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Starch is a vital energy molecule in plants that has a wide variety of uses in industry, such as feedstock for biomaterial processing and biofuel production. Plants employ a three enzyme cyclic pro...

To mimic the structure of the 1.8-fold more active (k(cat)) Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA was subjected to site-directed mutagenesis in the Trp170-Tyr175 segment of the third of the six well-conserved alpha-->alpha connecting loops of the catalytic (alpha/alpha)6-barrel. While the Trp170-->Phe, Gln172-->Asn and Tyr175-->Phe mutants showed an up to 1.7-fold increased k(cat) and Gly174-->Cys GA and approximately 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171-->Ser, Thr173-->Gly and A.niger wild-type GA had very similar kcat and K(m) values for the hydrolysis of isomaltose and the malto-oligosaccharides of DP 2-7. Crystal structures of pseudotetrasaccharide inhibitor complexes of Aspergillus awamori var. X100 GA, which is 94% identical to A.niger GA, indicate that Tyr175 is located at binding subsite 4, while the preceding target residues and the high-mannose type unit on Asn171 are at a larger distance from the site of catalysis. The mutations had a modest effect on thermostability; the temperature for 50% inactivation, Tm, was thus unchanged for Tyr175 -->Phe GA and reduced by 0.2-2.9 degrees C for the other mutants. The deletion of the N-linked high-mannose unit-in Asn171 -->Ser and Thr173-->Gly GAs-appeared to be of minor importance for enzyme activity and thermostability, and did not increase the sensitivity to proteolysis.

The thermodynamics of ligand binding to the starch-binding domain (SBD) of glucoamylase from Aspergillus niger has been studied using titration calorimetry. The ligand binding was studied both with the SBD fragment as well as glucoamylase G1 which contains both a catalytic domain and SBD. The ligands were beta-cyclodextrin and three thiopanose analogues [panose = alpha-D-Glcp-(1 leads to 6)-alpha-D-Glcp-(1 leads to 4)-D-Glcp] each including an alpha-(1 leads to 6) thioglycosidic linkage at the non-reducing end. beta-Cyclodextrin binds more strongly than the thiopanose analogues and these have a slightly increasing binding constant with chain length. The reactions are enthalpy-driven with unfavourable contributions from entropy and the variations in enthalpy and entropy compensate each other linearly. SBD was shown to have two binding sites that appear to bind identically and independently in the isolated binding domain, whereas they interact with each other in a negatively cooperative fashion when the catalytic domain of glucoamylase is present (glucoamylase G1). In glucoamylase G1 one site of SBD has an increased binding constant compared to the SBD fragment, whereas the other has the same association constant. The change in binding constant and induced cooperativity were not due to interactions with the catalytic binding site, since binding of beta-cyclodextrin was the same both when the catalytic site was occupied by the strong inhibitor acarbose and when the catalytic site was free.

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The authors investigated the effect of vascular endothelial growth factor (VEGF) on hypoxic injury of cultured rat hippocampal neurons. Treatment with glutamate receptor antagonists prevented hypoxic neuron death. The same magnitude of protection was observed in cultures treated with VEGF, which also reduced excitotoxic neuron death induced directly by an exposure to N-methyl-d-aspartate. Vascular endothelial growth factor did not alter the activation of the transcription factor nuclear factor-κB during hypoxia and protected cells in a PI-3-kinase-independent manner. Vascular endothelial growth factor failed to protect against staurosporine-induced, caspase-dependent apoptosis. These data suggest that VEGF-induced protection against hypoxic injury primarily involves the inhibition of excitotoxic processes.

The effect of the protein synthesis inhibitor II from barley seeds (Hordeum sp.) on protein synthesis was studied in rabbit reticulocyte lysates. Inhibitor treatment of the lysates resulted in a rapid decrease in amino acid incorporation and an accumulation of heavy polysomes, indicating an effect of the inhibitor on polypeptide chain elongation. The protein synthesis inhibition was due to a catalytic inactivation of the large ribosomal subunit with no effect on the small subparticle. The inhibitor-treated ribosomes were fully active in participating in the EF-1-dependent binding of [14C]phenylalanyl-tRNA to poly(U)-programmed ribosomes in the presence of GTP and the binding of radioactively labelled EF-2 in the presence of GuoPP[CH2]P. Furthermore, the ribosomes were still able to catalyse peptide-bond formation. However, the EF-1- and ribosome-dependent hydrolysis of GTP was reduced by more than 40% in the presence of inhibitor-treated ribosomes, while the EF-2- and ribosome-dependent GTPase remained unaffected. This suggests that the active domains involved in the two different GTPases are non-identical. Treatment of reticulocyte lysates with the barley inhibitor resulted in a marked shift of the steady-state distribution of the ribosomal phases during the elongation cycle as determined by the ribosomal content of elongation factors. Thus, the content of EF-1 increased from 0.38 mol/mol ribosome to 0.71 mol/mol ribosome, whereas the EF-2 content dropped from 0.20 mol/mol ribosome at steady state to 0.09 mol/mol ribosome after inhibitor treatment. The data suggest that the inhibitor reduces the turnover of ribosome-bound ternary EF-1 · GTP · aminoacyl-tRNA complexes during proof-reading and binding of the cognate aminoacyl-tRNA by inhibiting the EF-1-dependent GTPase.

International audience; The three-dimensional structure of barley malt alpha-amylase (isoform AMY2-2) was determined by multiple isomorphous replacement using three heavy-atom derivatives and solvent flattening. The model was refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement to an R-factor of 0.153 based on 18,303 independent reflections with F(o) > sigma(F(o)) between 10 and 2.8 A resolution, with root-mean-square deviations of 0.016 A and 3.3 degrees from ideal bond lengths and bond angles, respectively. The final model consists of 403 amino acid residues, three calcium ions and 153 water molecules. The polypeptide chain folds into three domains: a central domain forming a (beta alpha)8-barrel of 286 residues, with a protruding irregular structured loop domain of 64 residues (domain B) connecting strand beta 3 and helix alpha 3 of the barrel, and a C-terminal domain of 53 residues forming a five stranded anti-parallel beta-sheet. Unlike the previously known alpha-amylase structures, AMY2-2 contains three Ca2+ binding sites co-ordinated by seven or eight oxygen atoms from carboxylate groups, main-chain carbonyl atoms and water molecules, all calcium ions being bound to domain B and therefore essential for the structural integrity of that domain. Two of the Ca2+ sites are located only 7.0 A apart with one Asp residue serving as ligand for both. One Ca2+ site located at about 20 A from the other two was found to be exchangeable with Eu3+. By homology with other alpha-amylases, some important active site residues are identified as Asp179, Glu204 and Asp289, and are situated at the C-terminal end of the central beta-barrel. A starch granule binding site, previously identified as Trp276 and Trp277, is situated on alpha-helix 6 in the central (beta alpha)8-barrel, at the surface of the enzyme. This binding site region is associated with a considerable disruption of the (beta alpha)8-barrel 8-fold symmetry.The three-dimensional structure of barley malt alpha-amylase (isoform AMY2-2) was determined by multiple isomorphous replacement using three heavy-atom derivatives and solvent flattening. The model was refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement to an R-factor of 0.153 based on 18,303 independent reflections with F(o) > sigma(F(o)) between 10 and 2.8 A resolution, with root-mean-square deviations of 0.016 A and 3.3 degrees from ideal bond lengths and bond angles, respectively. The final model consists of 403 amino acid residues, three calcium ions and 153 water molecules. The polypeptide chain folds into three domains: a central domain forming a (beta alpha)8-barrel of 286 residues, with a protruding irregular structured loop domain of 64 residues (domain B) connecting strand beta 3 and helix alpha 3 of the barrel, and a C-terminal domain of 53 residues forming a five stranded anti-parallel beta-sheet. Unlike the previously known alpha-amylase structures, AMY2-2 contains three Ca2+ binding sites co-ordinated by seven or eight oxygen atoms from carboxylate groups, main-chain carbonyl atoms and water molecules, all calcium ions being bound to domain B and therefore essential for the structural integrity of that domain. Two of the Ca2+ sites are located only 7.0 A apart with one Asp residue serving as ligand for both. One Ca2+ site located at about 20 A from the other two was found to be exchangeable with Eu3+. By homology with other alpha-amylases, some important active site residues are identified as Asp179, Glu204 and Asp289, and are situated at the C-terminal end of the central beta-barrel. A starch granule binding site, previously identified as Trp276 and Trp277, is situated on alpha-helix 6 in the central (beta alpha)8-barrel, at the surface of the enzyme. This binding site region is associated with a considerable disruption of the (beta alpha)8-barrel 8-fold symmetry.

Glucoamylase (GA) from Aspergillus awamori (EC 3.2.1.3) is a secreted starch hydrolase with a large catalytic domain (aa 1–440), a starch-binding domain (aa 513–616), and a highly O- glycosylated region of 72 aa of unknown function that links the catalytic and starch-binding domains. We have genetically engineered a series of truncated forms of GA to determine how much of the highly O- glycosylated region is necessary for the activity or stability of GAII, a fully active form of the enzyme that lacks the starch-binding domain. Mutations were made by inserting stop-codon linkers into restriction sites within the coding region of the GA gene, and mutated genes were expressed in Saccharomyces cerevisiae for analysis of the truncated enzymes. Our results show that up to 30 aa from the C-terminal end of GAII can be deleted with little effect on the activity, thermal stability, or secretion of the enzyme. Further deletions resulted in diminution or loss of enzyme activity on starch plates, and loss of detectable enzyme in culture supernatants, indicating that these residues are essential for GAII function.

Structure-prediction and hydrophobic-cluster analysis of several starch hydrolases and related enzymes indicated the organization of eleven domain types. Most enzymes possess a catalytic (beta/alpha)8-barrel and a smaller C-terminal domain as seen in crystal structures of alpha-amylase and cyclodextrin glucanotransferase. Some also have a starch-granule-binding domain. Enzymes breaking or forming endo-alpha-1,6 linkages contain domains N-terminal to the (beta/alpha)8-barrel.

The porcine intestinal microvillar aminopeptidase (EC 3. 4. 11. 2) consists of three types of subunits, α, β and γ, of molecular weights determined to 168.000, 118,000 and 54.000, respectively. The isolated detergent-form of the enzyme was cross-linked in dilute solution in the presence of Triton X-100 by diimidates varying in chain length from five to twelve carbon atoms. The tendency of the subunits to participate in inter-protomer cross-linking depended on the chain length of the reagent as shown by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. Diimidates of chain lengths from seven to twelve carbon atoms caused formation of products of molecular weights up to about 660.000, whereas the six carbon atom diimidate generated complexes lower in molecular weight and the five carbon atom diimidate introduced very few inter-protomer cross-linkages. Cross-linking of brush border membrane vesicles, with dimethylsuberimidate, followed by Triton X-100 solubilization and isolation of the microvillar aminopeptidase by immunoadsorbent chromatography on antiaminopeptidase M-Sepharose, indicated a maximum molecular weight of the membrane-bound enzyme of 330.000. The β- and γ-polypeptide chains were previously demonstrated to be formed by limited proteolysis of the α-polypeptide chain. Peptide mapping excluded the possibility of the γ-chain being generated from the β-chain. The subunit structures of the membrane-bound enzyme were suggested as α2, αβγ, and β2γ2. Small amounts of a larger polypeptide chain of molecular weight 330.000 are present in preparations of microvillar aminopeptidase. Peptide mapping indicated structural homology between this component, termed 2α, and α,β and γ. It is suggested that the large polypeptide is a precursor of the intestinal aminopeptidase.

Peptides of the general structure Pro-Leu-X-Pro have been synthesized, with proline in l or d-form and with either glycine or sarcosine in the position of X. Their inhibitory action on the cleavage of the peptide Cbz-Gly-Pro-Leu-Gly-Pro by pure collagenase from Achromobacter iophagus was studied. Competitive inhibition was found in all cases, the most efficient by Pro-Leu-MeGly-Pro. One product of cleavage of the synthetic substrate, Cbz-Gly-Pro-Leu, also inhibits the enzyme.

International audience; alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nM. AMY2 is a (beta/alpha)8-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-strand 3 of the (beta/alpha)8-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2-BASI crystal structure [F. Vallee, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, B. Svensson & R. Haser (1998) Structure 6, 649-659] Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt-bridge with Lys140BASI. These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor. These mutations were performed in the well-expressed, inhibitor-sensitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with Ki = 0.33 nM, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403). The binding energy was reduced by 2.7-3.0 kcal.mol-1 as determined from Ki after the mutations R128Q and D142N. This corresponds to loss of a charged interaction between the protein molecules. In contrast, sensitivity to the inhibitor was gained (Ki = 7 microM) by the mutation T129R/K130P in the insensitive isozyme hybrid. Charge screening raised Ki 14-20-fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme-inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2-BASI complex.alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nM. AMY2 is a (beta/alpha)8-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-strand 3 of the (beta/alpha)8-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2-BASI crystal structure [F. Vallee, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, B. Svensson & R. Haser (1998) Structure 6, 649-659] Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt-bridge with Lys140BASI. These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor. These mutations were performed in the well-expressed, inhibitor-sensitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with Ki = 0.33 nM, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403). The binding energy was reduced by 2.7-3.0 kcal.mol-1 as determined from Ki after the mutations R128Q and D142N. This corresponds to loss of a charged interaction between the protein molecules. In contrast, sensitivity to the inhibitor was gained (Ki = 7 microM) by the mutation T129R/K130P in the insensitive isozyme hybrid. Charge screening raised Ki 14-20-fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme-inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2-BASI complex.

C-terminal processing of low pI barley alpha-amylase (AMY1) results in multiple forms in malt, aleurone protoplasts, and transformed yeast. Expression of an AMY1 cDNA in yeast thus leads to four secreted forms with distinct pI values between 4.7 and 5.1 and essentially identical Mr. AMY1-1 and AMY1-2 lacking the C-terminal Arg-Ser are generated by carboxypeptidase in vitro from AMY1-3 and AMY1-4, respectively. In vivo processing is due to the KEX1-encoded yeast carboxypeptidase. AMY1-2 and AMY1-4 are fully active, whereas AMY+-1 and AMY1-3 retain 3-4% activity toward p-nitrophenyl maltoheptaoside and have one fewer SH group, due to reaction with glutathione. AMY1-1-AMY1-4 are indistinguishable from malt AMY1 with respect to Ca(2+)-, substrate-, and beta-cyclodextrin-binding as well as recognition by three monoclonal antibodies and limited proteolysis by proteinase K. Transient AMY1 precursors present in barley aleurone protoplasts were trapped by addition of serine carboxypeptidase inhibitors, indicating that endogenous carboxypeptidase participates in the maturation of AMY1 during germination. Three pairs of precursor/mature AMY1 forms are recognized, presumably corresponding to the three genes encoding AMY1. Malt carboxypeptidase II can convert in vitro the precursors isolated from protoplasts into processed enzyme, and AMY1 from malt accordingly lacks the C-terminal heptapeptide. This report thus demonstrates posttranslational protein modification by carboxypeptidase in higher plants.

The mechanism of action on animal cell-free protein synthesis by the translation inhibitor II from barley seeds has been studied using the rabbit reticulocyte lysate protein synthesis system. No influence was detected on the ribosomal binding of initiator-tRNA, met-tRNAf, in the presence of barley hordein mRNA, or on the aminoacyl-tRNA synthetase catalyzed formation of aminoacyl-tRNA, suggesting the inhibitor to be without effect both on the protein synthesis initiation and on the supply of aminoacyl-tRNAs. Inhibition of reactions in the elongation cycle was examined by measurement of the transfer of the nascent peptide from peptidyl-tRNA to puromycin. The observed lack of influence on the puromycin reaction implies that the barley inhibitor neither blocked the peptidyltransferase nor the translocation activities. In addition, the high amount of Phe-tRNA bound to ribosomes in the presence of inhibitor and poly(U) suggested the aminoacyl-tRNA binding step to be unaffected by the inhibitor. These findings are compatible with inhibition of a reaction in the elongation cycle subsequent to the aminoacyl-tRNA binding but preceding the translocation.

This project focuses on applying microfluidic tissue culture for electrochemical or optical measurements during programmed cell death (PCD) in barley aleurone layer to increase understanding of the underlying mechanisms of PCD in plants.Microfluidic tissue culture enables in vitro experiments to approach in vivo conditions. Microfluidics also allow implementation of a wide range of electrochemical or optical assays for online, real-time, parallel analysis of important parameters such as redox activity, O2 and H2O2 concentration, extracellular pH, cell viability and enzyme activity1,2.Currently, we are optimising an intracellular whole-cell redox activity assay3 that detects changes in redox activity in barley aleurone layer during PCD. The assay uses a double mediator-system to electrochemically measure redox activity via changes in the NADP:NADPH ratio. Initial experiments assay show that the redox activity changes depending on phytohormone activation or inactivation of aleurone layer metabolism and subsequent PCD. This is similar to H2O2 concentration changes observed recently by Ishibashi et al4.We have also successfully detected PCD induced by phytohormones in barley aleurone layer using a double-fluorescent probe-system also used by Fath et al5.Future challenges include integrating both these systems into a microfluidic device for plant tissue culture.

alpha-Amylases belong to the alpha/beta-barrel protein family in which the active site is created by residues located at the C-terminus of the beta strands and in the helix-connecting loops extending from these ends. In the alpha-amylase family, a small separate domain B protrudes at the C-terminus of the third beta strand of the (beta/alpha)8-barrel framework. The 80% identical barley alpha-amylase isozymes 1 and 2 (AMY1 and AMY2, respectively) differ in substrate affinity and turnover rate, CaCl2 stimulation of activity, sensitivity to the endogenous 21-kDa alpha-amylase/subtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme-specific variations, AMY1-AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMY1-(1-90)-AMY2-(90-403) and AMY1-(1-161)-AMY2-(161-403) characterized in this study contain the 90-residue and 161-residue N-terminal sequences, respectively, of AMY1 and complementary C-terminal regions of AMY2. AMY1-(1-90)-AMY2-(90-403) comprises the 60-amino-acid domain B of AMY2 and resembles this isozyme in sensitivity to alpha-amylase/subtilisin inhibitor and its low affinity for the substrates p-nitrophenyl alpha-D-maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1-(1-161)-AMY2-(161-403) and AMY1, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaCl2. Domain B thus determines several functional and stability properties that distinguish the barley alpha-amylase isozymes.

Aspergillus niger glucoamylases GI and GII (E.C. 3.2.1.3) were isolated from a commercial enzyme preparation by ammonium sulfate precipitation followed by DEAE-cellulose ion exchange chromatography. Both enzymes consist of a single glycosylated polypeptide chain. The molecular weights of GI and GII were determined by sedimentation equilibrium ultracentrifugation to 52,000 and 46,000, respectively, and by molecular sieving to 65,000 and 55,000. The amino acid compositions of GI and GII were very similar. Furthermore, the N-terminal amino acid sequence of the intact GI and GII as well as of their cyanogen fragments were identical, suggesting great homology in the primary structure of the two forms. In addition the digests of GI and GII produced respectively by Armillaria mellea protease, Staphylococcus aureus V8 protease, and submaxillary protease were analyzed by high pressure gel permeation chromatography. The elution profiles were also consistent with GI and GII having similar polypeptide chains. However, digestion with carboxypeptidase Y showed different C-terminal residues of the two forms.

During the past few years great progress has been made in the understanding of the function of starch-degrading enzymes. Combining mutational analysis, multiple sequence alignment, and three dimensional structures of enzyme substrate analogue complexes formed the basis for developing amylolytic and related enzymes through protein engineering. In the case of barley a-amylase this has involved rational site-directed mutagenesis, regional random mutagenesis and isozyme sequence exchange as well as generation of isozyme chimeras. The approach has enabled modulation of enzyme specificity and identification of the functional roles of distinct residues and regions. The mutational analysis included the complex between barley α-amylase 2 and a proteinaceous inhibitor from barley seeds, BASI (barley a-amylase/subtilisin inhibitor). The interplay between the catalytic and the starch binding domains in glucoamylase from Aspergillus niger was investigated. The two domains are connected by a highly O-glycosylated linker and bind with 1:1 stoichiometry double-headed heterobifunctional synthetic analogues targeted to each domain. Glucoamylase is an inverting hydrolase and replacement of the catalytic base, a glutamic acid, by a cysteinesulfinic acid, gave rise to activity surpassing that of the wild-type enzyme, indicating a most surprising flexibility for the catalytic base.

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The complete amino acid sequence of the barley translation inhibitor II has been determined. Peptide fragments were generated by cleavage with either cyanogen bromide, hydroxylamine, o-iodosobenzoic acid, S. aureus V8 protease, endoproteinase Lys-C, clostripain, or trypsin. The fragments were separated by gel filtration and RP-HPLC and subjected to automated liquid phase sequencing, gas-phase sequencing or mass spectrometry. The translation inhibitor possessed a blocked N-terminus identified as acetylated alanine by mass spectrometry of an N-terminally blocked tetrapeptide generated by cleavage with cyanogen bromide. Barley translation inhibitor II consists of 280 amino acid residues in a single chain and shows a distant homology to the ricin A-chain.

Chymotrypsinogen A and alpha-chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N-acetyl-L-tryptophan methyl ester and N-acetyl-L-tyrosine ethyl ester catalyzed by alpha-chymotrypsin and delta-chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated alpha-chymotrypsin. Nitrated alpha-chymotrypsin, nitrated delta-chymotrypsin and delta-chymotrypsin could all bind N-acetyl-L-tryptophan methyl ester at alkaline pH, in contrast to alpha-chymotrypsin. The dissociation constant, Kd, of the complex of alpha-chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten-fold relative to the constant obtained with unmodified alpha-chymotrypsin. The nitrated delta-chymotrypsin and delta-chymotrypsin showed identical Kd values. The nitrated alpha-chymotrypsin is inactivated faster at pH 8.0 and 8.5 than alpha-chymotrypsin and apparently by a different mechanism.

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a β-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-β-d-xylopyranoside (p NPX), para-nitrophenyl-α-L-arabinofuranoside (p NPA), β-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2–4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I − V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for p NPA:p NPX of 1:25. BXA43 was stable below 40°C and at pH 4.0–8.0 and showed maximum activity at pH 5.5 and 50°C. K m and k cat for p NPX were 15.6 ± 4.2 mM and 60.6 ± 10.8 s-1, respectively, and substrate inhibition became apparent above 18 mM p NPX. Similar kinetic parameters and catalytic efficiency values were reported for β-d-xylosidase (XynB3) from Geobacillus stearothermophilus T‒6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to p NPX. BXA43 showed clearly metal inhibition for Zn2+ and Ag+, which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg505Tyr506 present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus.