Affinity chromatographic purification of human lysozyme, with special reference to human leukemia lysozyme.

Lysozyme [EC 3.2.1.17] was purified from human tears, serum, and urine of acute monocytic leukemia patients, renal disease patients, and residents in cadmium-polluted areas of Tsushima Island using an affinity adsorbent containing lysozyme-lysate of Micrococcus lysodeikticus cell walls as the ligand. By means of this procedure, leukemia lysozyme was purified 100- to 200-fold with an activity recovery of 80%. It was crystallized at pH 10. This purified preparation appeared homogeneous in disc electrophoresis and showed a specific activity 2.5-fold higher than that of crystalline lysozyme from hen egg-white. Tear lysozyme was also purified to a nearly homogeneous state while the enzymes from normal serum and urine of a nephrosis patient and of residents in cadmium-polluted area were still disc electrophoretically heterogeneous and showed low specific activity as compared with purified leukemia lysozyme.     

Affinity chromatography of alpha-chymotrypsin, subtilism, and metalloendopeptidases on carbobenzoxy-L-phenylalanyl-triehtylenetetraminyl-sepharose.

Carbobenzoxy-L-phenylalanyl-triethylenetetraminyl-Sepharose (Z-L-Phe-T-Sepharose) was found to be an effective affinity adsorbent for bovine pancreatic alpha-chymotrypsin [EC 3.4.21.1] as well as neutral [EC 3.4.24.4] and alkaline [EC 3.4.21.14] proteases of Bacillus species. These enzymes were adsorbed in the neutral pH range. alpha-Chymotrypsin was recovered by elution with 0.1 A acetic acid while neutral subtilopeptidase was eluted with 0.5 M NaCl at pH 0. Thermolysin and subtilisin were found in eluates with 1.5 and 2.0 M guanidine-HCl at pH 7.2, respectively. The resulting enzymes appeared homogeneous on disc-electrophoresis and showed higher specific activities than those of crystalline or highly purified preparations available commercially. Modifications of the active site serines of alpha-chymotrypsin and subtilisin by treatment with diisopropylfluorophosphate (DFP) or phenylmethanesulfonyl fluoride (PMSF) resulted in loss in their binding abilities to the adsorbent. Complexes of porcine alpha2-macroglobulin with each of these four enzymes and that of Streptomyces-subtilisin inhibitor (S-SI) with subtilisin were also found in nonadsorbed fractions.     

Differential Patterns of Bacteriolytic Activities in Potato in Comparison to Bacteriophage T4 and Hen Egg White Lysozymes

A comparison of the enzymatic activities of endogenous potato bacteriolytic enzymes with bacteriophage T4 and hen egg white lysozyme has been performed. Using Erwinia carotovora atroseptica and Pseudomonas solanacearum as substrates in, comparison to Micrococcus lysodeikticus a differential pattern of bacteriolytic activities could be detected. The expression pattern of endogenous potato lysozymes suggests that their functional activity against phytopathogenic bacteria in planta is unlikely. Antibacterial activities in transformed, T4 lysozyme expressing and non–transformed potato plants are evaluated.     

Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

Purification, amino acid sequence, and some properties of rabbit kidney lysozyme

The lysozyme (rabbit kidney lysozyme) from the homogenate of rabbit kidney (Japanese white) was purified by repeated cation-exchange chromatography on Bio-Rex 70. The amino acid sequence was determined by automated gas-phase Edman degradation of the peptides obtained from the digestion of reduced and S-carboxymethylated rabbit lysozyme with Achromobacter protease I (lysyl endopeptidase). The sequence thus determined was KIYERCELARTLKKLGLDGYKGVSLANWMCLAKWESSYNTRATNYNPGDKSTDYGIFQ INSRYWCNDGKTPRAVNACHIPCSDLLKDDITQAVACAKRVVSDPQGIRAWVAWRNHCQ NQDLTPYIRGCGV, indicating 25 amino acid substitutions from human lysozyme. The lytic activity of rabbit lysozyme against Micrococcus lysodeikticus at pH 7, ionic strength of 0.1, and 30 degrees C was found to be 190 and 60% of those of hen and human lysozymes, respectively. The lytic activity-pH profile of rabbit lysozyme was slightly different from those of hen and human lysozymes. While hen and human lysozymes had wide optimum activities at around pH 5.5-8.5, the optimum activity of rabbit lysozyme was at around pH 5.5-7.0. The high proline content (five residues per molecule compared with two prolines per molecule in hen or human lysozyme) is one of the interesting features of rabbit lysozyme. The transition temperatures for the unfolding of rabbit, human, and hen lysozymes in 3 M guanidine hydrochloride at pH 5.5 were 51.2, 45.5, and 45.4 degrees C, respectively, indicating that rabbit lysozyme is stabler than the other two lysozymes. The high proline content may be responsible for the increased stability of rabbit lysozyme.     

Calcium-binding and structural stability of echidna and canine milk lysozymes.

For echidna and canine milk lysozymes, which were presumed to be the calcium-binding lysozymes by their amino acid sequences, we have quantitated their calcium-binding strength and examined their guanidine unfolding profiles. The calcium-binding constants of echidna and canine lysozymes were determined to be 8.6 x 10(6) M(-1) and 8.9 x 10(6) M(-1) in 0.1 M KCl at pH 7.1 and 20 C, respectively. The unfolding of decalcified canine lysozyme proceeds in the same manner as that of alpha-lactalbumin, through a stable molten globule intermediate. However, neither calcium-bound nor decalcified echidna lysozyme shows a stable molten globule intermediate. This unfolding profile of echidna lysozyme is identical to that of conventional lysozymes and pigeon egg-white lysozyme, avian calcium-binding lysozyme. This result supports the suggestion of Prager and Jolles (Prager EM, Jolles P. 1996. Animal lysozymes c and g: An overview. In: Jolles P, ed. Lysozymes: Model enzymes in biochemistry and biology. Basel-Boston-Berlin: Birkhauzer Verlag. pp 9-31) that the lineage of avian and echidna calcium-binding lysozymes and that of eutherian calcium-binding lysozymes diverged separately from that of conventional lysozymes.     

Bacteriolytic enzymes produced by actinomycetes. I. The physicochemical properties of the enzymes and the spectrum of their lytic action

This review is devoted to the bacteriolytic enzymes produced by many actinomycetes, mainly by Streptomyces genus. The bacteriolytic enzymes hydrolyse the specific bonds in bacterial peptidoglycans and cause the solubilization of the cellular walls and the disintegration of the bacterial cells. Many of the enzymes are purified to the electrophoretic homogeneity. The actinomycetes form the endo-N-acetylmuramidases more often, then the endopeptidases follow according to the frequency of occurrence, while the amidases and endo-N-acetylglucosaminidases are met rather seldom among the streptomycete-producers. The known amidases and exo-enzymes which are also produced by some species of actinomycetes are not related to the lytic enzymes proper. Almost all known endopeptidases from streptomyces hydrolyse the bridge peptide bonds in which the carboxyl group of terminal D-alanyl of peptide chain is involved. The bacteriolytic spectra of the different muramidases differ from each other and essentially differ from the spectrum of the egg-white lysozyme. Some endomuramidases from streptomyces are able to hydrolyse streptococci and some other important from the practical point of view microorganisms resistant to the action of lysozyme.     

Some properties of the lysozymes in serum and colostrum from cows with high and low lytic power against Micrococcus lysodeikticus

Previous work has uncovered a dominant gene for high bacteriolytic activity of bovine serum against the test bacterium Micrococcus lysodeikticus. This major gene effect is also fully expressed in colostrum. In the present study the lytic power of serum and colostral whey from high and low level cows was subjected to a degree of characterization. It was found that the enzyme activities studied exhibited properties in accordance with those defined for a lysozyme (EC 3.2.1.17), i.e. (1) lysis of a suspension of M. lysodeikticus, (2) basic protein (pI = 10.0 and pI = 10.3 for bovine serum lysozyme (BSL) and bovine colostrum lysozyme (BCL), respectively), (3) molecular weight (MW) approximately 16 000 for both BSL and BCL, (4) liberation of free reducing sugars during action on cell wall peptidoglycan (the kinetics of BSL and BCL differed strongly), and (5) fairly heat stable, especially at acidic pH and relative labile at alkaline pH (BCL was far more sensitive to heating at alkaline pH than was BSL). The dramatic differences in activity between high and low level animals might be due to a major genetic mechanism influencing the amount of, or the activity of, circulating enzyme molecules, rather than a structural gene coding for a certain enzyme with a particular specific activity. This is also supported by the high correlation between the lytic capacity of BSL and BCL in spite of the different properties of these lysozymes (i.e. in respect of pI, enzyme kinetics and heat stability) reported in the present study.     

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.     

链霉菌G4溶菌酶的产生条件及特性

对链霉菌G4的产酶发酵条件和溶菌特性进行研究结果表明:蔗糖30 g/L、大豆蛋白胨12.5 g/L、牛肉膏2 g/L,对产酶最为有利;G4溶菌酶最适培养温度33 ℃,培养时间72 h,培养基初始pH 8.G4溶菌酶的最适作用温度和最适作用pH分别是55 ℃和6.5,多数金属离子会抑制G4溶菌酶的活性,其中Zn2+、Cu2+、Fe2+、 Pb2+几乎可以使其完全失活;对几种细菌、酵母菌的研究表明,G4溶菌酶对卵清溶菌酶不能作用的变形链球菌和金黄色葡萄球菌有很强的溶解活性.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme

Human airway lysozyme, purified from pathological bronchial secretions, is characterized by a specific activity 3-fold higher than that of hen egg-white lysozyme. The amino acid composition of human airway lysozyme is identical to that of other human lysozymes. The laser Raman spectra of human airway lysozyme and hen egg-white lysozyme in phosphate buffer solution (pH 7.2) are recorded in the range 300-1900 cm-1 at 488 nm. Drastic intensity differences are observed between the spectra analyzed in the ranges characteristic of the peptide backbone (e.g., beta-sheet; C alpha-C, C alpha-N), and of the aromatic side-chain vibrations (tyrosine, tryptophan). The deconvolution of the Raman amide I band gives secondary structures of 38% and 39% alpha-helix, 25% and 20% beta-sheet, and 37% and 41% undefined structure for the human and hen lysozymes, respectively.     

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.     

Purification and characterization of chitinase produced by Streptomyces erythraeus

A chitinase was purified from the culture filtrate of Streptomyces erythraeus (SE). The enzyme (SE chitinase) has a molecular weight of 30,000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl. SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of Micrococcus lysodeikticus. The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes. The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives. The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes.     

Process evaluations and economic analyses of recombinant human lysozyme and hen egg-white lysozyme purifications

Human lysozyme and hen egg-white lysozyme have antibacterial, antiviral, and antifungal properties with numerous potential commercial applications. Currently, hen egg-white lysozyme dominates low cost applications but the recent high-level expression of human lysozyme in rice could provide an economical source of lysozyme. This work compares human lysozyme and hen egg-white lysozyme adsorption to the cation exchange resin, SP-Sepharose FF, and the effect of rice extract components on lysozyme purification. With one exception, the dynamic binding capacities of human lysozyme were lower than those of hen egg-white at pH 4.5, 6, and 7.5 with ionic strengths ranging from 0 to 100 mM (5-20 mS). Ionic strength and pH had a similar effect on the adsorption capacities, but human lysozyme was more sensitive to these two factors than hen egg-white lysozyme. In the presence of rice extract, the dynamic binding capacities of human and hen egg-white lysozymes were reduced by 20-30% and by 32-39% at pH 6. Hen egg-white lysozyme was used as a benchmark to compare the effectiveness of human lysozyme purification from transgenic rice extract. Process simulation and cost analyses for human lysozyme purification from rice and hen egg-white lysozyme purification from egg-white resulted in similar unit production costs at 1 ton per year scale.     

Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori)

Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26°C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.     

Frog lysozyme. V. Isolation and some physical and immunochemical properties of lysozyme isozymes of the leopard frog, Rana pipiens.

Frog Lysozyme has been purified by sequential application of acid extraction, salt fractionation, CM-cellulose chromatography, heat treatment, and gel filtration. Eight isozymes of purified lysozyme were found to be stable during prolonged storage. Isozymes were separated by preparative polyacrylamide gel electrophoresis, Ninety percent of the lytic activity of frog ovarian egg was represented by forms 7 and 8, the most highly charged isozymes. Seventy-eight percent of frog liver lysozyme activity was that of form 4. Forms 7 and 8 differed from form 4 by being larger (apparent molecular weight of 18,000 vs. 16,000), by remaining active in more acidic environment, and by exhibiting a dependency upon NaCl for activity. Antiserum prepared against frog form 4 did not react with frog forms 7 and 8 and antiserum to chicken egg-white lysozyme did not react with any frog lysozymes. All frog lysozymes showed identical reversible binding to deaminated chitin. Apparent size differences and lack of immunological cross-reactivity suggest that at least some of the isozymes are non-allelic.     

Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica)

Lysozyme was purified from the plasma of eastern oysters (Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies. The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10. Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes. No similarity was found however between the N-terminal sequence of oyster plasma lysozyme and N-terminal sequences of other i-type lysozymes, suggesting that the N-terminal sequences of the i-type lysozymes may vary to a greater extent between species than reported in earlier studies. The optimal ionic strength, pH, cation concentrations, sea salt concentrations, and temperature for activity of the purified lysozyme were determined, as well as its temperature and pH stability. Purified oyster plasma lysozyme inhibited the growth of Gram-positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria (e.g., Escherichia coli, Vibrio vulnificus). This is a first report of a lysozyme purified from an oyster species and from the plasma of a bivalve mollusc.     

Characterization of lysozyme synthesized by differentiated mouse myeloid leukemia cells.

Lysozyme was induced by dexamethasone during normal differentiation of cultured mouse myeloid leukemia cells (M1) to macrophages and granulocytes. A large amount of lysozyme was produced by macrophage-like line cells (Mm-1), established from spontaneously differentiated macrophage-like cells from a clonal line of M1 cells. Lysozyme purified from the culture medium of these Mm-1 cells (Mm-1 lysozyme) had a molecular weight of 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and showed maximal activity at pH 6.6 with an optimal NaCl concentration of 0.04 M. Its mobility on polyacrylamide gel electrophoresis at pH 4.5 was distinctly lower than those of lysozymes from hen egg white and human urine. Rabbit anti-Mm-1 lysozyme serum inhibited the activities of lysozyme preparations from peritoneal macrophages of normal mice and rats and dexamethasone-induced differentiated M1 cells, but not those of preparations from hen egg white and human urine. Lysozyme was also purified from normal mouse lung, which is rich in alveolar macrophages and was found to be similar to lysozyme purified from the culture medium of Mm-1 cells in size and electrophoretic mobility and in its pH optimum, trypsin peptide map, and antigenicity. Thus the molecular structure of the lysozyme induced in differentiated mouse myeloid leukemia cells is similar to that of lysozyme produced by normal cells.     

Spin-label assay for lysozyme.

A spin-label assay for lysozyme, which is based on the enzymatic hydrolysis of spin-labeled peptidoglycan, is described. Hydrolysis of this polymer by lysozyme results in sharpening of the esr spectrum. The rate of spectral sharpening is a function of enzyme concentration. When the activities of hen egg-white and human lysozymes are compared by this method, human lysozyme is 3.5 times as active as the hen enzyme. The pH optima for both enzymes are pH 5.0. At this pH, the maximal activity for the hen egg-white lysozyme is observed at an ionic strength of 0.09. This assay is suitable for measuring lysozyme levels in biological fluids. It is a sensitive, continuous assay that measures muramidase activity on a defined substrate.