Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein.

Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp. The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose. Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8. The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8. Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C. At higher temperatures, the bound enzyme lost activity. The thermal stability of the fusion protein was greatly improved by immobilization. Immobilization did not alter the pH stability. Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme.     

Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.     

Specific Adhesion to Cellulose and Hydrolysis of Organophosphate Nerve Agents by a Genetically Engineered Escherichia coli Strain with a Surface-Expressed Cellulose-Binding Domain and Organophosphorus Hydrolase

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.     

温和气单孢菌YH311硫酸软骨素裂解酶的分离纯化与固定化

通过硫酸铵沉淀、QAESephadex-A50柱层析及Sephadex-G150凝胶过滤等纯化步骤,对源自温和气单孢菌YH311的ChSase进行了分离纯化。结果表明,ChSase经上述纯化步骤后被纯化了55倍,其最终纯度可达95%以上,比活为31.86u/mg。经SDSPAGE及IFE测定可知该酶的分子量约为80kD,等电点为4.3～4.8。将纯化后的ChSase用海藻酸钠或纤维素固定化后,ChSase的热稳定性及贮存稳定性均可得到大幅度的提高：固定化酶用80℃水浴处理120min或于4℃冰箱放置30d后仍可保留50%以上的相对活力;但固定化酶的收率较低,仅为18.56%和18.86%。     

New cationic exchanger support for reversible immobilization of proteins

New tailor-made cationic exchange resins have been prepared by covalently binding aspartic-dextran polymers (e.g. MW 15 000-20 000) to porous supports (aminated agarose and Sepabeads). More than 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans have been strongly adsorbed on these porous materials at pH 5. This interaction was stronger than in conventional carboxymethyl cellulose (e.g., at pH 7 and 25 degrees C, all proteins previously adsorbed at pH 5 were released from carboxymethyl cellulose, whereas no protein was released from the new supports under similar conditions). Ionic exchange properties of such composites were strongly dependent on the size of the aspartic-dextran polymers as well as on the exact conditions of the covalent coating of the solids with the polymer (optimal conditions: 100 mg aspartic-dextran 20 000/(mL of support); room temperature). Finally, some industrially relevant enzymes (Kluyveromices lactis, Aspergillus oryzae, and Thermus sp. beta-galactosidases, Candida antarctica B lipase, and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recovery and immobilization rates. After enzyme inactivation, the enzyme can be fully desorbed from the support and the support could be reused for several cycles.     

Effect of physical conditions and chemicals on the binding of a mini-CbpA from Clostridium cellulovorans to a semi-crystalline cellulose ligand

Aims:  To investigate the effect that environmental factors have on Clostridium cellulovorans cellulose binding domain (CBD) binding to a semi-crystalline cellulose ligand, namely Avicel.
Methods and Results:  The behaviour of a 58 kDa mini-CbpA protein containing the CBD from the scaffoldin protein of C. cellulovorans was studied in the presence of various environmental factors, in order to determine whether such factors promote or reduce CBD binding to its ligand, thus potentially affecting its activity on the substrate. The amount of binding was found to be dependent on the Avicel concentration and optimal binding occurred when the ligand concentration was 15 mg ml⁻¹. Optimal CBD binding occurred at pH 7·0 and at an incubation temperature of 28°C. The effects of dithiothreitol (DTT), 2-mercaptoethanol, acetone, butanol, ethanol and butyric acid were also investigated.
Conclusions:  Temperature, pH, DTT, 2-mercaptoethanol and solvents were shown to affect the binding of C. cellulovorans CBD to Avicel.
Significance and Impact of the Study:  Clostridium cellulovorans CBD binding to Avicel is affected by physical conditions and chemicals.     

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.     

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes was studied by incubating the membranes at different pH and temperatures and determining the bound bilirubin. At all pH values, the amount of membrane-bound bilirubin increased with the increase in bilirubin-to-albumin molar ratios (B/As), being highest at lower pH values in all cases. Further, linear increase in bound bilirubin with the increase in bilirubin concentration in the incubate was observed at a constant B/A and at all pH values. However, the slope value increased with the decrease in pH suggesting more bilirubin binding to membranes at lower pH values. Increase in bilirubin binding at lower pH can be explained on the basis of increased free bilirubin concentration as well as more conversion of bilirubin dianion to monoanion. Temperature dependence of bilirubin binding to membranes was observed within the temperature range of 7 degrees -60 degrees C, showing minimum binding at 27 degrees C and 37 degrees C which increased on either side. Increase in bilirubin binding at temperatures lower than 20 degrees C and higher than 40 degrees C can be ascribed to the change in membrane topography as well as bilirubin-albumin interaction.     

Functionalization of Recombinant Amelogenin Nanospheres Allows Their Binding to Cellulose Materials

Protein engineering to functionalize the self‐assembling enamel matrix protein amelogenin with a cellulose binding domain (CBD) is used. The purpose is to examine the binding of the engineered protein, rh174CBD, to cellulose materials, and the possibility to immobilize self‐assembled amelogenin nanospheres on cellulose. rh174CBD assembled to nanospheres ≈35 nm in hydrodynamic diameter, very similar in size to wild type amelogenin (rh174). Uniform particles are formed at pH 10 for both rh174 and rh174CBD, but only rh174CBD nanospheres showes significant binding to cellulose (Avicel). Cellulose binding of rh174CBD is promoted when the protein is self‐assembled to nanospheres, compared to being in a monomeric form, suggesting a synergistic effect of the multiple CBDs on the nanospheres. The amount of bound rh174CBD nanospheres reached ≈15 mg/g Avicel, which corresponds to 4.2 to 6.3 × 10^?7 mole/m². By mixing rh174 and rh174CBD, and then inducing self‐assembly, composite nanospheres with a high degree of cellulose binding can be formed, despite a lower proportion of rh174CBD. This demonstrates that amelogenin variants like rh174 can be incorporated into the nanospheres, and still retain most of the binding to cellulose. Engineered amelogenin nanoparticles can thus be utilized to construct a range of new cellulose based hybrid materials, e.g. for wound treatment.     

Immobilization of Lactococcus lactis to cellulosic material by cellulose‐binding domain of Cellvibrio japonicus

Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.     

Improved immobilization of fusion proteins via cellulose-binding domains

Cellulose-binding domains (CBDs) are structurally and functionally independent, noncatalytic modules found in many cellulose or hemicellulose degrading enzymes. Recent biotechnological applications of the CBDs include facilitated protein immobilization on cellulose supports. In some occasions there have been concerns about the stability of the CBD driven immobilization. Here we have studied the chromatographic behavior of variants of the Trichoderma reesei cellobiohydrolase I CBD belonging to family I. Both CBDs fused to antibody fragments and isolated CBDs were studied and compared. Tritium labeling by reductive methylation was used as a sensitive detection method. The fusion protein as well as the isolated CBD was found to leak from the column at a rate of 0.3-0.5% of the immobilized protein per column volume. However, the leakage could be overcome by using two CBDs instead of a single CBD for the immobilization. In this way leakage was reduced to less than 0.01% per column volume. The improved immobilization could also be seen as a decreased migration of the protein down the column in extended washes.     

Binding and processing of (125)I-ACTH by isolated rat splenic lymphocytes

The effect of incubation temperature and ligand competition was tested for (125)I-ACTH binding to isolated rat lymphocytes. AlphaMSH but not Agouti-like peptide was an effective competitive inhibitor for cell surface binding at 4 degrees C. Cells incubated with (125)I-ACTH at 37 degrees C rapidly associated ligand for 10 min and then gradually lost the radioactivity with time. Cells incubated with (125)I-ACTH at 4 degrees C accumulated ligand to only about half the maximal amount when compared to cells incubated at 37 degrees C for 10 min. Temperatures below 20 degrees C and toxins that block lysosomal degradation blocked the loss of cell-associated radioactivity. These results suggest the lymphocyte ACTH receptor is the Melanocortin 5 receptor and the receptor is internalized by endocytosis to deliver ligand to the lysosome.     

Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells

GH4C1 cells, a clonal strain of rat pituitary tumor cells, have high-affinity, functional receptors for the inhibitory hypothalamic peptide somatostatin (SRIF) and for epidermal growth factor (EGF). In this study we have examined the events that follow the initial binding of SRIF to its specific plasma membrane receptors in GH4C1 cells and have compared the processing of receptor-bound SRIF with that of EGF. When cells were incubated with [125I-Tyr1]SRIF at temperatures ranging from 4 to 37 degrees C, greater than 80% of the specifically bound peptide was removed by extraction with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. In contrast, the subcellular distribution of receptor-bound 125I-EGF was temperature dependent. Whereas greater than 95% of specifically bound 125I-EGF was removed by acid treatment after a 4 degrees C binding incubation, less than 10% was removed when the binding reaction was performed at 22 or 37 degrees C. In pulse-chase experiments, receptor-bound 125I-EGF was transferred from an acid-sensitive to an acid-resistant compartment with a half-time of 2 min at 37 degrees C. In contrast, the small amount of [125I-Tyr1]SRIF that was resistant to acid treatment did not increase during a 2-h chase incubation at 37 degrees C. Chromatographic analysis of the radioactivity released from cells during dissociation incubations at 37 degrees C showed that greater than 90% of prebound 125I-EGF was released as 125I-tyrosine, whereas prebound [125I-Tyr1]SRIF was released as a mixture of intact peptide (55%) and 125I-tyrosine (45%). Neither chloroquine (0.1 mM), ammonium chloride (20 mM), nor leupeptin (0.1 mg/ml) increased the amount of [125I-Tyr1]SRIF bound to cells at 37 degrees C. Furthermore, chloroquine and leupeptin did not alter the rate of dissociation or degradation of prebound [125I-Tyr1]SRIF. In contrast, these inhibitors increased the amount of cell-associated 125I-EGF during 37 degrees C binding incubations and decreased the subsequent rate of release of 125I-tyrosine. The results presented indicate that, as in other cell types, EGF underwent rapid receptor-mediated endocytosis in GH4C1 cells and was subsequently degraded in lysosomes. In contrast, SRIF remained at the cell surface for several hours although it elicits its biological effects within minutes. Furthermore, a constant fraction of the receptor-bound [125I-Tyr1]SRIF was degraded at the cell surface before dissociation. Therefore, after initial binding of [125I-Tyr1]SRIF and 125I-EGF to their specific membrane receptors, these peptides are processed very differently in GH4C1 cells.     

The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose.

CenA is a bacterial cellulase (beta-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide. The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed. CenA and CBD.PTCenA' (the CBD plus linker) adsorbed rapidly to cellulose at 30 degrees C, and no net desorption of protein was observed during the following 16.7 h. There was no detectable adsorption of the catalytic domain. Scatchard plots of adsorption data for CenA and for CBD.PTCenA were nonlinear (concave upward). The adsorption of CenA and CBD.PTCenA exceeded 7 and 8 mumol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed. A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues). A relative equilibrium association constant (Kr) of 40.5 and 45.3 liter.g cellulose-1 was estimated for CenA and CBD.PTCenA, respectively, according to this model. A similar Kr value (33.3 liter.g-1) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both beta 1,4-xylosidic and beta-1,4-glucosidic bonds. It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface.     

Stability analysis ofBacillus stearothermopilus L1 lipase fused with a cellulose-binding domain

This study was designed to investigate the stability of a lipase fused with a cellulose-binding domain (CBD) to cellulase. The fusion protein was derived from a gene cluster of a CBD fragment of a cellulase gene inTrichoderma hazianum and a lipase gene inBacillus stearothermophilus L1. Due to the CBD, this lipase can be immobilized to a cellulose material. Factors affecting the lipase stability were divided into the reaction-independent factors (RIF), and the reaction-dependent factors (RDF). RIF includes the reaction conditions such as pH and temperature, whereas substrate limitation and product inhibition are examples of RDF. As pH 10 and 50°C were found to be optimum reaction conditions for oil hydrolysis by this lipase, the stability of the free and the immobilized lipase was studied under these conditions. Avicel (microcrystal-line cellulose) was used as a support for lipase immobilization. The effects of both RIF and RDF on the enzyme activity were less for the immobilized lipase than for the free lipase. Due to the irreversible binding of CBD to Avicel and the high stability of the immobilized lipase, the enzyme activity after five times of use was over 70% of the initial activity.     

The effects of temperature, pH and growth rate on secondary metabolism in Streptomyces thermoviolaceus grown in a chemostat.

Streptomyces thermoviolaceus was grown in a chemostat under conditions of glutamate limitation. The effects of growth rate on production of the antibiotic granaticin, extracellular protein and protease activity as components of secondary metabolism were studied at 37, 45 and 50 degrees C. The amount of each secondary metabolite synthesized was highly dependent on growth rate and temperature. Granaticin yields were highest at growth rates of 0.1 to 0.15 h-1 at 37 degrees C, 0.175 h-1 at 45 degrees C and 0.045 h-1 at 50 degrees C. Protease activity of culture supernatants responded to low nutrient concentration and/or low growth rate. Measurements of extracellular protein revealed complex changes in amount which were dependent on growth rate and temperature. At 45 degrees C and a growth rate of 0.15 h-1, biomass yield was highest between pH 5.5 to 6.5 whereas granaticin synthesis was low at pH 5.5 and rose to highest values at between pH 6.5 and 7.5.     

海栖热袍菌内切葡聚糖酶Cel12B与木聚糖酶XynA CBD结构域融合基因的构建、表达及融合酶性质分析

海栖热袍菌内切葡聚糖酶Cel12B是极耐热胞外酶,氨基酸序列分析表明不含有纤维素结合结构域(CBD),对结晶纤维素无活性,但同样菌种来源的木聚糖酶XynA有催化结构域和纤维素结合结构城。用同样极耐热酶CBD区域和Cel12B融合构建重组质粒pET-20b-Cel12B-CBD,经诱导表达后,对结晶纤维素有活性,酶学特性研究表明:最适反应温度为100℃、最适pH为5.8、在pH4.5~7.0时酶活力稳定,90℃保温2h仍有87%的酶活。     

Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.

The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

Lipolytic activities of a lipase immobilized on six selected supporting materials

Six different types of materials including PVC, chitosan, chitin, agarose, Sepharose, and Trisacryl were evaluated for their lipase-coupling efficiencies. Among those tested, chitosan yielded the highest amount of lipase (79 mg/mL packed gel) immobilized but with lowest oil hydrolytic activity (0.03 mg eq/mL gel). The amount of lipase immobilized was affected by the length of the hydrocarbon chain attached to the PVC matrix but not by the pore size of the supports used. On the other hand, the specific activity of the immobilized lipase was affected by the pore size but not by the chain length of the hydrocarbon attached to the support. After immobilization, the optimal reaction pH was shifted from 7.5 to 8.5 and the optimal reaction temperature from 35 to 45-55 degrees C. Lipase immobilized on PVC exhibited higher thermal stability than that on agarose. The half-life of the PVC immobilized lipase operating at 30 degrees C in a packed-bed reactor was estimated to be about 400 h.