SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability

A quantitative linear model accurately (R² = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 °C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 °C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55–56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.SCHEMA is a computational approach to identifying blocks of sequence that minimize structural disruption when they are recombined in chimeric proteins (1). SCHEMA recombination of eight blocks from three fungal cellobiohydrolase class II (CBH II)² genes was used in our previous work to create a library of 3⁸ = 6,561 chimeric sequences, all having the native Hypocrea jecorina cellulose binding module and linker and observed to feature a degree of glycosylation similar to that found in native CBH IIs secreted by fungi (2). Synthesis and characterization of selected CBH II chimeras expressed in Saccharomyces cerevisiae revealed enzymes with thermostabilities and cellulose hydrolysis performance superior to those of the parent enzymes from Humicola insolens, H. jecorina, and Chaetomium thermophilum.Our prior analysis showed that a qualitative model based on sequence-stability data from 23 functional chimeras (categorizing blocks as destabilizing, stabilizing, or neutral) could identify highly stable chimeras in the SCHEMA library (2). When studying SCHEMA recombination of a bacterial cytochrome P450, we previously estimated that building a quantitative regression model would require stability measurements for at least 35 representative sequences (3). We therefore synthesized an additional 18 CBH II chimeras to further explore the sequences that the qualitative model predicted would encode the most thermostable chimeras. If sequence blocks contribute additively and independently of their context, as was found for SCHEMA chimeras of cytochrome P450 (3), then quantitative stability prediction would be possible based on stability data from a very limited sampling of the thousands of possible chimeras. Here we show that a quantitative CBH II chimera stability model can in fact be constructed and also that it was possible, using site-directed mutagenesis experiments, to pinpoint a single amino acid substitution that is responsible for the large stabilizing contribution of one of the SCHEMA blocks.Highly thermostable fungal CBH IIs are potentially useful for the degradation of cellulosic substrates in biofuels, textile, and other applications (4). High thermostability translates to longer half-lives at elevated hydrolysis temperatures, where viscosity and microbial contamination are reduced (5). We therefore investigated how selected thermostable CBH II chimeras perform in the hydrolysis of crystalline cellulose (Avicel) at elevated temperatures (up to 70 °C). All of the thermostable chimeras tested have specific activities on phosphoric acid swollen cellulose (PASC) at 50 °C that are comparable with the most active parent (H. jecorina CBH II) and hydrolyze Avicel at temperatures higher than any of the three parent enzymes, including the CBH II from the thermophilic fungus H. insolens.     

Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD

Enhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes. In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique. The library was constructed by in vitro recombination of the genes for EngB and non-cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous. To obtain thermostable clones without loss of activity, the library was screened by a combination of activity and thermostability screening. We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities. One of the mutants possessed a sevenfold higher thermostability than EngB. The possible mechanisms of thermostabilization are discussed.     

Exogenous cellulases of thermophilic micromycetes. II. Thermostability of enzyme preparations

The ability of a large number of higher fungi to form extracellular cellulases is investigated. Some representatives of these fungi grow at 40–50°C, and form extracellular cellulases exceeding cellulases of mesophilic fungi in thermostability. It is shown that cellulases of higher thermophilic fungi differ by their thermostability. The temperature optimum of cellulase action of higher fungi occurs within 60–62°C.     

Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.     

Lipopeptide produced from <Emphasis Type="Italic">Bacillus</Emphasis> sp. W112 improves the hydrolysis of lignocellulose by specifically reducing non-productive binding of cellulases with and without CBMs

Background

Surfactants have attracted increasing interest for their capability to improve the enzymatic hydrolysis of lignocellulosic biomass. Compared to chemical surfactants, biosurfactants have a broader prospect for industrial applications because they are more environmentally friendly and more effective in some researches. Commercial cellulase preparations are mainly composed of endoglucanases (EGs) and cellobiohydrolases (CBHs) that possess carbohydrate-binding modules (CBMs). However, the effects of lipopeptide-type biosurfactants on enzymatic saccharification of lignocellulose and adsorption behaviors of cellulases with CBMs remain unclear.

Results

In this study, we found that Bacillus sp. W112 could produce a lipopeptide-type biosurfactant from untreated biomass, such as wheat bran and Jerusalem artichoke tuber. The lipopeptide could enhance the enzymatic hydrolysis of dilute acid pretreated Giant Juncao grass (DA-GJG) by fungal and bacterial enzymes. The enhancement increased over a range of temperatures from 30 to 50 °C. Lipopeptide was shown to be more effective in promoting DA-GJG saccharification than chemical surfactants at low dosages, with a best stimulatory degree of 20.8% at 2% loading of the substrates (w/w). Lipopeptide increased the thermostability of EG and CBH in commercial cellulase cocktails. Moreover, the dual effects of lipopeptide on the adsorption behaviors of cellulases were found. It specifically lowered the non-productive binding of cellulases to lignin and increased the binding of cellulases to cellulose. In addition, we investigated the influence of lipopeptide on the adsorption behaviors of CBHs with CBMs for the first time. Our results showed that lipopeptide reduced the adsorption of CBM-deleted CBH to DA-GJG to a greater extent than that of intact CBH while the non-productive binding of intact CBH to lignin was reduced more, indicating that lipopeptide decreased the binding of CBMs onto lignin but not their combination with cellulose.

Conclusions

In this study, we found that lipopeptide from Bacillus sp. W112 promoted the enzymatic hydrolysis of DA-GJG at relative low loadings. The stimulatory effect could be attributed to increasing the cellulase thermostability, reducing non-productive adsorption of cellulases with CBMs caused by lignin and enhancing the binding of cellulases to cellulose.

     

Role of cysteine residues in thermal inactivation of fungal Cel6A cellobiohydrolases

Numerous protein engineering studies have focused on increasing the thermostability of fungal cellulases to improve production of fuels and chemicals from lignocellulosic feedstocks. However, the engineered enzymes still undergo thermal inactivation at temperatures well below the inactivation temperatures of hyperthermophilic cellulases. In this report, we investigated the role of free cysteines in the thermal inactivation of wild-type and engineered fungal family 6 cellobiohydrolases (Cel6A). The mechanism of thermal inactivation of Cel6A is consistent with disulfide bond degradation and thiol–disulfide exchange. Circular dichroism spectroscopy revealed that a thermostable variant lacking free cysteines refolds to a native-like structure and retains activity after heat treatment over the pH range 5–9. Whereas conserved disulfide bonds are essential for retaining activity after heat treatment, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A as well as Cel6A from Hypocrea jecorina and Humicola insolens.     

Characterization of Lignocellulolytic Activities from a Moderate Halophile Strain of Aspergillus caesiellus Isolated from a Sugarcane Bagasse Fermentation

A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese peroxidase (MnP) and esterases. No laccase activity was detected in the conditions we tested. The cellulase activity was thermostable, halostable, and no differential expression of cellulases was observed in media with different salt concentrations. However, differential band patterns for cellulase and xylanase activities were detected in zymograms when the fungus was grown in different lignocellulosic substrates such as wheat straw, maize stover, agave fibres, sugarcane bagasse and sawdust. Optimal temperature and pH were similar to other cellulases previously described. These results support the potential of this fungus to degrade lignocellulosic materials and its possible use in biotechnological applications.     

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a ''greener'' technology.     

Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel

We have developed a system to detect three hydrolytic enzymes (cellulase, lipase, and protease) using a single sodium dodecyl sulfate (SDS) gel and an electrotransfer system. After electrophoresis, proteins in the gel were transferred to three sandwiched substrate gels containing glycerol tributyrate, azo-carboxymethyl cellulose (Azo-CMC), and fibrin for detection of cellulase, lipase, and protease, respectively. We show that three cellulases (from a Paenibacillus sp. and two Bacillus sp. strains), one lipase (from a Staphylococcus sp.), and two proteases (from two Bacillus sp. strains) can be detected simultaneously with our zymogram system.     

A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle

Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (T_m > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF’s thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF’s thermostability by performing 4 μs of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.     

Expression of a thermostable cellulase gene from a thermophilic anaerobe in Saccharomyces cerevisiae

A cellulase gene from a thermophilic anaerobe was recloned in the yeast Saccharomyces cerevisiae. The maximum level of the gene expression in the recombinant yeast was 4.4 times higher than that in the Escherichia coli transformant harboring the same plasmid. Cellulase activity was observed only within the yeast cells. To compare the enzymatic properties of cellulase produced by the yeast and E. coli transformants, cellulases were purified to homogeneous state by only three purification steps of heat treatment, and cellulose affinity and ion exchange chromatographies. The molecular weights of the enzymes produced by the yeast and E. coli were 3.8 × 10⁴ and 4.0 × 10⁴, respectively by SDS-polyacrylamide gel electrophoresis. Neither of the enzymes was glycosylated. Although the molecular weights were slightly different, enzymatic properties and thermostability were almost indistinguishable between the enzymes produced by the yeast and E. coli transformants.     

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of β-glucosidase at 50 °C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive β-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive β-glucosidase. This is the first report on less thermostability of SSF derived β-glucosidase which is the main reason for getting less hydrolysis.     

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.     

Purification and biochemical characterization of glucose–cellobiose-tolerant cellulases from Scytalidium thermophilum

Two cellulases from Scytalidium thermophilum were purified and characterized, exhibiting tolerance to glucose and cellobiose. Characterization of purified cellulases I and II by mass spectrometry revealed primary structure similarities with an exoglucanase and an endoglucanase, respectively. Molecular masses were 51.2 and 45.6 kDa for cellulases I and II, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cellulases I and II exhibited isoelectric points of 6.2 and 6.9 and saccharide contents of 11 and 93 %, respectively. Optima of temperature and pH were 60–65 °C and 4.0 for purified cellulase I and 65 °C and 6.5 for purified cellulase II. Both cellulases maintained total CMCase activity after 60 min at 60 °C. Cysteine, Mn²⁺, dithiotreitol and ß-mercaptoethanol-stimulated cellulases I and II. The tolerance to cellulose hydrolysis products and the high thermal stabilities of Scytalidium cellulases suggest good potential for industrial applications.     

Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming

Thermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase from Bacillus sp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca²⁺. However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in the t₅₀ (time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in the t₅₀ value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.     

Production and characterization of thermostable cellulases from Streptomyces transformant T3-1

A total of 26 thermophilic isolates, selected from a compost of agricultural waste, which was mostly composed of vegetable, corncob and rice straw, were cultivated at 50 °C for further studies of thermostable cellulase production. The thermostable cellulase gene from the chromosomal DNA of actinomycetes isolate no. 10 was shotgun-cloned and transformed into Streptomyces sp. IAF 10-164. A transformant, T3-1, was found to be a good strain for the production of thermostable cellulases. Cultivation of T3-1 in modified Mandels–Reese broth containing 1% carboxymethylcellulose (CMC)-sodium salt and the optimal condition for microbial growth were studied. Batch cultivation in a flask revealed that CMCase and Avicelase production reached the maximum between the third to fifth day, whereas maximum -glucosidase production occurred on the ninth day. Microbial biomass increased from the first day to the fifth day and then decreased. The crude enzyme had the highest activity at 50 °C and at pH 6.5. The enzyme was shown to be a thermostable cellulase whose activities were stable at 50 °C for more than 7 days.     

Insertion of Endocellulase Catalytic Domains into Thermostable Consensus Ankyrin Scaffolds: Effects on Stability and Cellulolytic Activity

Degradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable α-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8; Clostridium thermocellum) and Cel12A (C12A, GH12; Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12A catalytic domain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.     

Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate

With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.     

Expression of a thermostable bacterial cellulase in transgenic tobacco plants

The bacterial gene of the thermostable endo-beta-1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased business and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.