Eva Nordberg Karlsson

Potential and utilization of thermophiles and thermostable enzymes in biorefining

In todays world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.

Structural and Functional Analyses of beta-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3.

Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized-that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 A resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (alpha/beta)(8) domain similar to a triose phosphate isomerase barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second beta-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the -1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the -1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.

Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na(+) and Ca(2+) ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and beta-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

Substituent Effects on in Vitro Antioxidizing Properties, Stability, and Solubility in Flavonoids

Antioxidants are widely used by humans, both as dietary supplements and as additives to different types of products. The desired properties of an antioxidant often include a balance between the antioxidizing capacity, stability, and solubility. This review focuses on flavonoids, which are naturally occurring antioxidants, and different common substituent groups on flavonoids and how these affect the properties of the molecules in vitro. Hydroxyl groups on flavonoids are both important for the antioxidizing capacity and key points for further modification resulting in O-methylation, -glycosylation, -sulfation, or -acylation. The effects of O-glycosylation and acylation are discussed as these types of substitutions have been most explored in vitro concerning antioxidizing properties as well as stability and solubility. Possibilities to control the properties by enzymatic acylation and glycosylation are also reviewed, showing that depending on the choice of enzyme and substrate, regioselective results can be obtained, introducing possibilities for more targeted production of antioxidants with predesigned properties.

Xylooligosaccharides from Hardwood and Cereal Xylans Produced by a Thermostable Xylanase as Carbon Sources for Lactobacillus brevis and Bifidobacterium adolescentis.

To compare xylans from forestry with agricultural origins, hardwood xylan (birch) and cereal arabinoxylan (rye) were hydrolyzed using two variants of the xylanase RmXyn10A, full-length enzyme and catalytic module only, from Rhodothermus marinus . Cultivations of four selected bacterial species, using the xylooligosaccharide (XOS) containing hydrolysates as carbon source, showed selective growth of Lactobacillus brevis DSMZ 1264 and Bifidobacterium adolescentis ATCC 15703. Both strains were confirmed to utilize the XOS fraction (DP 2-5), whereas putative arabinoxylooligosaccharides from the rye arabinoxylan hydrolysate were utilized by only B. adolescentis. Escherichia coli did not grow, despite its capability to grow on the monosaccharides arabinose and xylose. It was also shown that Pediococcus parvulus strain 2.6 utilized neither xylose nor XOS for growth. In summary, RmXyn10A or its catalytic module proved suitable for high-temperature hydrolysis of hardwood xylan and cereal arabinoxylan, producing XOS that could qualify as prebiotics for use in functional food products.

Cloning and sequence of a thermostable multidomain xylanase from the bacterium Rhodothermus marinus.

The gene (xyn1) encoding a Rhodothermus marinus xylanase has been cloned and expressed in Escherichia coli. The gene comprises 5 different domains in an unusual combination. The cellulose binding domains (CBDs) encoded by xyn1 are repeated in tandem at the N-terminus and show similarity with the CBD family IV. The xyn1-gene is the first example encoding a CBD family IV in combination with a xylan hydrolyzing catalytic domain of the glycosyl hydrolase family 10.

Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xyn1 from Rhodothermus marinus

The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli. The catalytic domain belongs to glycosyl hydrolase family 10. The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase. The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9. The pH and temperature optima for activity were determined to be 7.5 and 80 degrees C, respectively. At that temperature the enzyme had a half-life of 1 h 40 min. An addition of 1 mM calcium stabilized the activity of the enzyme at 80 degrees C. The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans). No exo- or endo-cellulase activity was observed. Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose. The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan. The R. marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase.

The structure of Rhodothermus marinus Cel12A, a highly thermostable family 12 endoglucanase, at 1.8 Å resolution

Cellulose is one of the most abundant polysaccharides in nature and microorganisms have developed a comprehensive system for enzymatic breakdown of this ubiquitous carbon source, a subject of much interest in the biotechnology industry. Rhodothermus marinus produces a hyperthermostable cellulase, with a temperature optimum of more than 90 degrees C, the structure of which is presented here to 1.8 A resolution. The enzyme has been classified into glycoside hydrolase family 12; this is the first structure of a thermophilic member of this family to have been solved. The beta-jelly roll fold observed has identical topology to those of the two mesophilic members of the family whose structures have been elucidated previously. A Hepes buffer molecule bound in the active site may have triggered a conformational change to an active configuration as the two catalytic residues Glu124 and Glu207, together with dependent residues, are observed in a conformation similar to that seen in the structure of Streptomyces lividans CelB2 complexed with an inhibitor. The structural similarity between this cellulase and the mesophilic enzymes serves to highlight features that may be responsible for its thermostability, chiefly an increase in ion pair number and the considerable stabilisation of a mobile region seen in S. lividans CelB2. Additional aromatic residues in the active site region may also contribute to the difference in thermophilicity.

Efficient production of truncated thermostable xylanases from Rhodothermus marinus in Escherichia coli fed-batch cultures

A cultivation strategy for the production of two truncated thermostable recombinant xylanases (Xyn1deltaN and Xyn1deltaNC) was developed. Fed-batch cultivations of Escherichia coli strain BL21(DE3) with a controlled exponential glucose feed led to high specific production of the recombinant proteins. Addition of complex nutrients (e.g. Tryptone Soya Broth (TSB)) to the media were shown to increase both the specific growth rate during the production phase and the production per cell. The final cell-mass concentration depended on the time of induction in relation to both the feed-start and the expected time at which the cultivation had to be terminated due to oxygen transfer limitations or cell lysis. The gene used for the genetic constructions (encoding Xyn1deltaN and Xyn1deltaNC) was originally isolated from Rhodothermus marinus. Recombinant protein expression was controlled by the T7 lac-promoter and induced in the fed-batch phase at low glucose concentrations by the single addition of either lactose or isopropyl-thio-beta-d-galactoside (IPTG). In lactose-induced cells, the production of recombinant xylanase was delayed for approximately 30 min in comparison with those induced with IPTG, but the specific product levels were comparable at 3 h after induction. At this time, approximately 35% of the intracellular protein content was constituted by recombinant xylanase. Under the cultivation conditions used, production of the shorter deletion derivative (Xyn1deltaNC) led to nonspecific leakage and cell lysis, starting 1.5 or 2 h after induction with IPTG or lactose, respectively. At 3 h after induction, 50% of the produced protein (Xyn1deltaNC) was found in the culture medium. This was not the case for the longer protein (Xyn1deltaN), where only 10% of the xylanase leaked into the medium.

Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.