Geoffrey P. Hazlewood

Bacterial cellulases and xylanases

Although this review focusses primarily on bacterial cellulases and xylanases, there is considerable overlap in the molecular biology and biochemistry of prokaryotic and fungal forms of these enzymes. Therefore, fungal plant cell wall hydrolases are discussed where comparisons with the corresponding bacterial enzymes are important. Cellulases and xylanases encompass a collection of enzymes whose primary function is to hydrolyse p1,4-glycosidic linkages in the major plant structural polysaccharides, cellulose and xylan. In converting cellulose and xylan to their constituent sugars, these enzymes play an essential role in the digestive processes of herbivores and in the recycling of photosynthetically fixed carbon. The past decade has seen a burgeoning interest in all aspects of the biochemistry and molecular biology of cellulases and xylanases, which at first glance is rather perplexing. As a paradigm for p1,4glycanases, lysozyme is the enzyme of choice; it has been studied in greater detail than cellulases or xylanases and has a clearly understood catalytic mechanism. The recent discoveries from analysis of gene sequences and the three-dimensional structure of cellulase proteins show, however, that there is much to be understood about the enzymic hydrolysis of plant carbohydrate polymers beyond what can be adduced from analogy with lysozyme. To really appreciate the rationale behind current studies of microbial cellulases and xylanases, it is necessary to take a broad view that takes into account not just their intrinsic interest, but also their undoubted commercial potential. The structural polysaccharides cellulose and hemicellulose (xylan being the major

Unusual Microbial Xylanases from Insect Guts

ABSTRACT Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted β-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a Mr, of 53000 and hydrolysed oat‐spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of Mr, 66192. The predicted primary structure of XYLA comprised an N‐terminal signal peptide followed by a 225‐amino‐acid repeated sequence, which was separated from a tandem 40‐residue C‐terminal repeat by a threonine/proline linker sequence. The large N‐terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full‐length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.

Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites.

BACKGROUND Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G. Family F enzymes show no sequence similarity with any known structure and their architecture is unknown. Studies of an inactive enzyme-substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases. RESULTS We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp. cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20. The structure was solved using an engineered catalytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal. We show that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base. Xylopentaose binds to five subsites A-E with the cleaved bond between subsites D and E. Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates. CONCLUSIONS The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates.

A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and α-galactosidase

Abstract Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and α-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached κ number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final κ number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by α-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA, gave the most effective κ number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability.

Release of ferulic acid dehydrodimers from plant cell walls by feruloyl esterases

Dehydrodimers of hydroxycinnamic acids, such as ferulic and p-coumaric acids, are important structural components which serve to cross-link polymers in plant cell walls. Dehydrodiferulate oligosaccharide diesters were solubilised from wheat bran or sugar beet pulp by treatment with Driselase and release of dehydrodiferulic free acids from this substrate by purified feruloyl esterases was analysed by HPLC. We detected 5-5diFA, 8-O-4diFA and 8-5BendiFA in saponified extracts from wheat bran and sugar beet pulp. Driselase-treatment solubilised 21 and 97% of the saponifiable dehydrodimers from wheat bran and sugar beet pulp, respectively, but only as dehydrodiferulate esters, not as dehydrodiferulic acids. At low concentrations, feruloyl esterase A (FAEA) from Aspergillus niger and an esterase (XylD) from Pseudomonas fluorescens released 93% and 36% of the saponifiable 5-5diFA from solubilised wheat bran, respectively, but only 12 and 15% from solubilised sugar beet pulp. At higher concentrations, only FAEA also released 8-O-4diFA (65%) from solubilised wheat bran, but not sugar beet pulp. We could not detect release of any dehydrodiferulic acids from solubilised wheat bran or sugar beet pulp using either a cinnamoyl ester hydrolase from Piromyces equi (CEH) or from A niger (CinnAE). Our results demonstrate that FAEA and Xy1D hydrolyse dehydrodiferulate diesters to release the free acids, which means they have the potential to break the putative cross-links present in graminaceous monocot and dicot cell walls, while two other feruloyl esterases (CinnAE and CEH) did not release dehydrodiferulic acids from plant cell walls.

Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement

ABSTRACT The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62°C for 1 h and 27% of its initial activity after 10 min at 85°C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.

An evolutionary route to xylanase process fitness

Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90°C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61°C to as high as 96°C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild‐type parent).

Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases.

Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non‐catalytic cellulose‐binding domain (CBD). To establish whether CBDs are common features of plant cell‐wall hydroiases from C. fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of Mr 68000. The deduced primary sequence of XYLD was confirmed by the size (64kDa) and N‐terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endo‐acting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an /V‐terminal signal peptide followed by a 190‐residue domain that exhibited significant homology to Family‐G xylanases. Truncated derivatives of xynD, encoding the W‐terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190‐residue N‐terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90‐residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197‐amino‐acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/prolineor novel glycine‐rich linker regions. Although full‐length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C‐terminal CBD hydrolysed xylan but did not bind to cellulose. Fusion of the C‐terminal domain to glutathione‐Stransferase generated hybrid proteins that bound to crystalline cellulose, but not to amorphous cellulose or xylan. The location of CBDs in a C. fimi xylanase indicates that domains of this type are not restricted to cellulases, but are widely distributed between hemicellutases also, and therefore play a pivotal role in the activity of the whole repertoire of plant cell‐wall hydrolases. The role of the NodB homologue in XYLD is less certain.

The resistance of cellulases and xylanases to proteolytic inactivation.

The sensitivity of a range of cellulases and xylanases to proteolytic inactivation was investigated. The xylanases, all the Clostridium thermocellum cellulases and cellulase E from Pseudomonas fluorescens subsp. cellulosa exhibited no decrease in catalytic activity during a 3-h incubation with proteinases of the small intestine. Under these conditions, the control Escherichia coli enzymes analysed had half-lives of 4.3–13.5 min. The addition of substrate significantly decreased the sensitivity of proteinase-labile enzymes to inactivation. The significance of these data in relation to the use of cellulases and xylanases for improving animal nutrition is discussed.