)gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR.It was cloned into the expression vector pGAPZαA.The resultant recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after being linearized by BspHI digestion.The recombinant Pichia pastoris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation condition and the characteristics of the recombinant EG II were also explored.

Constitutive expression of codon optimized Trichoderma reesei TrCel5A in Pichia pastoris using GAP promoter

To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.