Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution.

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

Identification and Characterization of a New Alkaline Thermolysin-Like Protease, BtsTLP1, from Bacillus thuringiensis Serovar Sichuansis Strain MC28.

Thermolysin and its homologs are a group of metalloproteases that have been widely used in both therapeutic and biotechnological applications. We here report the identification and characterization of a novel thermolysin-like protease, BtsTLP1, from insect pathogen Bacillus thuringiensis serovar Sichuansis strain MC28. BtsTLP1 is extracellularly produced in Bacillus subtilis, and the active protein was purified via successive chromatographic steps. The mature form of BtsTLP1 has a molecule mass of 35.6 kDa as determined by mass spectrometry analyses. The biochemical characterization indicates that BtsTLP1 has an apparent Km value of 1.57 mg/ml for azocasein and is active between 20°C and 80°C. Unlike other reported neutral gram-positive thermolysin homologs with optimal pH around 7, BtsTLP1 exhibits an alkaline pH optimum around 10. The activity of BtsTLP1 is strongly inhibited by EDTA and a group of specific divalent ions, with Zn(2+) and Cu(2+) showing particular effects in promoting the enzyme autolysis. Furthermore, our data also indicate that BtsTLP1 has potential in cleaning applications.

Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea.

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10°C higher melting temperature (T(m) of 72.5°C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65°C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 Šresolution; R(work) and R(free) of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A.

The crystal structure of the core domain of a cellulose induced protein (Cip1) from Hypocrea jecorina, at 1.5 Å resolution.

In an effort to characterise the whole transcriptome of the fungus Hypocrea jecorina, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major H. jecorina cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1). This protein consists of a glycoside hydrolase family 1 carbohydrate binding module connected via a linker region to a domain with yet unknown function. After cloning and expression of Cip1 in H. jecorina, the protein was purified and biochemically characterised with the aim of determining a potential enzymatic activity for the novel protein. No hydrolytic activity against any of the tested plant cell wall components was found. The proteolytic core domain of Cip1 was then crystallised, and the three-dimensional structure of this was determined to 1.5 Å resolution utilising sulphur single-wavelength anomalous dispersion phasing (sulphor-SAD). A calcium ion binding site was identified in a sequence conserved region of Cip1 and is also seen in other proteins with the same general fold as Cip1, such as many carbohydrate binding modules. The presence of this ion was found to have a structural role. The Cip1 structure was analysed and a structural homology search was performed to identify structurally related proteins. The two published structures with highest overall structural similarity to Cip1 found were two poly-lyases: CsGL, a glucuronan lyase from H. jecorina and vAL-1, an alginate lyase from the Chlorella virus. This indicates that Cip1 may be a lyase. However, initial trials did not detect significant lyase activity for Cip1. Cip1 is the first structure to be solved of the 23 currently known Cip1 sequential homologs (with a sequence identity cut-off of 25%), including both bacterial and fungal members.

Expression of novel ß-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts.

ß-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of ß-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or ß-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for ß-1,3-1,4 glucans, showing maximum activity on barley ß-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley ß-glucan and lichean were 3-O-ß-cellotriosyl-D-glucose and 3-O-ß-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for ß-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase.

Hypocrea jecorina CEL6A protein engineering.

The complex technology of converting lignocellulose to fuels such as ethanol has advanced rapidly over the past few years, and enzymes are a critical component of this technology. The production of effective enzyme systems at cost structures that facilitate commercial processes has been the focus of research for many years. Towards this end, the H. jecorina cellobiohydrolases, CEL7A and CEL6A, have been the subject of protein engineering at Genencor. Our first rounds of cellobiohydrolase engineering were directed towards improving the thermostability of both of these enzymes and produced variants of CEL7A and CEL6A with apparent melting temperatures above 70°C, placing their stability on par with that of H. jecorina CEL5A (EG2) and CEL3A (BGL1). We have now moved towards improving CEL6A- and CEL7A-specific performance in the context of a complete enzyme system under industrially relevant conditions. Achievement of these goals required development of new screening strategies and tools. We discuss these advances along with some results, focusing mainly on engineering of CEL6A.

Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces and secretes profuse quantities of enzymes that act synergistically to degrade cellulase and related biomass components. We partially sequenced over 5100 random T. reesei cDNA clones. Among the sequences whose predicted gene products had significant similarity to known proteins, 12 were identified that encode previously unknown enzymes that likely function in biomass degradation. Microarrays were used to query the expression levels of each of the sequences under different conditions known to induce cellulolytic enzyme synthesis. Most of the genes encoding known and putative biomass-degrading enzymes were transcriptionally co-regulated. Moreover, despite the fact that several of these enzymes are not thought to degrade cellulase directly, they were coordinately overexpressed in a cellulase overproducing strain. A variety of additional sequences whose function could not be ascribed using the limited sequence available displayed analogous behavior and may also play a role in biomass degradation or in the synthesis of biomass-degrading enzymes. Sequences exhibiting additional regulatory patterns were observed that might reflect roles in regulation of cellulase biosynthesis. However, genes whose products are involved in protein processing and secretion were not highly regulated during cellulase induction.

Cloning and relational analysis of 15 novel fungal endoglucanases from family 12 glycosyl hydrolase.

Cellulases belong to the large family of glycosyl hydrolases (GHs) and are produced by a variety of bacteria and fungi. These extracellular enzymes act as endoglucanases (EGs), cellobiohydrolases or beta-glucosidases. In this paper, we describe molecular screening for EGs from the GH family 12. Using three homologous sequence boxes deduced from five previously known members of the family, we analysed 22 cellulase-producing fungal strains obtained from a diverse area of the fungal kingdom. Polymerase chain reactions using degenerate primers designed to the homologous protein boxes were used to identify the family 12 homologues. Several fungi showed the presence of multiple versions of the gene, while amino acid sequence analysis showed diversity in 15 novel members of the family, ranging from 26% to 96% similarity. Our sequence analysis shows that the phylogenetic tree of family 12 EGs can be divided into four subfamilies: 12-1 (fungal group I), 12-2 (fungal group II), 12-3 ( Streptomyces group in which Rhodothermus marinus fits) and 12-4 ( Thermophiles group). Erwinia carotovora may form a new subgroup.