Improvement of the lytic properties of a β-1,3-glucanase by directed evolution.
Salazar, O., Basso, C., Barba, P., Orellana, C. & Asenjo, J. A. (2006). Molecular Biotechnology, 33(3), 211-219.
BGLII is a bacterial endoglucanase that hydrolyzes the β-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.
Endo-β-1, 3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family.
Sakamoto, Y., Nakade, K. & Konno, N. (2011). Applied and Environmental Microbiology, 77(23), 8350-8354.
The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
Production, purification and application-relevant characterisation of an endo-1,3(4)-β-glucanase from Rhizomucor miehei.
Boyce, A. & Walsh, G. (2007). Applied Microbiology and Biotechnology, 76(4), 835-841.
Growth on a wheat bran media induced production of an extracellular β-glucanase by Rhizomucor miehei (DSM 1330). The enzyme was purified to homogeneity. Substrate specificity studies coupled with protein database similarity searching using mass spectrometry-derived sequence data indicate it to be an endo-1,3(4)-β-glucanase (EC 18.104.22.168). The enzyme was characterised in terms of potential suitability for use in animal (poultry) feed. Significant activity was observed over the entire pH range typical of the avian upper digestive tract (pH 2.6–6.5). The enzyme was also found to be more thermostable than current commercialized β-glucanases, particularly when heated at a high enzyme concentration, and retained twice as much residual activity as the latter upon exposure to simulated avian digestive tract conditions. There are no previous reports of the production, purification or characterization of a β-glucanase from a Rhizomucor, and the enzyme’s application-relevant physicochemical characteristics render it potentially suited for use in animal feed.
Immunity or Digestion: Glucanase activity in a Glucan-binding protein family from Lepidoptera.
Pauchet, Y., Freitak, D., Heidel-Fischer, H. M., Heckel, D. G. & Vogel, H. (2009). Journal of Biological Chemistry, 284(4), 2214-2224.
The cell surfaces of microorganisms display distinct molecular patterns formed from lipopolysaccharides, peptidoglycans, or β1,3-glucans. Binding of these surfaces by pattern recognition proteins such as β1,3-glucan recognition proteins (βGRPs) activates the immune response in arthropods. We identified a 40-kDa β1,3-glucan-binding protein with sequence similarity to previously characterized lepidopteran βGRPs from hemolymph, but unlike these it is secreted into the larval gut lumen and is an active β1,3-glucanase. This glucanase was not detected in hemolymph. Its mRNA is constitutively and predominantly expressed in the midgut and is induced there when larvae feed on a diet containing bacteria. Homologs of this predominantly midgut-expressed gene from many Lepidoptera possess key residues shown to be part of the active site of other glucanases, and form a cluster that is distinct from previously described βGRPs. In addition, this group includes proteins from insects such as the Anopheles gambiae GNBP subgroup B for which a catalytic role has not been previously suspected. The current domain classification does not distinguish between the catalytic and noncatalytic clades, and should be revised. The noncatalytic βGRPs may be evolutionarily derived from this newly described enzyme family that continues to function catalytically in digestion and/or pathogen defense.
Higher order structures of a bioactive, water-soluble (1→3)-β-D-glucan derived from Saccharomyces cerevisiae.
Qin, F., Sletmoen, M., Stokke, B. T. & Christensen, B. E. (2013). Carbohydrate Polymers, 92(2), 1026-1032.
Water-soluble (1→3)-β-D-glucans with 1,6-linked branches (SBG), originally isolated from the cell walls of Saccharomyces cerevisiae and partially depolymerised to a weight average degree of polymerisation (DPw) in the range 120–160 for optimal performance in wound healing applications, were studied by dynamic light scattering (DLS), SEC MALLS and AFM. Results indicate that dilute aqueous SBG solutions (1 µg/ml to 3 mg/ml) contain higher order structures with a very wide size distribution in water (10–500 nm), corresponding to a mixture of single chains, multi-chain aggregates including triple-stranded motifs, and particulate materials. The latter were enriched in longer chains compared to non-particulate fractions. The size distribution of SBG aggregates shifted to slightly lower values upon heating, but showed hysteresis upon cooling. AFM images prepared from very dilute aqueous solution (1–5 µg/ml) analysis showed by comparison to other (1→3)-β-D-glucans that some of the structures were the triple helical species coexisting with larger aggregates and single chains, in contrast to carboxymethylated SBG, which contained predominantly single chains. The ability to control the aggregation behaviour of SBG enables tailoring of the physical, and possibly bioactive, properties of SBG preparations.
Conserved Cys residue influences catalytic properties of potato endo-(1→3)-β-glucanase GLUB20-2.
Witek, A. I., Witek, K. & Hennig, J. (2008). Acta Biochimica Polonica, 55(4), 791-797.
The synthesis and degradation of (1→3)-β-glycosidic bonds between glucose moieties are essential metabolic processes in plant cell architecture and function. We have found that a unique, conserved cysteine residue, positioned outside the catalytic centre of potato endo-(1→3)-β-glucanase — product of the gluB20-2 gene, participates in determining the substrate specificity of the enzyme. The same residue is largely responsible for endo-(1→3)-β-glucanase inhibition by mercury ions. Our results confirm that the spatial adjustment between an enzyme and its substrate is one of the essential factors contributing to the specificity and accuracy of enzymatic reactions.
A novel glycosylphosphatidylinositol-anchored glycoside hydrolase from Ustilago esculenta functions in β-1,3-glucan degradation.
Nakajima, M., Yamashita, T., Takahashi, M., Nakano, Y. & Takeda, T. (2012). Applied and Environmental Microbiology, 78(16), 5682-5689.
A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of β-1,3-glucan after its release in the extracellular medium.
Chain length distribution and aggregation of branched (1→3)-β-D-glucans from Saccharomyces cerevisae.
Qin, F., Aachmann, F. L. & Christensen, B. E. (2012). Carbohydrate Polymers, 90(2), 1092-1099.
Water-soluble (1→3)-β-D-glucans with 1,6-linked branches (SBG), originally isolated from the cell walls of Saccharomyces cerevisiae and partially depolymerised for optimal performance in wound healing applications, were studied by size exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) detector and a viscosity detector at both high and ambient column temperatures. The strongly aggregating materials could be dispersed as single chains in water following partial carboxymethylation (degree of substitution (DS) 0.51 or higher). Lower DS (0.23) also dispersed as single chains provided a column temperature of 80°C was applied. Reduction of reducing ends prior to carboxymethylation was required to avoid alkaline peeling and hence to obtain correct molecular weight distributions of the native material. DS was determined using 13C NMR and potentiometric titration (range 0.23–0.91). Further analysis of CM-SBG in the single chain state suggested a randomly coiled behaviour with marginal influence of the branches in terms of macromolecular dimensions, which were close to those of CM-curdlan. The result of the investigation is a simple and reliable protocol for preparing undegraded and un-aggregated SBG derivatives, which are well suited as a standard analysis of the molecular weight distribution of SBG-like molecules.
A xyloglucan-specific family 12 glycosyl hydrolase from Aspergillus niger: recombinant expression, purification and characterization.
Master, E. R., Zheng, Y., Storms, R., Tsang, A. & Powlowski, J. (2008). Biochem. J, 411, 161-170.
A new GH12 (glycosyl hydrolase 12) family XEG [xyloglucan-specific endo-β-1,4-glucanase (EC 22.214.171.124)] from Aspergillus niger, AnXEG12A, was overexpressed, purified and characterized. Whereas seven xyloglucanases from GH74 and two xyloglucanases from GH5 have been characterized previously, this is only the third characterized example of a GH12 family xyloglucanase. GH12 enzymes are structurally and mechanistically distinct from GH74 enzymes. Although over 100 GH12 sequences are now available, little is known about the structural and biochemical bases of xyloglucan binding and hydrolysis by GH12 enzymes. Comparison of the AnXEG12A cDNA sequence with the genome sequence of A. niger showed the presence of two introns, one in the coding region and the second one in the 333-nt-long 3´-untranslated region of the transcript. The enzyme was expressed recombinantly in A. niger and was readily purified from the culture supernatant. The isolated enzyme appeared to have been processed by a kexin-type protease, which removed a short prosequence. The substrate specificity was restricted to xyloglucan, with cleavage at unbranched glucose in the backbone. The apparent kinetic parameters were similar to those reported for other xyloglucan-degrading endoglucanases. The pH optimum (5.0) and temperature resulting in highest enzyme activity (50–60°C) were higher than those reported for a GH12 family xyloglucanase from Aspergillus aculeatus, but similar to those of cellulose-specific endoglucanases from the GH12 family. Phylogenetic, sequence and structural comparisons of GH12 family endoglucanases helped to delineate features that appear to be correlated to xyloglucan specificity.