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

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

Purification and biochemical characterization of novel α-amylase and cellulase from Bacillus sp. PM06.

Bacillus sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate's crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/min/mg, 1.154 ± 0.1 mM, and 589 ± 3.5/(s mM), using starch as a substrate. Whereas cellulase showed 556.3 ± 1.3 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(s mM) of Vmax, Km, Kcat/Km, respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted α-amylase and cellulase activities. Cu2+ and Ni2+ both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 ± 1.7 and 147.5 ± 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified α-amylase and cellulase. A more significant amount of 397.7 ± 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes.

Screening, cloning, enzymatic properties of a novel thermostable cellulase enzyme, and its potential application on water hyacinth utilization.

Cellulose is the cheapest, natural, renewable organic substance that is used as a carbon source in various fields. Water hyacinth, an aquatic plant rich in cellulose, is often used as a raw material in fuel production. However, natural cellulase can be hardly used in industrial production on account of its low thermal stability and activity. In this study, a metagenomic library was constructed. Then, a new cellulase gene, cel1029, was screened by Congo red staining and expressed in the prokaryotic system. Enzymatic properties of Cel1029 were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions, and salt solutions. Finally, its ability of degrading water hyacinth was identified and evaluated. Cel1029 displayed high homology with endoglucanase in the glycoside hydrolase family 5 (GH5) and had high stability across a broad temperature range. More than 86% of its enzymatic activities were retained between 4 and 60 °C after 24 h of incubation. Single-factor analysis and orthogonal design were further conducted to determine the optimal conditions for the highest reducing sugar yield of water hyacinth. Interestingly, Cel1029 efficiently transformed water hyacinth with a reducing sugar yield of 430.39 mg/g in 22 h. These findings may open the door for significant industrial applications of a novel GH5 cellulase (NCBI Reference Sequence: MK051001, Cel1029) and help identify more efficient methods to degrade cellulose-rich plants.

Overexpression and characterization of TnCel12B, a hyperthermophilic GH12 endo-1,4-ß-glucanase cloned from Thermotoga naphthophila RKU-10T.

A putative cellulolytic gene (825 bp) from Thermotoga naphthophila RKU-10T was overexpressed as an active soluble endo-1,4-ß-glucanase (TnCel12B), belongs to glycoside hydrolase family 12 (GH12), in a mesophilic expression host. Heterologous expression and engineered bacterial cell mass was improved through specific strategies (induction and cultivation). Hence, intracellular activity of TnCel12B was enhanced in ZYBM9 modified medium (pH 7.0) by 8.38 and 6.25 fold with lactose (200 mM) and IPTG (0.5 mM) induction, respectively; and 6.95 fold was increased in ZYP-5052 auto-inducing medium after 8 h incubation at 26 °C (200 rev min-1). Purified TnCel12B with a molecular weight of ~32 kDa, was optimally active at 90 °C and pH 6.0; and exhibited prodigious stability over a wide range of temperature (50-85 °C) and pH (5.0-9.0) for 8 h TnCel12B displayed great resistance towards different chemical modulators, though activity was improved by Mg2+, Zn2+, Pb2+ and Ca2+. Purified TnCel12B had affinity with various substrates but peak activity was observed toward barley ß-glucan (1664 U mg-1) and carboxymethyl cellulose (736 U mg-1). The values of Km, Vmax, kcat, and kcatKm-1 were found to be 4.63 mg mL-1, 916 µmol mg-1min-1, 1326.7 s-1 and 286.54 mL mg-1 s-1, respectively using CMC substrate. All noteworthy features of TnCel12B make it an appropriate industrial candidate for bioethanol production and various other potential applications.

Non-chromatographic purification of thermostable endoglucanase from Thermotoga maritima by fusion with a hydrophobic elastin-like polypeptide.

Endoglucanase EG12B from Thermotoga maritima is a thermophilic cellulase that has great potential for industrial applications. Here, to enable the selective purification of EG12B in a simple and efficient manner, an elastin-like polypeptide (ELP), which acts as a thermally responsive polypeptide, was fused with EG12B to enable its inverse phase transition cycling (ITC). A small gene library comprising ELPs from ELP5 to ELP50 was constructed using recursive directional ligation by plasmid reconstruction. ELP50 was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP50, which was transformed into Escherichia coli BL21 (DE3) to enable the expression of fusion protein via IPTG induction. Gray scanning analysis revealed that the EG12B-ELP50 expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP50 was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP50 purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP50 had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP50 could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification.

Purification and Biochemical Characterization of Alkalophilic Cellulase from the Symbiotic Bacillus subtilis BC1 of the Leopard Moth, Zeuzera pyrina (L.) (Lepidoptera: Cossidae).

In the current study, an extracellular cellulase belonging to symbiotic Bacillus subtilis Bc1 of the leopard moth is purified and characterized. The molecular mass of enzyme was 47.8 kDa using SDS-PAGE. The purified enzyme had optimum activity in temperature and pH around 60 °C and 8, respectively. The purified cellulase was introduced as a stable enzyme in a wide variety of temperature (20-80 °C) and pH (4-10) and remained active to more than 74% at 80 °C for 1 h. Moreover, the cellulase extremely was stabled in the presence of metal ions and organic solvents and its activity was increased by acetone (20% v/v), CaCl2 and CoCl2 and inhibited by MnCl2 and NiCl2. The values of enzyme's Km and Vmax were found to be 1.243 mg/mL and 271.3 µg/mL/min, respectively. The purified cellulase hydrolyzed cellulose, avicel and carboxymethyl cellulose (CMC) and the final product of CMC hydrolysis was cellobiose using thin-layer chromatography analysis. Consequently, owing to exo/endoglucanase activity and organic solvent, temperature and pH stability of the purified cellulase belong to B. subtilis BC1, it can be properly employed for various industrial purposes.

Enhanced purification of histidine-tagged carboxymethylcellulase produced by Escherichia coli BL21/LBH-10 and comparison of its characteristics with carboxymethylcellulase without histidine-tag.

To enhance purification yield of the carboxymethylcellulase (CMCase) of P. aquimaris LBH-10, E. coli BL21/LBH-10 was constructed to produce the six histidine-tagged CMCase (CMCase with a His-tag). The purification yield of the CMCase with a His-tag produced by E. coli BL21/LBH-10 was 44.4%. The molecular weight of the CMCase with a His-tag was determined as 56 kDa. Its Km and Vmax were 7.4 g/L and 70.9 g/L min, respectively. The CMCase with a His-tag hydrolyzed avicel, carboxymethylcellulose (CMC), filter paper, pullulan, and xylan but did not hydrolyze cellobiose and p-nitrophenyl-ß-D-glucopyranoside. The optimal temperature for reaction was 50 °C and more than 75% of its original activity was maintained at broad temperatures ranging from 20 to 70 °C after 24 h. The optimal pH was 4.0 and more than 60% of its original activity was maintained at pH ranging from 4.0 to 7.0. The activity of the CMCase with a His-tag was enhanced by CoCl2, KCl, PbCl2, RbCl2, and SrCl2 until the concentration of 100 mM, but inhibited by EDTA, HgCl2, MnCl2, and NiCl2. The characteristics of the CMCase with a His-tag produced by E. coli BL21/LBH-10 were little different from the CMCase without a His-tag, which seemed to resulted from the conformational change in the structure due to a His-tag. The purification yield of the CMCase with a His-tag using affinity chromatography from the cell broth after cell breakdown was proven to be more economic than that from the supernatant with its low concentration of cellulase.

Enzyme production by solid-state fermentation on soybean meal: A comparative study of conventional and ultrasound-assisted extraction methods.

Protease, cellulase, and α-amylase producing Bacillus subtilis strain was cultivated by solid-state fermentation technique using soybean meal as a substrate. The aim of the present study was to establish a highly efficient enzymes' extraction method as a first stage in downstream processing. The conventional extraction procedure was optimized by determining pH, stirring rate, solid/liquid ratio, and time of extraction on enzymes' recoveries from fermented soybean meal. Yields of leached enzymes were compared to the amounts of enzymes that are achieved with ultrasound-assisted extraction (UAE). UAE was established to be superior method for obtaining higher yields of proteases (up to 330 IU) and α-amylases (825 IU), under significantly shorter extraction time and gaining more concentrated product. However, the obtained model predicts that conventional process led to a product with a higher cellulolytic activity (≥7.5 IU).

Cellulolytic enzyme production from agricultural residues for biofuel purpose on circular economy approach.

This study evaluated the production of cellulolytic enzymes from different agricultural residues. The crude enzyme extract produced was characterized and applied for saccharification of some agricultural residues. Maximum cellulolytic activities were obtained using soybean hulls. All enzymatic activities were highly stable at 40 °C at a pH range of 4.5-5.5. For stability at low temperatures, the enzyme extract was stored at freezing temperature and cooling for about 290 days without major loss of activity. The Km values found for total cellulase (FPase), endoglucanase (CMCase), and xylanase were 19.73 mg ml-1, 0.65 mg ml-1, and 22.64 mg ml-1, respectively, and Vmax values were 0.82 mol min-1 mg-1, 0.62 mol min-1 mg-1, and 104.17 mol min-1 mg-1 to cellulose, carboxymethyl cellulose, and xylan, respectively. In the saccharification tests, the total amount of total reducing sugars (TRS) released from 1 g of soybean hulls catalyzed by the enzymes present in the crude enzyme extract was 0.16 g g-1 dry substrate.

Haloferax sulfurifontis GUMFAZ2 producing xylanase-free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa-India.

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.

Cold-tolerant endoglucanase producing ability of Mrakia robertii A2-3 isolated from cryoconites, Hamtha glacier, Himalaya.

A psychrotolerant yeast strain Mrakia robertii A2-3 isolated from cryoconites of Hamtah glacier, Himalaya, India was investigated for the production of cold-tolerant endoglucanase. Optimum endoglucanase production was found at 15°C with an initial pH of 5.5, and potent inducers were 1% wt/vol of xylose and KNO3 and 0.1% wt/vol of NaCl. Under optimum conditions, the enzyme production was 1.81-fold higher than the unoptimized conditions. Crude enzyme was partially purified by ammonium sulfate precipitation followed by dialysis. The enzyme was purified to 2.53-fold and the yield was 6.03% with specific activity of 17.38 U/mg and molecular weight ~57 kDa. The Km and Vmax values of the partially purified enzyme were found to be 1.57 mg/ml and 142.85 U/mg, respectively. The characterization study revealed that the best temperature was 15°C for activity and stability. Furthermore, the enzyme showed the highest activity at pH 11.0 and was stable at pH 6.0. Fe2+ , Mn2+ , Na2+ , Cu2+ , Co2+ , Ca2+ proved to be activators of endoglucanase. Ethylenediamine tetraacetic acid showed very low effect on the enzyme activity whereas it was active with Tween-80 and sodium deoxycholate. The present study successfully produced a cold-active endoglucanase with novel properties making it promising as a biocatalyst for industrial processes.

Efficient enzymatic saccharification of macroalgal biomass using a specific thermostable GH 12 endoglucanase from Aspergillus terreus JL1.

Stranded green macroalgae represents an important and renewable biomass that remains under valorized despite the numerous environmental problems generated by their accumulation in coastal regions. This work describes the isolation of a filamentous thermophile fungus identified as Aspergillus terreus JL1 that produces an efficient cellulolytic activity for green macroalgae saccharification. The characterization of the endoglucanase activity obtained after submerged fermentation showed a differential induction depending on the carbon source used with a unique isoform released when Ulva lactuca was used as inducer. The crude extract obtained hydrolyzed efficiently the untreated algal biomass (70.5%) compared to other cellulolytic extracts. The unique endoglucanase released was then purified to homogeneity (Yield: 49.6%; Specific activity: 30.1 U/mg; Purification fold: 4.36) and characterized biochemically. Its peptidic sequence was then determined and showed its belonging to the GH12. The described enzyme represents a promising biotechnological tool for algal biomass conversion.

Biochemical and structural analyses of a bacterial endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

ß-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite ß-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear ß-1,2-glucan. To this end, we purified an endo-ß-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo-ß-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis The Cpin_6279 protein specifically hydrolyzed linear ß-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an endo-ß-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-ß-1,2-Glc-ß-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)6-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other ß-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris.

BACKGROUND: Cellulases are of great significance for full utilization of lignocellulosic biomass. Termites have an efficient ability to degrade cellulose. Heterologous production of the termite-origin cellulases is the first step to realize their industrial applications. The use of P. pastoris for the expression of recombinant proteins has become popular. The endoglucanase from Reticulitermes speratus (RsEG), belonging to glycoside hydrolase family 9 (GHF9), has not been produced in P. pastoris yet. RESULTS: A mutant RsEGm (G91A/Y97W/K429A) was successfully overexpressed in P. pastoris. RsEGm, with optimum pH 5.0, was active over the pH range of 4.0 to 9.0, and exhibited superior pH stability over between pH 4.0 and pH 11.0. It displayed the highest activity and good stability at 40 °C, but lost activity quickly at 50 °C. The apparent kinetic parameters of RsEGm against Carboxymethyl Cellulose (CMC) were determined, with K m and V max of 7.6 mg/ml and 5.4 µmol/min•mg respectively. Co2+, Mn2+ and Fe2+ enhanced the activity of RsEGm by 32.0, 19.5 and 11.2% respectively, while Pb2+ and Cu2+ decreased its activity by 19.6 and 12.7% separately. CONCLUSIONS: RsEGm could be overexpressed in P. pastoris. It was stable between pH 4.0 and pH 11.0, and exhibited higher stability at temperatures ≤ 40 °C. This endoglucanase may have potential to be used in the field of laundry, textile and lignocellulose-based biofuels and chemicals.

The ectomycorrhizal basidiomycete Laccaria bicolor releases a secreted ß-1,4 endoglucanase that plays a key role in symbiosis development.

In ectomycorrhiza, root ingress and colonization of the apoplast by colonizing hyphae is thought to rely mainly on the mechanical force that results from hyphal tip growth, but this could be enhanced by secretion of cell-wall-degrading enzymes, which have not yet been identified. The sole cellulose-binding module (CBM1) encoded in the genome of the ectomycorrhizal Laccaria bicolor is linked to a glycoside hydrolase family 5 (GH5) endoglucanase, LbGH5-CBM1. Here, we characterize LbGH5-CBM1 gene expression and the biochemical properties of its protein product. We also immunolocalized LbGH5-CBM1 by immunofluorescence confocal microscopy in poplar ectomycorrhiza. We show that LbGH5-CBM1 expression is substantially induced in ectomycorrhiza, and RNAi mutants with a decreased LbGH5-CBM1 expression have a lower ability to form ectomycorrhiza, suggesting a key role in symbiosis. Recombinant LbGH5-CBM1 displays its highest activity towards cellulose and galactomannans, but no activity toward L. bicolor cell walls. In situ localization of LbGH5-CBM1 in ectomycorrhiza reveals that the endoglucanase accumulates at the periphery of hyphae forming the Hartig net and the mantle. Our data suggest that the symbiosis-induced endoglucanase LbGH5-CBM1 is an enzymatic effector involved in cell wall remodeling during formation of the Hartig net and is an important determinant for successful symbiotic colonization.

Expression and characterisation of a thermophilic endo-1,4-ß-glucanase from Sulfolobus shibatae of potential industrial application.

An endo-1,4-ß-D-glucanase gene was cloned from the thermophilic archaea Sulfolobus shibatae and expressed in E. coli. The recombinant enzyme was purified by heat denaturation and affinity chromatography prior to characterisation. The purified recombinant enzyme exhibited maximum activity at 95-100 °C and displayed a broad pH profile with over 91% of its maximum activity observed at pH 3-5. Upon assessment of enzyme thermal stability, full activity was observed after 1 h incubation at 75, 80 and 85 °C while 98%, 90% and 84% of original activity was detected after 2 h at 75, 80 and 85 °C, respectively. Maximum activity was observed on barley ß-glucan and lichenan and the purified enzyme also hydrolysed CMC and xylan. Endoglucanase activity was confirmed by viscometric assay with a rapid decrease in substrate viscosity observed immediately upon incubation with barley ß-glucan or CMC. The crude enzyme released reducing sugars from acid-pretreated straw at 75-85 °C. The thermophilic nature and biochemical properties of the enzyme indicate its potential suitability in industrial applications undertaken at high temperature, such as the production of second-generation bioethanol from lignocellulosic feedstocks and in the brewing industry. This is the first known report of an endoglucanase from S. shibatae.

Expression, purification and molecular characterization of a novel endoglucanase protein from Bacillus subtilis SB13.

Bacillus subtilis strain SB13 which is isolated in our previous work was confirmed to produce endoglucanase. In this study, a novel endoglucanase gene (accession number: KX576676) was identified and cloned from SB13. Compared with other consensus sequence of reported endoglucanase genes in the GenBank database, this gene displays five differences (including T740C,A874G,A983G, T1210G and T1301C), which leading to five amino acid changes. Homology modeling has indicated that these five changes were located in the α-helix and random coil regions of the glycosyl hydrolase family 5 (GH5) domain, the random coil and ß-sandwich of the type 3 carbohydrate-binding module (CBM3) domain, and the random coil domain. Aprokaryotic expression vector pET30a-endoglucanase was constructed and the endoglucanase was induced to express. The expressed endoglucanase was confirmed by liquid chromatography-tandem mass spectrometry (LC-MSMS) and detected via reaction with carboxymethyl cellulose. In order to obtain the highest expression level of endoglucanase, the expression conditions including IPTG concentration, temperature and pH were optimized. The recombinant endoglucanase protein was purified using a Ni-NTA column, and the 6 × His-tag was removed with thrombin. The results showed that both the modified and unmodified purified endoglucanase had high activity (7.65 ± 0.35 U and 15.05 ± 1.81 U, respectively), thus demonstrating the potential use of this enzyme in various industrial applications. The substitutions of L247P,N292D, F404V and L434P might contribute to the activity of the endoglucanase, and the insertion of a 6 × His-tag at the N-terminal of the endoglucanase might also affect its activity.

High-level expression and purification of a molluskan endoglucanase from Ampullaria crossean in Pichia pastoris.

EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the mollusk Ampullaria crossean. In this study, the mature EG27I peptide gene fused to the HFBII secretion signal of Trichoderma reesei was expressed under the GAP promoter of Pichia pastoris in SMD1163 strain. A bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into our culture medium. The respective final OD600 and hydrolytic activity were 333 and 1.28 U/mL when high-cell-density fermentation of the recombinant P. pastoris was performed in a 7.5 L fermenter through a fed-batch strategy for 132 h. The recombinant protein concentration of the fermentation supernatant was 47.7 mg/L. EG27I was consecutively purified from the fermentation supernatant through ultrafiltration, cation exchange, and hydrophobic interaction. The specific activity of the recombinant EG27I was 26.8 U/mg, and the optimal pH and temperature of the enzyme were 5 and 50 °C, respectively. The half-life of the enzyme activity at 100 °C could reach 40 min. The N-terminal amino acid sequence analysis of the purified recombinant protein confirmed that the amino terminal sequence was consistent with the natural structure. The high quantity and purity of the EG27I provide a basis for future structural and functional studies.

Comparison of transplastomic Chlamydomonas reinhardtii and Nicotiana tabacum expression system for the production of a bacterial endoglucanase.

The bulk production of recombinant enzymes by either prokaryotic or eukaryotic organisms might contribute to replace environmentally non-friendly chemistry-based industrial processes with enzyme-based biocatalysis, provided the cost of enzyme production is low. In this context, it is worth noting that the production of recombinant proteins by photosynthetic organisms offer both eukaryotic (nuclear) and prokaryotic (chloroplast) alternatives, along with the advantage of an autotrophic nutrition. Compared to nuclear transformation, chloroplast transformation generally allows a higher level of accumulation of the recombinant protein of interest. Furthermore, among the photosynthetic organisms, there is a choice of using either multicellular or unicellular ones. Tobacco, being a non-food and non-feed plant, has been considered as a good choice for producing enzymes with applications in technical industry, using a transplastomic approach. Also, unicellular green algae, in particular Chlamydomonas reinhardtii, have been proposed as candidate organisms for the production of recombinant proteins. In the light of the different features of these two transplastomic systems, we decided to make a direct comparison of the efficiency of production of a bacterial endoglucanase. With respect to the amount obtained, 14 mg g-1 of biomass fresh weight equivalent to 8-10% of the total protein content and estimated production cost, 1.5-2€ kg-1, tobacco proved to be far more favorable for bulk enzyme production when compared to C. reinhardtii which accumulated this endoglucanase at 0.003% of the total protein.

Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.

In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V max and catalytic efficiency (k cat/K M) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.