Chimeric lactase capable of spontaneous and strong immobilization on cellulose and development of a continuous-flow system for lactose hydrolysis at high temperatures.
ABSTRACT: Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.
Project description:The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, ?-(1?3,1?4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30% and on insoluble crystalline as well as amorphous cellulose by over 50%.
Project description:BACKGROUND:Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra- and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose. RESULTS:Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U.mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2:1 or 4:1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber. CONCLUSION:Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD.
Project description:In this study, the interaction forces between different cellulosic nanomaterials and a protein domain belonging to cellulose binding modules family 1 (CBM1) were investigated at the molecular scale. Cellulose binding modules are protein domains found in carbohydrate active enzymes having an affinity toward cellulosic materials. Here, the binding force of a fusion protein containing a cellulose binding module (CBM1) produced recombinantly in E. coli was quantified on different cellulose nanocrystals immobilized on surfaces. Adhesion of the CBM on cellulose with different degrees of crystallinity as well as on chitin nanocrystals was examined. This study was carried out by single molecule force spectroscopy using an atomic force microscope, which enables the detection of binding force of individual molecules. The study contains a preliminary quantification of the interactions at the molecular level that sheds light on the development of new nanocellulose-based nanocomposites with improved strength and elasticity.
Project description:Many methods have been used to detect heavy metals in herbal medicines, while few are developed to remove them. In this study, a novel genetically engineered fusion protein composed of metallothionein (MT), cellulose binding module (CBM), and superfolder GFP (sfGFP) was designed to remove heavy metals. MT, a kind of cysteine-rich protein, was used to chelate heavy metals with high specific affinity. The CBM facilitated the fusion protein MT-CBM-sfGFP binding to cellulose specifically, which made the purification and immobilization in one step. The sfGFP was used to detect the fusion protein MT-CBM-sfGFP easily during the process of expression and immobilization. The MT from Cancer pagurus (MTCap) and the CBM from Cellulomonas fimi (CBMCef) were used as an example and the fusion protein (MTCap-CBMCef-sfGFP) was expressed in Escherichia coli. Then, the cell lysates were mechanically mixed with cellulose to create biosorbent MTCap-CBMCef-sfGFP@cellulose. The efficiency of the biosorbent MTCap-CBMCef-sfGFP@cellulose for Pb2+ removal was evaluated using the water decoction of Honeysuckle as a model. Results suggested that MTCap-CBMCef-sfGFP@cellulose had high efficiency for Pb2+ removal from the water decoction of Honeysuckle without affecting its active ingredients. The low-cost, easy production, and high efficiency of the biosorbent enable it to have many applications in heavy metal removal from aqueous solutions of herbal medicines and food.
Project description:Four mini-scaffoldins were constructed from modules derived from the Clostridium thermocellum cellulosome-integrating protein CipA. Cip7 and Cip6 contained one and two cohesin modules respectively. Cip14 and Cip16, also containing one and two cohesin modules respectively, were flanked by a cellulose-binding domain. Endoglucanase CelD formed stable complexes with all mini-scaffoldins. Analytical ultracentrifugation of the complexes showed that 1 mol of CelD bound per mol of Cip14, and 2 mol of CelD bound per mol of Cip16. Under the conditions used for assaying cellulase activity, 96% of CelD alone bound to Avicel. Association with Cip14 or Cip16 increased the cellulose binding of CelD to 99%, while association with Cip7 or Cip6 decreased binding to 79 and 75% respectively. The hydrolytic activity of CelD against Avicel was increased 3-fold in complexes with Cip14 and Cip16, but remained substantially the same in complexes with Cip6 and Cip7. Addition of whole CipA also enhanced the efficiency of Avicel hydrolysis by CelD. However, even at an optimal ratio of the components, CelD-CipA complexes were somewhat less active than complexes of CelD with Cip14 or Cip16. These results suggest that the synergism observed between CelD and Cip14 or Cip16 is mostly due to the presence of the cellulose-binding domain, which promotes productive binding of the enzyme.
Project description:Background:Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WTintact), the catalytically deficient mutant Cel7A E212Q (E212Qintact) and their proteolytically isolated catalytic domains (CD) (WTcore and E212Qcore, respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy. Results:Binding of WTintact and E212Qintact on the recalcitrant algal cellulose (AC) showed two bound populations: ~?85% bound with shorter residence times of <?15 s while ~?15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WTcore and E212Qcore, exhibited three binding populations on AC: ~?75% bound with short residence times of ~?15 s (similar to the intact enzymes), ~?20% bound for <?100 s and ~?5% that were effectively immobilized. Conclusions:Cel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.
Project description:The cohesin-dockerin interaction in Clostridium thermocellum cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA. Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of C. thermocellum endoglucanase CelD (1,4-beta-d endoglucanase, EC) toward various cellulosic substrates. The seventh cohesin domain of CipA was fused to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBD(CBH1) and CBD(CBH2)) (1,4-beta-d glucan-cellobiohydrolase, EC), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBD(Cex)) (beta-1,4-d glucanase, EC), and from C. thermocellum CipA (CBD(CipA)). The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes. Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBD(CBH1) < CBD(CBH2) approximately CBD(Cex) < CBD(CipA). In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD. Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs. Furthermore, burst of activity also was observed upon adding new batches of CelD-CBD complexes that contained a CBD differing from the first one. This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping. The only exception was CBD(CipA), whose sites appeared to overlap all of the other sites.
Project description:Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.
Project description:We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3' to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter-1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.
Project description:Bacterial cellulose spheres subjected to amination and inlaid modification with superparamagnetic molecules were analyzed with regard to possibility of their application as an immobilization carrier of Lecitase® Ultra (LU) enzyme. The starting point to obtain the carrier was synthesis of bacterial cellulose spheres performed in shaking cultures of Komagataeibacter xylinus. These spheres were subsequently subjected to a multi-stage modification to increase the efficiency of the immobilization process and to separate product from the reaction medium. Maximal yield of Lecitase® Ultra immobilization equaled 70%. It was also found that immobilization process did not affect the pH and LU temperature optimum. Moreover, immobilized enzyme exhibited similar temperature stability profile as its native form. The immobilization process did not significantly affect the enzyme KM value. The immobilized enzyme retained over 70% of its initial activity after 8 cycles of use. The immobilized enzyme displayed good storage stability and retained 80% of its initial activity after 4 weeks at 4 °C. The potential application of such modified cellulose-based carrier may be correlated with lower costs of process thanks to higher enzyme's reusability in comparison to unbound enzyme. Moreover, data presented in the current study may serve as proof of a concept of cellulose-based carrier utilization for immobilization of enzymes other than LU and of high industrial importance.