Lytic Enzyme from Streptomyces globisporus 1829 Strain

The maximum yield of lytic enzyme was obtained from shake flask cultures of Streptomyces globisporus 1829 which were grown at 30°DC for 48 hr in a medium containing 2% dextrin, 0.5% soybean meal, 0.2% polypeptone, 0.5% Na₂HP0₄. 12H₂0, 0.1% KH₂P0₄, 0.1% MgS0₄·7H₂0, 1.0% NaCI and 0.02% CaCl₂, pH 7.5. The activity of successively transferred substrains of St. globisporus 1829 gradually decreased. However, a mutant strain obtained by ultra-violet irradiation has been shown not to have lost any lytic activity for 2 years. The enzyme exhibited maximum activity at 60°C in the pH range of 6 to 6.5 and was lytic against the intact cells of Streptococci, Lactobacilli and Bacilli but inert against the intact cells of Staphylococcus aureus and Micrococcus lysodeikticus.     

Serratia marcescens-Lytic Enzyme Produced by Micromonospora sp. Strain No. 152

A strain of Micromonospora sp. producing a lytic enzyme toward Serratia marcescens was isolated from soil. The lytic enzyme, called 152-enzyme, was purified from the culture filtrate by salting-out with ammonium sulfate, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-75. The molecular weight of 152-enzyme was 17,000 and the isoelectric point was pH 7.3. The 152-enzyme showed lytic activity toward S. marcescens, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Bacillus subtilis, but was completely intert toward Staphylococcus aureus. The enzyme also showed caseinolytic activity. The lytic and caseinolytic activities of 152-enzyme were maximum around pH 11.0 and at 60°C. Both activities were inhibited by DFP and API-2c. Liberation of amino groups from cell walls of P. aeruginosa by incubation with 152-enzyme suggested that the enzyme was a kind of cell wall-lytic peptidase.     

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH₄)₂SO₄ and soybean flour as nitrogen sources and KH₂PO₄ at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.     

Recovery of soil streptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> pathogens

A total of 161 different Streptomyces isolates were recovered from 5 soil samples representing the driest habitats of Jordan. These were then characterized and assessed for their antagonistic activity against four clinical multi-drug resistant Pseudomonas aeruginosa test pathogens. Results indicated that only 3 strains out of 139 and 6 out of 22 isolated at 27°C and 45°C, respectively, were active against at least three strains of pathogenic Pseudomonas. However, three Streptomyces strains (J_2b, J₄, and J₁₂) that were isolated at 45°C inhibited all of the tested pathogens with an inhibition zone ranging between 5 and 16 mm in diameter. Data obtained from comparing the inhibition activity of these unique Streptomyces strains toward multi-resistant Pseudomonas pathogens with standard used antibiotics revealed that these isolates produce possible different inhibitory bioactive compounds other than the standard antibiotics.     

Purification and Properties of Mycodextranase from Streptomyces sp. J-13-3

An enzyme hydrolyzing nigeran (alternating α-l,3-and α-l,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-I00. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS–PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50°C. The enzyme activity was inhibited significantly by Hg⁺, Hg²⁺, and p-chloromercuribenzoic acid. The K_m (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the α-l,4-glucosidic linkages in nigeran.     

Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2

Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO₄, 1.4% K₂HPO₄, 0.02% CaCl₂·2H₂O, 0.002% FeSO₄·7H₂O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F₂ fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70°C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90°C, the t_1/2 values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90°C, the enzyme retained its activity up to 8 h in the presence of 1mM MnCl₂. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The K _{m app} values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the V_max values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F₂ chitosanase at 24 h incubation (70°C) were pentasaccharide (GlcN)₅ and hexasaccharide (GlcN)₆. The prelimiaary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium. Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.     

Microbial Production of L-Tyrosine by an n-Paraffin-grown Bacterium

Four mannanases (Mannanases I, II, III, and IV) were isolated from the culture filtrate of a Streptomyces sp. by ion exchange chromatography. Mannanase IV was the main component and accounted for 64.4% of the total activity of the four mannanases. Mannanase IV was further purified by gel filtration, and the purified Mannanase IV was homogeneous on disc-gel electrophoretic analysis.

Optimum pH and temperature for the activity of Mannanase IV were 6.8 and 57°C, respectively. It was stable at temperatures up to 45°C when examined at pH 6.8 for 30min, and lost only 15% of its activity at 70°C for 30min at pH 6.8. The isoelectric point and molecular weight were pH 3.65 and 42,900, respectively. The enzyme was strongly inactivated by Al³⁺, Hg²⁺, Fe²⁺, Fe³⁺, Cd²⁺, Ag⁺, Sn²⁺, and Cu²⁺, and completely inhibited by iodoacetic acid and N-bromosuccinimide. The enzyme hydrolyzed mannotriose to mannose and mannobiose, but did not hydrolyze mannobiose.     

Lysis of Pediococcus and Brevibacterium Cells by Cell-Wall Lytic Enzymes,L3, L11, ALE and Lysozyme

Several species belonging to the genera Pediococcus and Brevibacterium, which have resistant cell walls against the usual cell-disrupting methods, were effectively attacked by new cell-wall lytic enzymes, L₃, L₁₁ and ALE which were obtained from a Streptomyces sp., a Flavobacterium sp., and a Staphylococcus sp., respectively. Among them, the L₃ enzyme was mostly effective to all pediococcal and brevibacterial strains.     

Production of L-leucine aminopeptidase by selected Streptomyces isolates

Aims: To screen various Streptomyces cultures producing l ‐leucine aminopeptidase (LAP). Methods and Results: Twenty‐one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B‐3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8·0–8·5 and the temperature optima varied between 50 and 65°C. LAP of S. mobaraensis was stable at 60°C and pH 8·5 for 60 min. Metal ion salts, CoCl₂.6H₂O and ZnSO₄.7H₂O in 0·7 mmol l^?1 concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. Conclusions: Streptomyces mobaraensis NRRL B‐3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. Significance and Impact of the Study: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N‐terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Optimization of protease production by Streptomyces sp. A6 using statistical approach for reclamation of shellfish waste

Protease producing Streptomyces sp. A6 was isolated from intertidal zone of the coast of Diu (Gujarat, India). Plackett–Burman method was applied to identify important factors (shrimp waste, FeCl₃, ZnSO₄ and pH) influencing protease production by Streptomyces sp. A6. Further optimization was done by response surface methodology using central composite design. The concentrations of medium components for higher protease production as optimized using the above approach were (g l^?1): Shrimp waste, 14; FeCl₃, 0.035; ZnSO₄, 0.065 and pH, 8.0. This statistical optimization approach led to production of 129.02 ± 2.03 U ml^?1 of protease which was 4.96 fold higher compared to that obtained using the unoptimized medium. The protease production was scaled to 3 l in a 5-l bench fermenter using optimized medium which further increased the production by 63.4%. Deproteinization and chitin recovery obtained at the end of fermentation was 85.12 ± 4.7 and 70.58 ± 1.33%, respectively. The present study is the first report on statistical optimization of medium components for production of protease by Streptomyces species using cheaper raw material such as shrimp waste. The study also explored the possibility Streptomyces sp. A6 for reclamation of shrimp wastes.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Characterization of Polyphosphate Glucokinase SCO5059 from Streptomyces coelicolor A3(2)

SCO5059, encoded in Streptomyces coelicolor A3(2), was identified as a polyphosphate glucokinase. The K _m values of SCO5059 for glucose and polyphosphate (poly(P)₆) were estimated to be 12 and 4 µM, respectively, and the k _cat value was 0.3 s^?1 at pH 7.7 at 28 °C. SCO5059 homologs are highly conserved among Streptomyces, and can work as polyphosphate glucokinase as well.     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.     

Development of urea biosensor using non-covalent complexes of urease with aldehyde derivative of PEG and analysis on serum samples

Abstract

Non-covalent complexes of urease/polyethylene glycol (PEG)-aldehyde were synthesized using regular molar ratios of urease and PEG-aldehyde at room temperature. The physical properties of the non-covalent complexes were analyzed in order to investigate the impact of coupling ratio, temperature, pH, storage stability, and thermal stability. Urease activity was analyzed by UV–Vis spectrophotometer at 630?nm. The results showed that the strongest thermal resistance was obtained using n_U/n_PEG:1/1 (mg/mL) complex within all molar ratios tested. The enzymatic activity of n_U/n_PEG:1/1 complex doubled the activity of the free enzyme. Therefore, this complex was chosen to be used in the analyses. When coupled with PEG-aldehyde, urease exhibited improved activity between pH 4.0–9.0 and the optimum pH was found to be 7.0. The thermal inactivation results of the complex demonstrated that higher activity remained (40%) when compared with the free enzyme (10%) at 60?°C. The storage stability of the non-covalent complex was 4 weeks which was greater than the storage stability of the free enzyme. A kinetic model was suggested in order to reveal the mechanism of enzymatic conversion. Potentiometric urea biosensor was prepared using two different membranes: carboxylated poly vinyl chloride (PVC) and palmitic acid containing PVC. The potentiometric responses of both sensors were tested against pH and temperature and the best results were obtained at pH 7.0 and 20–30?°C. Also, selectivity of the suggested biosensors toward Na⁺, Li⁺ Ca²⁺, and K⁺ ions was evaluated and the reproducibility responses of the urea biosensors were measured with acceptable results.     

Characterization of the staphylococcal bacteriophage lysin CHAP(K)

Aims: To develop an efficient purification strategy for the bacteriophage lysin CHAP_K. To evaluate its antibacterial spectrum_, enzymatic properties, optimal reaction conditions and lytic activity against live Staphlyococcus aureus. Methods and Results: Recombinant CHAP_K was purified to homogeneity by cation exchange chromatography, with yields of up to 10 mg from 1 l of Escherichia coli culture. The lytic spectrum of CHAP_K includes all staphylococcal species and also members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Carnobacterium. The enzyme was active from pH 6 to 11 with an optimum activity at pH 9, from 5 to 40°C, with an optimum activity at 15°C. When cell lysis by CHAP_K and lysostaphin was compared over a concentration range of 2·5–10 μg ml^?1 using live Staph. aureus for 5 min at 37°C, CHAP_K gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin. Conclusions: This study describes in detail the purification and characteristics of the novel phage‐derived enzyme CHAP_K demonstrating that it has excellent biochemical properties as an anti‐staphylococcal agent. Significance and Impact of the Study: Currently, there is a need for new antimicrobial agents due to the increasing worldwide prevalence of antibiotic resistance. Our findings demonstrate the potential for development of CHAP_K as an alternative therapeutic against pathogenic staphylococci including MRSA.     

Purification and Properties of Phage HM 7-Induced Lytic Enzyme of Clostridium saccharoperbutylacetonicum

A lytic enzyme was isolated from phage HM 7-induced lysate of Clostridium saccharoperbutylacetonicum, and purified about 200-fold by precipitation with ammonium sulfate, gel filtration with Sephadex G–75 and ampholine isoelectric focusing. The purified lytic enzyme had an apparent homogeneity on disc-electrophoresis, and the character of acidic protein showing isoelectric point at pH 4.0. The molecular weight of lytic enzyme was estimated to be about 100,000 from the result of SDS-polyacrylamide gel electrophoresis. The optimum pH for the lytic enzyme activity was 6.5. Maximum activity occurred at 30 to 35°C, and at the ionic strength of 0.04 m or above. The lytic enzyme activity was stimulated about 140% by 10?³ m EDTA. The lytic enzyme lysed the living cells, but it had a narrow specificity which was restricted to a certain species of Clostridium such as Cl. saccharoperbutylacetonicum, Cl. butyricum, Cl. botulinum, Cl. sporogenes, and Cl. thiaminolyticum.     

Production,purification, and characterization of acetyl esterase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. PC22 and its action in cooperation with xylanolytic enzymes on xylan degradation

Acetyl esterase was produced by Streptomyces sp. PC22 at comparable levels of about 0.3 U ml⁻¹ using either 1.0% (w/v) birchwood xylan or 1.5% (w/v) corn husks as a carbon source and cultivating at 45 °C, at pH 9 for 3 or 2 days, respectively. The enzyme was purified from culture filtrate to about 54-fold purity by ammonium sulfate precipitation, followed by consecutive chromatography using a Macro-Prep DEAE, t-butyl hydrophobic interaction and hydroxyapatite, respectively. The approximate molecular weight of the purified enzyme was 155 kDa as analyzed by gel filtration, and it contained four identical 34 kDa subunits, as assessed by SDS-PAGE. It had K _m and V _max values for p-nitrophenyl acetate of 0.43 mM and 70.78 U mg⁻¹ and 7.8 mM and 1,027 U mg⁻¹ for α-naphthyl acetate, respectively. Its optimal pH and temperature were 6.5–7.0 and 50 °C, respectively. It was stable for 30 min at a broad range of pH values, from 5.0 to 9.0, and at temperatures up to 60 °C. The purified enzyme had no other xylanolytic activities. It showed cooperative action on birchwood xylan degradation, when used in combination with xylanase from the same strain and β-xylosidase from Streptomyces sp. CH7. Enhancement was 1.4-fold, compared to the expected amount of individual enzymes alone. This indicates that the enzyme has potential industrial applications, especially for utilizing renewable hemicelluloses containing acetyl xylan for the production of biofuels or other fermentation products.     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

Biolytic extraction of poly(3-hydroxybutyrate) from Bacillus megaterium Ti3 using the lytic enzyme of Streptomyces albus Tia1

The applicability of Streptomyces sp. cell lytic enzymes for devising a simple and competent biological polyhydroxyalkanoate (PHA) recovery approach from Bacillus megaterium cells was investigated. B. megaterium strain Ti3 produced 50% (w/w) PHA using glucose as carbon source. The intracellular PHA was recovered employing a non-PHA accumulating actinomycetes (Tia1) identified as Streptomyces albus, having potent lytic activity against living and heat inactivated B. megaterium. Interestingly, maximum biomass (2.53?±?0.6?g/L by 24?h) of the lytic actinomycete was obtained in PHA production medium itself thus circumventing the prior actinomycete acclimatization just by co-inoculation with B. megaterium as an inducer. Maximum lytic activity was observed at pH 6.0, 40?°C, 220?mg of biomass and 33.3?mL of concentrated culture filtrate in a 100?mL reaction mixture. Preliminary biochemical investigations confirmed the proteolytic and caseinolytic nature of the lytic enzyme. PHA yield of 0.55?g/g by co-inoculation extraction approach was comparable with the conventional sodium hypochlorite based extraction method. Interestingly, S. albus also demonstrated a broad spectrum lytic potential against varied Gram-negative and Gram-positive PHA producers highlighting the extensive applicability of this biolytic PHA recovery approach. The lytic enzyme retained almost 100% relative activity on storage at ?20?°C upto two months. ¹H Nuclear magnetic resonance analysis of the extracted polymer confirmed it as a homopolymer composed of 3-hydroxybutyrate monomeric units. This is the first report on Streptomyces sp. based biological and eco-friendly, intracellular PHA recovery from Bacillus spp.     

Characterization,cloning, sequencing,and expression of an aminopeptidase N from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TH-4

The aminopeptidase N (TH-4AP) of Streptomyces sp. TH-4 was purified from a culture supernatant. The purified enzyme had a molecular mass of 95 kDa. The gene encoding TH-4AP was cloned and sequenced. The primary structure of the protein possessed the PepN-conserved motif GxMEN and the zinc-binding motif HExxHx₁₈E, and showed 88% identity with that of PepN from Streptomyces lividans strain 66. We succeeded in overproducing a His-tagged recombinant enzyme using Escherichia coli. The enzyme had a 1.5-fold higher activity in the presence of cobalt ions than in their absence. To evaluate the possible application of TH-4AP to decrease the content of bitter peptides, we investigated the ability of Streptomyces aminopeptidases to hydrolyze synthetic peptides by a coupling method using l-amino acid oxidase and peroxidase. The substrate specificity of TH-4AP toward synthetic peptides was significantly different from that toward aminoacyl-p-nitroanilide derivatives.