Regenerated Amorphous Cellulose Research Articles

Expression of β-Glucosidases from the Yak Rumen in Lactic Acid Bacteria: A Genetic Engineering Approach.

β-glucosidase derived from microorganisms has wide industrial applications. In order to generate genetically engineered bacteria with high-efficiency β-glucosidase, in this study two subunits (bglA and bglB) of β-glucosidase obtained from the yak rumen were expressed as independent proteins and fused proteins in lactic acid bacteria (Lactobacillus lactis NZ9000). The engineered strains L. lactis NZ9000/pMG36e-usp45-bglA, L. lactis NZ9000/pMG36e-usp45-bglB, and L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB were successfully constructed. These bacteria showed the secretory expression of BglA, BglB, and Bgl, respectively. The molecular weights of BglA, BglB, and Bgl were about 55 kDa, 55 kDa, and 75 kDa, respectively. The enzyme activity of Bgl was significantly higher (p < 0.05) than that of BglA and BglB for substrates such as regenerated amorphous cellulose (RAC), sodium carboxymethyl cellulose (CMC-Na), desiccated cotton, microcrystalline cellulose, filter paper, and 1% salicin. Moreover, 1% salicin appeared to be the most suitable substrate for these three recombinant proteins. The optimum reaction temperatures and pH values for these three recombinant enzymes were 50 °C and 7.0, respectively. In subsequent studies using 1% salicin as the substrate, the enzymatic activities of BglA, BglB, and Bgl were found to be 2.09 U/mL, 2.36 U/mL, and 9.4 U/mL, respectively. The enzyme kinetic parameters (Vmax, Km, Kcat, and Kcat/Km) of the three recombinant strains were analyzed using 1% salicin as the substrate at 50 °C and pH 7.0, respectively. Under conditions of increased K+ and Fe2+ concentrations, the Bgl enzyme activity was significantly higher (p < 0.05) than the BglA and BglB enzyme activity. However, under conditions of increased Zn2+, Hg2+, and Tween20 concentrations, the Bgl enzyme activity was significantly lower (p < 0.05) than the BglA and BglB enzyme activity. Overall, the engineered lactic acid bacteria strains generated in this study could efficiently hydrolyze cellulose, laying the foundation for the industrial application of β-glucosidase.

Engineering processive cellulase of Clostridium thermocellum to divulge the role of the carbohydrate-binding module.

The processive cellulase (CelO) is an important modular enzyme of Clostridium thermocellum. To study the effect of the carbohydrate-binding module (CBM3b) on the catalytic domain of CelO (GH5), four engineered derivatives of CelO were designed by truncation and terminal fusion of CBM3b. These are CBM at the N-terminus, native form (CelO-BC, 62 kDa); catalytic domain only (CelO-C, 42 kDa); CBM at the C-terminus (CelO-CB, 54 kDa) and CBM attached at both termini (CelO-BCB, 73 kDa). All constructs were cloned into pET22b (+) and expressed in Escherichia coli BL21 (DE3) star. The expression levels of CelO-C, CelO-CB, CelO-BC, and CelO-BCB were 35%, 35%, 30%, and 20%, respectively. The enzyme activities of CelO-C, CelO-CB, CelO-BC, and CelO-BCB against 1% regenerated amorphous cellulose (RAC) were 860, 758, 985, and 1208 units per μmole of the enzyme, respectively. The enzymes were partially purified from the lysate of E. coli cells by heat treatment followed by anion exchange FPLC purification. Against RAC, CelO-C, CelO-CB, CelO-BC, and CelO-BCB showed KM values of 32, 33, 45, and 43 mg⋅mL-1 and Vmax values of 3571, 3846, 3571, and 4545 U⋅min-1 , respectively. CBM3b at the N-terminus of GH5 linked through a P/T-rich linker was found to enhance the catalytic activity and thermostability of the enzyme.

Cloning and expression of a novel trans-anethole oxygenase gene from Paraburkholderia sp. MR185.

trans-Anethole oxygenase (TAO) is the key enzyme responsible for the oxidation of trans-anethole to p-anisaldehyde. A strain, Paraburkholderia sp. MR185, was isolated from soil in Yulin star anise-planting regions using trans-anethole as a sole carbon source and a gene which encodes a protein with high similarities to a hypothetical protein of Paraburkholderia sp. MM5384-R2 which shows 61.27% identies with TAO from Pseudomonas putida JYR-1 was cloned and sequenced. The gene, tao, was expressed in E. coli cells and its protein product was purified by affinity chromatography through regenerated amorphous cellulose (RAC). SDS-PAGE analysis indicated a clear band of recombinant protein TAO, and its molecular weight, 38.3 kDa, was consistent with the theoretical value. Its enzyme activity of producing p-anisaldehyde from trans-anethole was detected by DNPH (2,4-dinitrophenylhydrazine) chromogenic reaction and HPLC, and the specific activity of TAO reached 3.93 U/mg protein. Immobilized TAO on RAC was used to catalyze the production of p-anisaldehyde from trans-anethole, and the enzyme retained more than 60% of its initial activity after 10 uses. This is the first report on Paraburkholderia TAO.

Oxidized Product Profiles of AA9 Lytic Polysaccharide Monooxygenases Depend on the Type of Cellulose.

Lytic polysaccharide monooxygenases (LPMOs) are essential for enzymatic conversion of lignocellulose-rich biomass in the context of biofuels and platform chemicals production. Considerable insight into the mode of action of LPMOs has been obtained, but research on the cellulose specificity of these enzymes is still limited. Hence, we studied the product profiles of four fungal Auxiliary Activity family 9 (AA9) LPMOs during their oxidative cleavage of three types of cellulose: bacterial cellulose (BC), Avicel PH-101 (AVI), and regenerated amorphous cellulose (RAC). We observed that attachment of a carbohydrate-binding module 1 (CBM1) did not change the substrate specificity of LPMO9B from Myceliophthora thermophila C1 (MtLPMO9B) but stimulated the degradation of all three types of cellulose. A detailed quantification of oxidized ends in both soluble and insoluble fractions, as well as characterization of oxidized cello-oligosaccharide patterns, suggested that MtLPMO9B generates mainly oxidized cellobiose from BC, while producing oxidized cello-oligosaccharides from AVI and RAC ranged more randomly from DP2–8. Comparable product profiles, resulting from BC, AVI, and RAC oxidation, were found for three other AA9 LPMOs. These distinct cleavage profiles highlight cellulose specificity rather than an LPMO-dependent mechanism and may further reflect that the product profiles of AA9 LPMOs are modulated by different cellulose types.

Biofilm-Mediated Immobilization of a Multienzyme Complex for Accelerating Inositol Production from Starch.

Bacterial biofilm, as a natural and renewable material, is a promising architecture for enzyme immobilization. In this study, we have demonstrated the feasibility of an Escherichia coli biofilm to immobilize a self-assembly multienzyme complex by the covalent interaction between a peptide SpyTag and its protein partner SpyCatcher. The SpyTag-labeled biofilm is displayed on the surface of E. coli by overexpressing the recombinant CsgA-SpyTag, in which CsgA is capable of forming biofilms. This SpyTag bearing biofilm is used to bind with SpyCatcher bearing synthetic mini-scaffoldin, which also contains a carbohydrate-binding module 3 (CBM3), and four different cohesins from different microorganisms. CBM3 was used to bind with cellulose, while the four different cohesins were used to recruit four dockerin-containing cascade enzymes, which were subsequently applied to convert starch to myo-inositol. Compared to the free enzyme mixture, the biofilm-immobilized enzyme complex exhibited a 4.28 times increase in initial reaction rate in producing myo-inositol from 10 g/L maltodextrin (a derivative of starch). Additionally, this biofilm-immobilized enzyme complex showed much higher recycle ability than the enzyme complex which was immobilized on a regenerated amorphous cellulose (RAC) system. In conclusion, the biofilm-mediated immobilization of the enzymatic biosystem provides a promising strategy to increase the reaction rate and enhance the stability of an in vitro enzymatic biosystem, exhibiting high potential to improve the efficiency of an in vitro biosystem.

CalkGH9T: A Glycoside Hydrolase Family 9 Enzyme from Clostridium alkalicellulosi

Glycoside hydrolase family 9 (GH9) endoglucanases are important enzymes for cellulose degradation. However, their activity on cellulose is diverse. Here, we cloned and expressed one GH9 enzyme (CalkGH9T) from Clostridium alkalicellulosi in Escherichia coli. CalkGH9T has a modular structure, containing one GH9 catalytic module, two family 3 carbohydrate binding modules, and one type I dockerin domain. CalkGH9T exhibited maximal activity at pH 7.0–8.0 and 55 °C and was resistant to urea and NaCl. It efficiently hydrolyzed carboxymethyl cellulose (CMC) but poorly degraded regenerated amorphous cellulose (RAC). Despite strongly binding to Avicel, CalkGH9T lacked the ability to hydrolyze this substrate. The hydrolysis of CMC by CalkGH9T produced a series of cello-oligomers, with cellotetraose being preferentially released. Similar proportions of soluble and insoluble reducing ends generated by hydrolysis of RAC indicated non-processive activity. Our study extends our knowledge of the molecular mechanism of cellulose hydrolysis by GH9 family endoglucanases with industrial relevance.

Sophorolipid Production Using Lignocellulosic Biomass by Co-culture of Several Recombinant Strains of Starmerella bombicola with Different Heterologous Cellulase Genes from Penicillum oxalicum.

One of the reasons hindering large-scale application of sophorolipids (SLs) is high production cost. In this study, six recombinant strains of Starmerella bombicola, sbEG1, sbEG2, sbCBH1, sbCBH1-2, sbBGL1, and sbCBH2 expressing cellulase genes eg1, eg2, cbh, cbh1-2, bgl1, and cbh2 from Penicillium oxalicum were respectively constructed. Four strains showed cellulase activities and were co-cultivated in fermentation media containing 2% glucose, 1% Regenerated Amorphous Cellulose (RAC), 2% glucose, and 1% RAC, respectively. After 7days' cultivation, concentration of SLs in medium with 1% RAC (g/L) reached 1.879g/L. When 2% glucose and 1% of RAC were both contained, the titer of SLs increased by 39.5% than that of control strain and increased by 68.8% than that in the medium with only 2% glucose. Results demonstrated that cellulase genes from filamentous fungi in S. bombicola can function to degrade lignocellulosic cellulose to produce SLs.

Enhanced Thermostability and Enzymatic Activity of Cel6A Variants from Thermobifida fusca by Empirical Domain Engineering (Short Title: Domain Engineering of Cel6A).

Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (cel6A) of Thermobifida fusca to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of cel6A.CBC which revealed thermostability of cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass.

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity.

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes.

Engineer multi-functional cellulase/xylanase/β-glucosidase with improved efficacy to degrade rice straw

Plant lignocellulosic biomass can be enzymatically degraded and converted into biofuel. We have studied a bifunctional cellulase/xylanase from thermophilic Clostridium thermocellum (CtCel5E), which can digest two major plant cell wall polysaccharides, cellulose (a polymer of glucose) and xylan (a major hemicellulose composed of xylose), into disaccharides cellobiose and xylobiose, respectively. In the study presented here, the bifunctional CtCel5E was fused with a β-glucosidase from Clostridium cellulovorans (CcBglA) to convert cellulose into glucose with improved activities toward carboxymethyl cellulose (CMC), regenerated amorphous cellulose (RAC), and alkali-treated rice straw as compared to the mixture of two individual enzymes by eliminating accumulation of cellobiose intermediate. The engineered CtCel5E-CcBglA was secreted from yeast to degrade alkali-treated rice straw into glucose that sustained cell growth. Our study provides a strategy of consolidating enzymes for more efficient biomass processing in vitro and by yeast cells.

Comparative study of the insoluble and soluble Ulp1 protease constructs as Carrier free and dependent protein immobilizates

In this study, we analyzed and compared the properties of yeast Ulp1 protease in active inclusion bodies (IBs) as special protein immobilizate, and the soluble Ulp1 via oriented immobilization. Fusion of the N-terminal self-assembling peptide GFIL8 to the Ulp1 increased production of active IBs in Escherichia coli. Attachment of the N-terminal cellulose-binding module facilitated the constructed protein immobilized on the regenerated amorphous cellulose (RAC) with a binding capacity up to about 235mg protein per gram of RAC. Compared with the immobilized soluble construct, the insoluble Ulp1 showed higher resistance to limited proteolysis with trypsin digestion, lower leaky amount at different storage temperatures, but more rapid decrease in cleavage activity after stored at 4°C for 8 days. The immobilized soluble Ulp1 maintained about 42% initial cleavage activity with repetitive use successively, whereas the aggregated Ulp1 lost its cleavage capacity after cleaving the protein substrate once. Crosslinking of IBs mediated by glutaraldehyde inactivated the Ulp1. Freshly prepared and used IBs showed similar resistance to protease-K digestion, and comparable binding capacity of Congo red and thioflavin T. Taken together, due to different advantages, the Ulp1 constructs as carrier-free and carrier-dependent immobilizates are used under different conditions.

A Novel Monomodular and Multifunctional Processive β‐1,4‐Endoglucanase Has Been Identified and Characterized from Porcine Gut Microbiome

Cellulose‐degrading enzymes play an important role in dietary fibre digestion of humans and animals, as well as in multiple industrial applications. Herein, a plasmid metagenomic expression library was constructed for mining cellulose‐degrading enzymes from the pig hindgut microbiota that was modulated by feeding a purified crystalline cellulose supplemented diet (Journal of Animal Science, 2012, 90 (Suppl. 4):400–402). One of the cellulase genes screened from this library was localized to a 2949‐base pair insert fragment p4818. Within the sequence preceding this enzyme‐coding region, ‐10 and ‐35 promoter elements were identified along with three transcription‐factor binding sites. The enzyme possesses a catalytic domain consistent with those of the glycoside hydrolase family 5 subfamily 2 (GH5_2), thus named as p4818Cel5_2A. The protein was overexpressed, purified, and characterized; and it displayed optimal enzymatic activity at pH 6.0 and 50 °C. The enzyme also exhibited resilience to trypsin digestion, where &gt;70 % activity was retained after 6.0 h incubation with trypsin at 37 °C. p4818Cel5_2A exhibited a broad substrate profile with the highest specific activity towards β‐glucan, carboxymethyl cellulose (CMC), followed by hydroxyethyl‐cellulose and regenerated amorphous cellulose (RAC). The enzyme was also active on other substrates including crystalline cellulose substrates such as Avicel and Solkafloc, as well as the hemicelluloses of glucomannan and xyloglucan. Furthermore, based on viscosity and hydrolysis product analyses, the enzyme behaved like a typical endo‐β‐1,4‐glucanase, and displayed processivity towards RAC. In summary, p4818Cel5_2A exhibited novel enzyme properties for potential industrial applications.Support or Funding InformationRelated research activities have been supported by grants (to M.Z.F.) from the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) New Directions &amp; Alternative Renewable Fuels Research Program; and the University of Guelph Gryphon's LAAIR (Leading to Accelerated Adoption of Innovative Research) program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The PT/S-Box of Modular Cellulase AcCel12B Plays a Key Role in the Hydrolysis of Insoluble Cellulose

Cellulases play key roles in the degradation of lignocellulosic materials. The function and mechanism of the catalytic domain (CD) and carbohydrate-binding module (CBM) of cellulases were earlier revealed by analysis and characterization of protein structure. However, understanding of the catalytic mechanism of the entire enzyme, and the analysis of the catalytic model, were inadequate. Therefore, the linker chain between CD and CBM has been extensively studied to bridge this gap. Cellulase AcCel12B and three mutants with different linker lengths (with no or 1–3 PT/S-box units) were successfully constructed and purified. Results showed that the activity of cellulases on Avicel and regenerated amorphous cellulose (RAC) increased with the number of PT/S-box units. Furthermore, the desorption of AcCel12B and its mutants from RAC and Avicel were significantly different. The energy of desorption of wild-type and mutant AcCel12B from cellulose decreased with the number of PT/S-box units. Thus, AcCel12B containing more PT/S-box units was more easily desorbed and had more opportunity to hydrolyze cellulose than other samples. The number of PT/S-box units in endocellulase affected the desorption of the enzyme, which is possibly responsible for the differences in the activity of wild-type and mutant AcCel12B on Avicel and RAC.

A Simple Method for Beta-glucosidase Immobilization and Its Application in Soybean Isoflavone Glycosides Hydrolysis

In this study, a simple, inexpensive and fast β-glucosidase immobilization system was constructed and evaluated in isoflavone glycosides hydrolysis. A β-glucosidase gene from Thermoascus aurantiacus IFO9748 was recombinantly expressed in Pichia pastoris KM71H and immobilized on regenerated amorphous cellulose (RAC) by fused cellulose binding module 3. Through simple mixing cellulose and crude enzyme for 15 min under room temperature, 96.04% β-glucosidase was immobilized onto RAC. The optimum temperature for β-glucosidase activity was increased by 5oC after immobilization. The half-life (t½) of heat inactivation of immobilized enzyme at 60oC was improved over 8 folds. After 30 rounds recycled at 40oC, 96.9% daidzin and 98.9% genistin could still be hydrolyzed. A continuous hydrolysis system was also constructed, and at the flow rate of 0.2 mL/min after 30 h hydrolysis, 95.6% genistin and 90.2% daidzin can still be hydrolyzed. Combined the simple and high efficient enzyme immobilization procedure and inexpensive cellulose, this scalable and practical system may have broad prospects for industrial utilization.

Tobacco etch virus protease mediating cleavage of the cellulose-binding module tagged colored proteins immobilized on the regenerated amorphous cellulose.

In this study, four fusion proteins were designed, in which the N-terminal cellulose-binding module as the affinity tag was immobilized on the regenerated amorphous cellulose (RAC), and the release of the C-terminal colored proteins was detected easily and rapidly after on-resin cleavage using the free tobacco etch virus protease (TEVp) variant, or the immobilized cognate protease with a binding capacity of up to 220mg protein per gram of RAC. The enhanced stability and repetitive use of the immobilized TEVp compensated slight loss of the catalytic efficiency toward the soluble protein substrate. On-resin cleavage and purity of the released target proteins are related to the context of the fusion tag, the incorporated linker composition, and the colored protein. Owing to low cost and high binding capacity of the RAC, the TEVp immobilized on the resin is an ideal alternative for removing fusion tag. The colored proteins are easily monitored in the on-resin process of fusion proteins, and rapid separation from RAC.

Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2

GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic, and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was purified and immobilized in a fusion form, and its reusability was investigated. The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB-gadB, which contained promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum) coding sequence, the gadB gene from L. fermentum YS2 coding for GAD, and the T7 terminator. After a one-step purification of CBM-GAD using regenerated amorphous cellulose (RAC) as an adsorbent, SDS-PAGE analysis revealed a clear band of 71 kDa; the specific activity of the purified fusion protein CBM-GAD reached 83.6 ± 0.7 U·mg-1. After adsorption onto RAC, the immobilized GAD with CBM3 tag was repeatedly used for GABA synthesis. The protein-binding capacity of RAC was 174 ± 8 mg·g-1. The immobilized CBM-GAD could repeatedly catalyze GABA synthesis, and 8% of the initial activities was retained after 10 uses. We tested the conversion of monosodium glutamate to GABA by the immobilized enzyme; the yield reached 5.15 g/L and the productivity reached 3.09 g/L·h. RAC could be used as an adsorbent in one-step purification and immobilization of CBM-GAD, and the immobilized enzyme could be repeatedly used to catalyze the conversion of glutamate to GABA.

The Development of Leucine Dehydrogenase and Formate Dehydrogenase Bifunctional Enzyme Cascade Improves the Biosynthsis of L-tert-Leucine

Leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) were assembled together based on a high-affinity interaction between two different cohesins in a miniscaffoldin and corresponding dockerins in LDH and FDH. The miniscaffoldin with two enzymes was further absorbed by regenerated amorphous cellulose (RAC) to form a bifunctional enzyme complex (miniscaffoldin with LDH and FDH adsorbed by RAC, RSLF) in vitro. The enzymatic characteristics of the bifunctional enzyme complex and free enzymes mixture were systematically compared. The synthesis of L-tert-leucine by the RSLF and free enzyme mixture were compared under different concentrations of enzymes, coenzyme, and substrates. The initial L-tert-leucine production rate by RSLF was enhanced by 2-fold compared with that of the free enzyme mixture. Ninety-one grams per liter of L-tert-leucine with an enantiomeric purity of 99% e.e. was obtained by RSLF multienzyme catalysis. The results indicated that the bifuntional enzyme complex based on cohesin-dockerin interaction has great potential in the synthesis of L-tert-leucine.

Using cellulose binding module of CBM3A as the purification tag for secreted Endoglucanase A (CelA) in Bacillus subtilis

Traditional methods of recombinant protein purification are uneconomic and inconvenient to the secreted proteins at large-volume. CBM3a, a module from cellulosome’s scaffoldin of Clostridium thermocellum, directs the binding of the cellulase complex on the cheap cellulose substrate. Most of previous studies about CBM3a fused with cellulases as the purification tag were conducted in intracellular Escherichia coli system. In this research, we used the extracellular Bacillus subtilis WB800N expression system to investigate the CBM3a-tag fused with endoglucanase CelA into plasmid pHT. The results indicated that protein CelA was secrected and purified by CBM3a-tag binding on the Regenerated Amorphous Cellulose (RAC) subtrate. This can be used for further improvement in protein purification tag designing.

Extra carbohydrate binding module contributes to the processivity and catalytic activity of a non-modular hydrolase family 5 endoglucanase from Fomitiporia mediterranea MF3/22.

FmEG from Fomitiporia mediterranea is a non-modular endoglucanase composed of a 24-amino acids extension and 13-amino acids linker-like peptide at the N-terminus and a 312-amino acids GH5 catalytic domain (CD) at the C-terminus. In this study, six FmEG derivatives with deletion of N-terminal fragments or fusion with an extra family 1 carbohydrate-binding module (CBM1) was constructed in order to evaluate the contribution of CBM1 to FmEG processivity and catalytic activity. FmEG showed a weak processivity and released cellobiose (G2) and cellotriose (G3) as main end products, and cellotriose (G4) as minor end product from filter paper (FP), but more amount of G4 was released from regenerated amorphous cellulose (RAC). All derivatives had similar activity on carboxymethylcellulose (CMC) with the same optimal pH (7.0) and temperature (50°C). However, fusing an extra CBM1 to FmEG△24 or FmEG△37 with flexible peptide significantly improved its processivity and catalytic activity to FP and RAC. Overall, 1.79- and 1.84-fold increases in the soluble/insoluble product ratio on FP, and 1.38- and 1.39-fold increases on RAC, compared to FmEG△24, were recorded for CBM1-FmEG△24 and CBM1-linker-FmEG△24, respectively. Meanwhile, they displayed 2.64- and 2.67-fold more activity on RAC, and 1.68- and 1.77-fold on FP, respectively. Similar improvement was also obtained for CBM1-linker-FmEG△37 as compared with FmEG△37. Interestingly, fusion of an extra CBM1 with FmEG also caused an alteration of cleavage pattern on insoluble celluloses. Our results suggest that such improvements in processivity and catalytic activity may arise from CBM1 binding affinity. The N-terminal 24- or 37-amino acids may serve as linker for sufficient spatial separation of the two domains required for processivity and catalytic activity. In addition, deletion of the N-terminal 24- or 37-amino acids led to significant reduction in thermostability but not the enzymatic activity.

Cloning, Expression, and Characterization of a Thermophilic Endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B.

The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg−1, respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL−1 and 131.75 U·mg−1, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future.