Torben Vedel Borchert

Industrial enzyme applications.

The effective catalytic properties of enzymes have already promoted their introduction into several industrial products and processes. Recent developments in biotechnology, particularly in areas such as protein engineering and directed evolution, have provided important tools for the efficient development of new enzymes. This has resulted in the development of enzymes with improved properties for established technical applications and in the production of new enzymes tailor-made for entirely new areas of application where enzymes have not previously been used.

DNA shuffling of subgenomic sequences of subtilisin

DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.

Protein engineering of bacterial α-amylases

Abstract α-Amylases constitute a very diverse family of glycosyl hydrolases that cleave α1→4 linkages in amylose and related polymers. Recent structural and mutagenic studies of archeael, mammalian and bacterial α-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class. Because of their high thermo-stability, the Bacillus α-amylases have found widespread use in industrial processes, and much attention has been devoted to optimising these enzymes for the very harsh conditions encountered there. Stability has been a major area of focus in this respect, and several remarkably stable bacterial α-amylases have been produced by bioengineering techniques. Protein engineering studies of pH-activity profiles and of substrate specificities have also been initiated, although without much success. In the coming years it is likely, however, that the focus of α-amylase engineering will shift from engineering stability to these new areas.

Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently.

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.

Characterization and Three-Dimensional Structures of Two Distinct Bacterial Xyloglucanases from Families Gh5 and Gh12.

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed “hemicellulose.” One such hemicellulose is xyloglucan, which displays a β-1,4-linked d-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (β/α)8 and β-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the β-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (β/α)8 GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

Understanding How Diverse β-Mannanases Recognize Heterogeneous Substrates

The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity toward heterogeneous substrates, in which the sequence of sugars is variable, is unclear. An excellent example of such heterogeneity is provided by the plant structural polysaccharide glucomannan, which comprises a backbone of beta-1,4-linked glucose and mannose units. beta-Mannanases, located in glycoside hydrolase (GH) families 5 and 26, hydrolyze glucomannan by cleaving the glycosidic bond of mannosides at the -1 subsite. The mechanism by which these enzymes select for glucose or mannose at distal subsites, which is critical to defining their substrate specificity on heterogeneous polymers, is currently unclear. Here we report the biochemical properties and crystal structures of both a GH5 mannanase and a GH26 mannanase and describe the contributions to substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived from Bacillus agaradhaerens, can accommodate glucose or mannose at both its -2 and +1 subsites, while the GH26 Bacillus subtilis mannanase, BsMan26A, displays tight specificity for mannose at its negative binding sites. The crystal structure of BaMan5A reveals that a polar residue at the -2 subsite can make productive contact with the substrate 2-OH group in either its axial (as in mannose) or its equatorial (as in glucose) configuration, while other distal subsites do not exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is able to hydrolyze glucomannan in which the sequence of glucose and mannose is highly variable. The crystal structure of BsMan26A in light of previous studies on the Cellvibrio japonicus GH26 mannanases CjMan26A and CjMan26C reveals that the tighter mannose recognition at the -2 subsite is mediated by polar interactions with the axial 2-OH group of a (4)C(1) ground state mannoside. Mutagenesis studies showed that variants of CjMan26A, from which these polar residues had been removed, do not distinguish between Man and Glc at the -2 subsite, while one of these residues, Arg 361, confers the elevated activity displayed by the enzyme against mannooligosaccharides. The biological rationale for the variable recognition of Man- and Glc-configured sugars by beta-mannanases is discussed.

Between-Species Variation in the Kinetic Stability of TIM Proteins Linked to Solvation-Barrier Free Energies

Theoretical, computational, and experimental studies have suggested the existence of solvation barriers in protein unfolding and denaturation processes. These barriers are related to the finite size of water molecules and can be envisioned as arising from the asynchrony between water penetration and breakup of internal interactions. Solvation barriers have been proposed to play roles in protein cooperativity and kinetic stability; therefore, they may be expected to be subject to natural selection. We study the thermal denaturation, in the presence and in the absence of chemical denaturants, of triosephosphate isomerases (TIMs) from three different species: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania mexicana. In all cases, denaturation was irreversible and kinetically controlled. Surprisingly, however, we found large differences between the kinetic denaturation parameters, with T. cruzi TIM showing a much larger activation energy value (and, consequently, much lower room-temperature, extrapolated denaturation rates). This disparity cannot be accounted for by variations in the degree of exposure to solvent in transition states (as measured by kinetic urea m values) and is, therefore, to be attributed mainly to differences in solvation-barrier contributions. This was supported by structure-energetics analyses of the transition states and by application of a novel procedure to estimate from experimental data the solvation-barrier impact at the entropy and free-energy levels. These analyses were actually performed with an extended protein set (including six small proteins plus seven variants of lipase from Thermomyces lanuginosus and spanning a wide range of activation parameters), allowing us to delineate the general trends of the solvation-barrier contributions. Overall, this work supports that proteins sharing the same structure and function but belonging to different organisms may show widely different solvation barriers, possibly as a result of different levels of the selection pressure associated with cooperativity, kinetic stability, and related factors.

Structure of a pullulanase from Bacillus acidopullulyticus

The industrial hydrolysis of polysaccharides continues to be of considerable interest and, with the drive towards green chemical processes and renewable sources of fuel, the enzymatic hydrolysis of plant carbohydrates is only set to increase. One of the most widely used plant derived polysaccharides are the starches; predominantly a-1,4 linked D-glucopyranoside polymers with, to varying degrees, a-1,6 branches. Complete starch hydrolysis requires a consortium of enzymes including endo-amylases, glucoamylases and a-glucosidases as well as diverse a-1,6 cleaving enzymes including ‘‘pullulanases’’. Many of these a-glucan active enzymes are found in the largest of the CArbohydrate-active enZYmes (CAZY) sequence families, family GH13 (see www.cazy.org1,2 recently reviewed in a historical context in Ref. 3). Considering the diversity of GH13 enzymes4 and their burgeoning applications in environmental-friendly processes, there remains a need to study these enzymes further particularly to probe their 3-D structures and often complex modular architectures. Here we report the 3-D structure, refined in two crystal forms at resolutions of 1.7 and 2.1 A, respectively, of a pullulanase from the bacterium Bacillus acidopullulyticus. The substrate, pullulan, is a polysaccharide in which repeating maltotriosyl units (a-1,4 linked) are joined through a-1,6 links, Figure 1. Pullulanases cleave the a1,6 links to liberate maltotriose. In industrial applications, these enzymes find use not merely for the breakdown of pullulan but also for the hydrolysis of the a-1,6 linkages in amylopectin starch. The B. acidopullulyticus enzyme (hereafter BaPul13A) thus finds commercial application in the starch industry where its ‘‘debranching’’ activity is utilized in the production of high fructose corn syrup5 and in the production of high maltose content syrups but also in the brewing industry, especially in the production of low calorie and ‘‘light’’ beers where it allows more complete attenuation (i.e., more complete fermentation of the mash and less residual sugar). The 3-D structure of BaPul13A was solved by single isomorphous replacement using an ‘‘in-house’’ uranyl derivative and subsequently refined in two different crystal forms. Pullulanases are frequently characterized by complex multi-domain architectures in which the catalytic module is appended to several carbohydrate-binding domains (CBMs) as well as many domains of unknown function (termed ‘‘X’’ modules). The structure of BaPul13a indeed forms an unusual domain organization, in which the N-terminal CBM41 domain is disordered/partially absent in-crystal but in which the X45a-X25-X45b-CBM48-GH13 multi-domain architecture is both clear and not, to our knowledge, previously observed. Comparison with the known Klebsiella pneumoniae pullulanase6 and the pullulanase/debranching enzyme from Bacillus subtilis reveals a conserved active centre and similar polysaccharide binding surfaces consistent with a1,6 hydrolase activity within the a-amylase fold.

Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase.

Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B. subtilis. The enzyme was purified in two steps to homogeneity prior to crystallization. Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions. The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0. They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A. A complete data set was collected to 2 A from a single native crystal using synchrotron radiation.

Structure of two fungal β-1,4-galactanases: Searching for the basis for temperature and pH optimum

β‐1,4‐Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal β‐1,4‐galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing ∼56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 Å and 2.14 Å resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 Å resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the α‐helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.