Action of lysozyme on oligosaccharides from peptidoglycan N-unacetylated at glucosamine residues

On N-acetylmuramyl-L-alanine-amidase treatment followed by lysozyme digestion, peptidoglycan N-unacetylated partially at glucosamine residues yielded three oligosaccharides with N-unacetylated glucosamine residues, GlcN-MurNAc-GlcNAc-MurNAc¹, GlcN-MurNAc-GlcN-MurNAc-GlcNAc-MurNAc, and GlcNAc-MurNAc-GlcN-MurNAc-GlcNAc-MurNAc. Lysozyme can not hydrolyze the former two saccharides, whereas it cleaves the latter one into GlcNAc-MurNAc and GlcN-MurNAc-GlcNAc-MurNAc. This confirms requirement for the acetamido group of the N-acetylglucosamine in the interaction with subsite C of lysozyme.     

Peptidoglycan of Rhodopseudomonas viridis: partial lack of N-acetyl substitution of glucosamine

A lack of at least 70% of N-acetyl substitution of glucosamine in the glycan strands of the peptidoglycan from the gram-negative bacterium Rhodopseudomonas viridis is reported. A disaccharide, very likely GlcN beta(1 leads to 4) Mur, was observed in hydrolysates of the isolated peptidoglycan. The disaccharide was not observed when peptidoglycan was N-acetylated before hydrolysis. The peptidoglycan of R. viridis was resistant to lysozyme but became sensitive after N-acetylation with acetic anhydride. The disaccharide was found with peptidoglycan from all R. viridis strains investigated, as well as with R. sulfoviridis P1 and R. palustris strains, but not with peptidoglycan from R. gelatinosa, Rhodospirillum tenue, and Pseudomonas diminuta NCTC 8545.     

Structure of the rigid-layer of rhizobium cell wall. II. Evidence for a covalent bond between peptidoglycan and cellodextrins

Covalent linkages between peptidoglycan and cellodextrins in the cell walls of Rhizobium were defined by the analysis of lysozyme split products. Digestion of peptidoglycan with lysozyme resulted in the liberation, beside disaccharide tetrapeptide fragments composed of glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in a molar ratio 1:1:2:1:1, also significant amounts of glucose and its polymers. The neutral carbohydrates composed of glucose, were further purified and determined as cellobiose, cellotriose and cellotetrose. Peptidoglycans pretreated with cellulase, which librated glucose and cellobiose, still contains glucose linked by lysozyme sensitive but cellulase insensitive bond.     

A three dimensional model of the digestion of peptidoglycan by lysozyme

The digestion of single peptidoglycan chains of the recently proposed conformation (Formanek et al., 1974) can be described with the same enzymatic mechanism as proposed by Phillips for a hexasaccharide consisting of alternating N-acetylglucosamine, N-acetylmuramic acid residues (Phillips, 1966). It is shown by model building, that in a peptidoglycan lysozyme complex the peptide chains do not exhibit any sterical hindrance. The digestion of the peptidoglycan sacculus by lysozyme may occur at latice defects of its paracrystalline structure. A slit of about 30 A length and 10--15 A width between peptidoglycan micells may be sufficient for the attachment of lysozyme.     

A three dimensional model of the digestion of peptidoglycan by lysozyme

The digestion of single peptidoglycan chains of the recently proposed conformation (Formanek et al., 1974) can be described with the same enzymatic mechanism as proposed by Phillips for a hexasaccharide consisting of alternating N-acetylglucosamine, N-acetylmuramic acid residues (Phillips, 1966). It is shown by model building, that in a peptidoglycan lysozyme complex the peptide chains do not exhibit any sterical hindrance.The digestion of the peptidoglycan sacculus by lysozyme may occur at lattice defects of its paracrystalline structure. A slit of about 30 å lenght and 10–15 å width between peptidoglycan micells may be sufficient for the attachment of lysozyme.     

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.     

Chemical composition and turnover of peptidoglycan in Neisseria gonorrhoeae.

The peptidoglycan of all four colonial types of a number of strains of Neisseria gonorrhoeae constituted 1 to 2% of the dry weight of the cell. The chemical composition of cell types examined was similar with molar ratios of 1:1:2:1:1 for muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid, respectively. Ninety-six percent of the mass of the peptidoglycan was composed of these compounds. A lipoprotein analogous to that observed in Escherichia coli was not detected. The chain length of the glycan varied from 80 to 110 disaccharide units. The peptide contained equimolar amounts of D- and L-alanine. The rate of turnover of peptidoglycan in strain RD5 was 50% per generation. Turnover proceeded without a lag and followed first-order kinetics.     

Molecular organization of heparan sulphate from human skin fibroblasts.

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.     

Purification and characterization of lysozyme produced by Streptomyces erythraeus.

A species of lysozyme (SE lysozyme) was purified from culture filtrate of Streptomyces erythraeus. The enzyme has a molecular weight of 18,500 as determined by ultracentrifugation. Its isoelectric point is 9.5, and it shows optimal activity at pH 4.0 with an optimal ionic strength of 0.1. Investigation of the substrate specificity showed SE lysozyme to be an N-acetyl-muramidase. The simplest product in the digest of cell walls of Micrococcus lysodeikticus was identified as a disaccharide, [GlcNAcbeta(1 leads to 4) MurNAc]. While S. aureus as well as M. lysodeikticus was lysed by this lysozyme, chitin and its derivatives were not.     

Characterization of the structure of antithrombin-binding heparan sulfate generated by heparan sulfate 3-O-sulfotransferase 5

The 3-O-sulfation of glucosamine is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). Both heparan sulfate 3-O-sulfotransferase -1 (3-OST-1) and 3-O-sulfotransferase-5 (3-OST-5) transfer sulfate to the 3-OH group of glucosamine to generate antithrombin-binding heparan sulfate (HS(act)). Here, we reported the isolation and characterization of the antithrombin-binding HS oligosaccharides generated by 3-OST-5 (3-OST-5 oligo(act)). (3)H-labeled HS of Chinese hamster ovary cells was exhaustively modified by 3-OST-1 to remove the 3-OST-1 modification sites followed by antithrombin-affinity fractionation. The non-antithrombin-binding fraction of 3-OST-1 pretreated HS was further modified by 3-OST-5 to generate additional antithrombin-binding HS, which was designated as 3-OST-5 HS(act). Structural analysis of 3-OST-5 HS(act) revealed that the antithrombin-binding site of 3-OST-5 HS(act) is located within a domain clustered with N-sulfated glucosamine units. We also isolated 3-OST-5 antithrombin-binding oligosaccharides (3-OST-5 oligo(act)) from high pH nitrous acid degraded 3-OST-5 HS(act). A disaccharide analysis revealed that 3-OST-5 oligo(act) were composed of multiple 3-O-sulfated glucosamine units. Our results provide additional insights on the relationship between the anticoagulant activity and structure of HS.     

Regulation of the functional activity of lysozyme by alkylhydroxybenzenes

In our study, we investigated the capacity of alkylhydroxybenzenes (AHB), which are microbial anabiosis autoinducers, for alteration of the enzymatic activity of the hen egg-white lysozyme, as well as the efficiency of hydrolysis of specific (peptidoglycan) and nonspecific (chitin) substrates catalyzed by lysozyme. AHB homologues (C7-AHB and C12-AHB), which differ in their hydrophobicity and effects in their interaction with lysozyme, were used as modifying agents. C7-AHB stimulated enzymatic activity within the whole range of concentrations used (10^?7?10^?3 M). More hydrophobic C12-AHB exhibited this ability only at low concentrations and inhibited fermentative activity at high concentrations, acting as a mixed-type inhibitor. Both AHB homologues caused changes in the hydrophobicity of lysozyme molecules. An increase in the affinity level between the C7-AHB-modified enzyme and the nonspecific substrate (colloidal chitin or cell wall polymers of Saccharomyces sp.) was observed, which manifested itself in the enhancement of the hydrolysis rate by 200–500% (as compared to the native enzyme). A significant effect on the efficiency of the lysozyme-catalyzed modifications of the substrate (peptidoglycan, colloidal chitin) structure as a result of its complexation with AHB was demonstrated. A stabilizing effect of C7-AHB and C12-AHB was revealed, which ensured a high level of activity of the AHB-modified enzyme (as compared to the control) after heat treatment (functional stability), as well as at nonoptimal temperatures of catalysis (operational stability). The biological significance of lysozyme modification with AHB and the practical aspects of its application are discussed.     

Isolation and characterization of the bacteriophage T4 tail-associated lysozyme.

Direct evidence has been obtained that the tail-associated lysozyme of bacteriophage T4 (tail-lysozyme) is gp5, which is a protein component of the hub of the baseplate. Tails were treated with 3 M guanidine hydrochloride containing 1% Triton X-100, and the tail-lysozyme was separated from other tail components by preparative isoelectric focusing electrophoresis as a peak with a pI of 8.4. The molecular weight as determined from sodium dodecyl sulfate electrophoresis was 42,000. The tail-lysozyme was unambiguously identified as gp5 when the position of the lysozyme was compared with that of gp5 of tube-baseplates from 5ts1/23amH11/eL1ainfected Escherichia coli cells by two-dimensional gel electrophoresis. The tail-lysozyme has N-acetylmuramidase activity and the same substrate specificity as gene e lysozyme; the optimum pH is around 5.8, about 1 pH unit lower than for the e lysozyme. We assume that the tail-lysozyme plays an essential role in locally digesting the peptidoglycan layer to let the tube penetrate into the periplasmic space. The tail-lysozyme is presumably also responsible for "lysis from without."     

The antiproliferative activity of arterial heparan sulfate resides in domains enriched with 2-O-sulfated uronic acid residues.

Heparan sulfate isolated from bovine arterial tissue by a multistep purification procedure or from arterial tissue proteoheparan sulfate by beta-elimination exhibits antiproliferative activity toward arterial smooth muscle cells when added to subconfluent cell cultures in a concentration of 50-100 micrograms/ml medium. Enzymatic disintegration of heparan sulfate by heparitinases I and II and isolation of the resulting oligosaccharides indicate that the antiproliferative activity of the heparan sulfate molecule resides in a sulfate-rich octa/decasaccharide domain which is separated by longer sequences of sulfate-free or sulfate-poor N-acetylglucosamine containing disaccharide units. The octa/decasaccharide fraction has a 3-4-fold higher antiproliferative activity than the native heparan sulfate molecule and contains 45% of a disulfated disaccharide which consists of 2-O-sulfated uronic acid and N-sulfated glucosamine (UA(2S)-GlcNS and 12% of a trisulfated disaccharide (UA(2S)-GlcNS(6S). A sulfate-rich hexasaccharide fraction containing 14% of the disulfated disaccharide but 18% of the trisulfated disaccharide has negligible antiproliferative activity. The results indicate the presence of specific structural determinants in the arterial heparan sulfate molecule which may have the function of an endogenous inhibitor of arterial smooth muscle cell growth.     

Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[HO3S-6]Gal(beta 1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.     

p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme

p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.     

Isolation and characterization of sulphated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-beta-galactosidase

A series of oligosaccharides has been isolated from the keratan sulphate peptidoglycan (3 M NaCl fraction) of bovine cornea after digestion with the endo-beta-galactosidase of Bacteroides fragilis. Structural information on the major oligosaccharides was obtained from (a) their susceptibilities to endo-beta-galactosidase before and after desulphation, (b) their elution positions on a column of Bio-Gel P-4 and retention times on a high-performance anion-exchange column and (c) negative-ion fast-atom-bombardment mass spectrometry. More than 75% of the oligosaccharides were sulphated unbranched poly(N-acetyllactosamine) sequences, (-3/4GlcNAc beta 1-3Gal beta 1-)n, and approximately 3% was the neutral disaccharide, GlcNAc beta 1-3Gal. The sulphated disaccharide, GlcNAc-SO-3 beta 1-3Gal, accounted for almost 35% of the oligosaccharide material while 40% consisted of four oligosaccharides, unbranched tetra-, hexa-, octa- and decasaccharides of poly(N-acetyllactosamine) type, having 3, 5, 7 and 9 sulphate residues respectively. Proton nuclear magnetic resonance studies at 500 MHz (Hounsell, E. F., et al. following paper in this journal) have shown that a sulphate residue is attached to the C-6 position of each N-acetylglucosamine and each internal galactose residue of these four oligosaccharides which express to varying degrees the antigenic determinants recognised by three monoclonal antibodies to keratan sulphate (Mehmet, H. et al., paper which follows the next paper in this journal).     

Structure of the linkage units between ribitol teichoic acids and peptidoglycan.

The structure of the linkage regions between ribitol teichoic acids and peptidoglycan in the cell walls of Staphylococcus aureus H and 209P and Bacillus subtilis W23 and AHU 1390 was studied. Teichoic acid-linked saccharide preparations obtained from the cell walls by heating at pH 2.5 contained mannosamine and glycerol in small amounts. On mild alkali treatment, each teichoic acid-linked saccharide preparation was split into a disaccharide identified as N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol residues. The Smith degradation of reduced samples of the teichoic acid-linked saccharide preparations from S. aureus and B. subtilis gave fragments characterized as 1,2-ethylenediol phosphate-(glycerolphosphate)3-N-acetylmannosaminyl beta(1----4)N- -acetylxylosaminitol and 1,2-ethylenediolphosphate-(glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylxylosaminitol, respectively. The binding of the disaccharide unit to peptidoglycan was confirmed by the analysis of linkage-unit-bound glycopeptides obtained from NaIO4 oxidation of teichoic acid-glycopeptide complexes. Mild alkali treatment of the linkage-unit-bound glycopeptides yielded disaccharide-linked glycopeptides, which gave the disaccharide and phosphorylated glycopeptides on mild acid treatment. Thus, it is concluded that the ribitol teichoic acid chains in the cell walls of the strains of S. aureus and B. subtilis are linked to peptidoglycan through linkage units, (glycerol phosphate)3-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and (glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine, respectively.     

Cell walls from Actinopolyspora halophila,an extremely halophilic actinomycete

Cell wall components were prepared from Actinopolyspora halophila (strain wt), an extremely halophilic actinomycete requiring a minimum 12% NaCl concentration for growth, and from an erythromycin-resistant strain of A. halophila (strain ER) that required only 6% NaCl for growth. Both cell wall preparations contained glutamic acid, alanine, and diaminopimelic acid in a 1:2:1 molar ratio. On the basis of muramic acid content, peptidoglycans from the wt and ER strains contained 255 and 245 disaccharide units per mg dry weight respectively. In addition, both cell wall preparations contained from 10 to 20% more glucosamine than muramic acid, and equimolar amounts of d-galactose and d-arabinose. Analysis of cell walls before and after digestion with Myxobacter AL-1 protease indicated that nearly all glycan disaccharide units were peptide-substituted and that peptide cross-bridging was facilitated by direct peptide linkages between N-diaminopimelic acid and C-terminal alanine. While the peptidoglycan of A. halophila wt was 50% peptide cross-linked, that from A. halophila ER was approximately 67% peptide cross-linked. Chemical modifications involving substitution of non-N-acetylated hexosamines of the cell walls greatly enhanced their sensitivity to lysozyme. Although differences in peptidoglycan structure between the two strains of A. halophila were observed, these probably do not account for the reduced salt requirement for growth of the erythromycin-resistant strain.Issued as NRCC 25165     

Oligosaccharide chains of herpes simplex virus type 2 glycoprotein gG.2

gG.2 glycoprotein was purified by H966 monoclonal antibodies linked to Sepharose from herpes simplex virus type 2-infected HEp-2 cells labeled with [3H] glucosamine. The glycoprotein was subjected to Pronase digestion and the glycopeptides were fractionated by Con A-Sepharose in a major fraction (88.5% of total radioactivity) unbound to the lectin gel and in a minor species which bound to the lectin as a N-linked diantennary oligosaccharide. Mild and strong acid hydrolysis of Con A-unbound and Con A-bound fractions revealed that (i) both species were highly sialylated; (ii) the Con A-unbound fraction contained mainly labeled N-acetylgalactosamine, as is the case for O-linked oligosaccharides; and (iii) the Con A-bound fraction carried the vast majority of the labeled N-acetylglucosamine present in gG.2. Three size classes of oligosaccharides were separated from mild alkaline borohydride-treated Con A-unbound glycopeptides, which accounted for about 80% of the radioactivity present in the fraction. Galactosaminitol was recovered as the major labeled product in the strong acid hydrolyzates of the oligosaccharides generated by reductive beta-elimination, indicating that they were O-glycosidically linked to the peptide backbone. Thin-layer and DEAE-Sephacel chromatography of the three O-linked oligosaccharide species indicated that disialylated tetrasaccharides and monosialylated trisaccharides were the major components, whereas neutral disaccharide was a minor component. Digestion with neuraminidase and beta-galactosidase of the O-linked oligosaccharides supported the idea that the common disaccharide core was mainly of the structure beta-galactosyl-N-acetylgalactosamine. The large occurrence of O-linked oligosaccharides differentiates this type 2-specific herpes simplex virus glycoprotein from the type-common herpesvirus glycoproteins gB, gC, and gD.     

Peptidoglycan of free-living anaerobic spirochetes

Electron microscope examination of negatively stained or thin-sectioned cells of Spirochaeta stenostrepta treated with penicillin or lysozyme showed that the peptidoglycan was present as a thin, electron-dense layer adjacent and external to the cytoplasmic membrane. The peptidoglycan was isolated from cells of S. stenostrepta and Spirochaeta litoralis by a procedure including treatments with sodium lauryl sulfate and Pronase. Hydrolysates of the isolated S. stenostrepta and S. litoralis peptidoglycans contained glucosamine, muramic acid, glutamic acid, l-ornithine, and alanine in molar ratios of 0.90:0.85:1.00:1.00:1.40 and of 0.63:0.63:0.99:1.00:1.41, respectively. Determination of N-terminal residues suggested that nearly 50% of the ornithine in S. stenostrepta and S. litoralis peptidoglycans was involved in peptide cross-linkage. The peptidoglycan layer of S. stenostrepta was sensitive to lysozyme and myxobacter AL-1 protease.