STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : II. THE RELATION BETWEEN THE SOLUBILITY OF CASEIN AND ITS CAPACITY TO COMBINE WITH BASE. THE SOLUBILITY OF CASEIN IN SYSTEMS CONTAINING THE PROTEIN AND SODIUM HYDROXIDE.

1. The solubility in water of purified, uncombined casein has previously been reported to be 0.11 gm. in 1 liter at 25°C. This solubility represents the sum of the concentrations of the casein molecule and of the soluble ions into which it dissociates. 2. The solubility of casein has now been studied in systems containing the protein and varying amounts of sodium hydroxide. It was found that casein forms a well defined soluble disodium compound, and that solubility was completely determined by (a) the solubility of the casein molecule, and (b) the concentration of the disodium casein compound. 3. In our experiments each mol of sodium hydroxide combined with approximately 2,100 gm. of casein. 4. The equivalent combining weight of casein for this base is just half the minimal molecular weight as calculated from the sulfur and phosphorus content, and one-sixth the minimal molecular weight calculated from the tryptophane content of casein. 5. From the study of systems containing the protein and very small amounts of sodium hydroxide it was possible to determine the solubility of the casein molecule, and also the degree to which it dissociated as a divalent acid and combined with base. 6. Solubility in such systems increased in direct proportion to the amount of sodium hydroxide they contained. 7. The concentration of the soluble casein compound varied inversely as the square of the hydrogen ion concentration, directly as the solubility of the casein molecule, Su, and as the constants Ka₁ and Ka₂ defining its acid dissociation. 8. The product of the solubility of the casein molecule and its acid dissociation constants yields the solubility product constant, Su·Ka₁·Ka₂ = 2.2 x 10^–12 gm. casein per liter at 25°C. 9. The solubility of the casein molecule has been estimated from this constant, and also from the relation between the solubility of the casein and the sodium hydroxide concentration, to be approximately 0.09 gm. per liter at 25°C. 10. The product of the acid dissociation constants, Ka₁ and Ka₂, must therefore be 24 x 10^–12N. 11. It is believed that these constants completely characterize the solubility of casein in systems containing the protein and small amounts of sodium hydroxide.     

Nematicidal Activity of α -Chaconine: Effect of Hydrogen-ion Concentration

α-Chaconine, a steroid-glycoalkaloid from Solanum tuberosum L., was increasingly more toxic to a free-living nematode, Panagrellus redivivus, with decreasing acidity from about pH 5 to 7. A study of the toxicity to adult nematodes at three concentrations of α-chaconine in buffer from pH 4 to 7.5 indicated that the free base is the nematicidal form of the compound. The median effective doses (ED₅₀) of α-chaconine to inhibit the motility of P. redivivus were estimated as 85 μg/ml at pH 6.7, 170 μg/ml at pH 6.5, and 340 μg/ml at pH 6.2.     

In vitro stability of nitrate reductase from wheat leaves: I. Stability of highly purified enzyme and its component activities

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH₂)-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes—methylviologen-nitrate reductase and NADH-ferricyanide reductase—also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH₂-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH₂-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t_½ [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH₂ do not donate electrons to the same site of the nitrate reductase protein.     

General Biochemical Characterization of Thermostable Pullulanase and Glucoamylase from Clostridium thermohydrosulfuricum

Cell extracts of Clostridium thermohydrosulfuricum, an anaerobic bacterium which ferments starch into ethanol at 65°C, contained both pullulanase and glucoamylase activities. The general physiochemical and catalytic properties of these enzyme activities were compared. Pullulanase and glucoamylase activities were stable and optimally active at 85 and 75°C, respectively. The pH optima for activity and pH stability ranges were, respectively, 5.5 to 6 and 4.5 to 5.5 for pullulanase and 4 to 6 and 5 to 6 for glucoamylase. The apparent [S]_0.5v and V_max for pullulanase activity on pullulan were 0.33 mg/ml and 2.6 U/mg of protein. The apparent [S]_0.5v and V_max for glucoamylase activity on starch were of 0.41 mg/ml and 0.31 U/mg of protein. These enzymes were active and stable in the presence of air or 10% (vol/vol) ethanol. These enzyme activities allowed the organism to actively degrade raw starch into glucose in the absence of significant α-amylase activity.     

Microbiological Survey of Adirondack Lakes with Various pH Values

Nine high-altitude oligotrophic Adirondack lakes in upstate New York having water of pH 4.3 to 7.0 were surveyed for total bacterial numbers and possible adaptation of the microbial communities to environmental pH. The number of heterotrophic bacteria from water samples recoverable on standard plate count agar were low (10¹ to 10³ per ml) for most of the lakes. Acridine orange direct counts were approximately two orders of magnitude higher than plate counts for each lake. Sediment aerobic heterotrophs recovered on standard plate count agar ranged from 1.4 × 10⁴ to 1.3 × 10⁶ per g of sediment. Direct epifluorescence counts of bacteria in sediment samples ranged from 3.0 × 10⁶ to 1.4 × 10⁷ per g. Low density values were consistent with the oligotrophic nature of all the lakes surveyed. There were no apparent differences in numbers of bacteria originally isolated at pH 5.0 and pH 7.0 between circumneutral lakes (pH > 6.0) and acidic lakes (pH < 5.0). Approximately 1,200 isolates were recultured over a range of pH from 3.0 to 7.0. Regardless of the original isolation pH (pH 5.0 or pH 7.0), less than 10% of the isolates grew at pH < 5.0. Those originally isolated at pH 5.0 also grew at pH 6.0 and 7.0. Those originally isolated at pH 7.0 preferred pH 7.0, with 98% able to grow at pH 6.0 and 44% able to grow at pH 5.0. A chi-square contingency test clearly showed (P < 0.005) that two distinct heterotrophic populations had been originally isolated at pH 5.0 and pH 7.0, although there is undoubtedly some overlap between the two populations.     

Usnic Acid Potassium Salt: An Alternative for the Control of Biomphalaria glabrata (Say, 1818)

In Brazil, the snail Biomphalaria glabrata is the most important vector of schistosomiasis due to its wide geographical distribution, high infection rate and efficient disease transmission. Among the methods of schistosomiasis control, the World Health Organization recommends the use of synthetic molluscicides, such as niclosamide. However, different substances of natural origin have been tested as alternatives for the control or eradication of mollusks. The literature describes the antitumor, antimicrobial and antiviral properties of usnic acid as well as other important activities of common interest between medicine and the environment. However, usnic acid has a low degree of water solubility, which can be a limiting factor for its use, especially in aquatic environments, since the organic solvents commonly used to solubilize this substance can have toxic effects on aquatic biota. Thus, the aim of the present study was to test the potassium salt of usnic acid (potassium usnate) with regard to molluscicidal activity and toxicity to brine shrimp (Artemia salina). To obtain potassium usnate, usnic acid was extracted with diethyl ether isolated and purified from the lichen Cladonia substellata. Biological assays were performed with embryos and adult snails of B. glabrata exposed for 24 h to the usnate solution solubilized in dechlorinated water at 2.5; 5 and 10 µg/ml for embryos, 0.5; 0.9; 1;5 and 10 µg/ml for mollusks and 0.5; 1; 5; 10 µg/ml for A. salina. The lowest lethal concentration for the embryos and adult snails was 10 and 1 µg/ml, respectively. No toxicity to A. salina was found. The results show that modified usnic acid has increased solubility (100%) without losing its biological activity and may be a viable alternative for the control of B. glabrata.     

Influence of the Natural Microbial Flora on the Acid Tolerance Response of Listeria monocytogenes in a Model System of Fresh Meat Decontamination Fluids

Depending on its composition and metabolic activity, the natural flora that may be established in a meat plant environment can affect the survival, growth, and acid tolerance response (ATR) of bacterial pathogens present in the same niche. To investigate this hypothesis, changes in populations and ATR of inoculated (10⁵ CFU/ml) Listeria monocytogenes were evaluated at 35°C in water (10 or 85°C) or acidic (2% lactic or acetic acid) washings of beef with or without prior filter sterilization. The model experiments were performed at 35°C rather than lower (≤15°C) temperatures to maximize the response of inoculated L. monocytogenes in the washings with or without competitive flora. Acid solution washings were free (<1.0 log CFU/ml) of natural flora before inoculation (day 0), and no microbial growth occurred during storage (35°C, 8 days). Inoculated L. monocytogenes died off (negative enrichment) in acid washings within 24 h. In nonacid (water) washings, the pathogen increased (approximately 1.0 to 2.0 log CFU/ml), irrespective of natural flora, which, when present, predominated (>8.0 log CFU/ml) by day 1. The pH of inoculated water washings decreased or increased depending on absence or presence of natural flora, respectively. These microbial and pH changes modulated the ATR of L. monocytogenes at 35°C. In filter-sterilized water washings, inoculated L. monocytogenes increased its ATR by at least 1.0 log CFU/ml from days 1 to 8, while in unfiltered water washings the pathogen was acid tolerant at day 1 (0.3 to 1.4 log CFU/ml reduction) and became acid sensitive (3.0 to >5.0 log CFU/ml reduction) at day 8. These results suggest that the predominant gram-negative flora of an aerobic fresh meat plant environment may sensitize bacterial pathogens to acid.     

THE SOLUBILITY OF TYROSINE IN ACID AND IN ALKALI

Measurements have been made of the solubility at 25°C. of tyrosine in hydrochloric acid and in sodium hydroxide solutions varying from 0.001 to 0.05 M, and also in distilled water. The pH of the saturated solutions was measured with the hydrogen electrode. The following values for the ionization constants of tyrosine have been obtained from the measurements: k_b = 1.57 x 10^–12, k_a1 = 7.8 x 10^–10, k_a2 = 8.5 x 10^–11. The changes in solubility with pH can be satisfactorily explained by the use of these ionization constants.     

Effects of High Pressure Homogenization on the Activity,Stability, Kinetics and Three-Dimensional Conformation of a Glucose Oxidase Produced by Aspergillus niger

High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5–6.0 and a remarkable activity increase (30–300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.     

Effects of pH and temperature on the wild-type and a mutant form of Neurospora glutamate dehydrogenase

1. We confirm the observation of Bürk (1965) that Neurospora crassa NADP-linked glutamate dehydrogenase normally exists in an inactive form below pH7·0 and in a fully active form above pH8·0 in either tris or orthophosphate buffer. At pH7·4 the enzyme is about half activated at 25°. 2. The variety of the enzyme produced by the mutant am^2l shows a similar behaviour except that the transition is shifted about one pH unit in the alkaline direction. 3. The am^2l enzyme has previously been reported to be activated by brief warming to 30° in phosphate buffer at pH8·0. The wild-type enzyme shows a similar effect at pH7·0. In tris buffer this effect is much less pronounced. 4. The am^2l enzyme is extremely unstable at 47° at pH7·0; its stability is somewhat greater at lower pH, and is markedly increased by increasing the pH in the range 7·0–8·7. The wild-type enzyme also shows an indication of a stability minimum at pH7·0, but a temperature of 60° is needed for a measurable rate of inactivation. 5. The inactive form of the enzyme is much more subject to thermal irreversible denaturation than is the active form.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : VI. THE ACTIVITY COEFFICIENTS OF THE IONS IN CERTAIN OXYHEMOGLOBIN SOLUTIONS.

1. The solvent action of a neutral salt upon a protein, oxyhemoglobin, has been found identical to the solvent action of a neutral salt upon a bi-bivalent or uni-quadrivalent compound. 2. The solubility of oxyhemoglobin in phosphate solutions of varying ionic strength has been defined by the equation: log See PDF for Equation in which µ is the ionic strength, and S ₀ is the solubility in the absence of salt. 3. The values of S ₀ have been calculated to be 12.2, 11.2, and 13.1 gm. per liter respectively at pH 6.4, 6.6, and 6.8. 4. The relatively great solubility of oxyhemoglobin in water has been ascribed to the strong affinity constants for acid and base of certain groups in oxyhemoglobin. 5. The small change in the solubility of oxyhemoglobin effected by neutral salts suggests that but few such groups are dissociated in oxyhemoglobin in the state in which it crystallizes near its isoelectric point. 6. Certain of the other properties of oxyhemoglobin, such as its low viscosity, are considered in the light of its molecular weight and its valence type.     

Liquid Chromatographic Determination of Dipicolinic Acid from Bacterial Spores

Dipicolinic acid was determined by reverse-phase liquid chromatography. Elution was with 0.2 M potassium phosphate, pH 1.8, containing 1.5% tert-amyl alcohol or higher concentrations of lower alcohols or acetonitrile. The normal analytical range was 50 to 1,000 μM, which is equivalent to 0.1 to 1 mg of spores per ml with a relative standard error of 2 to 4% and a detection limit of <100 pmol. Dipicolinic acid was fully extracted from spores by heating at pH 1.8 for 10 min at 100°C. Sporulating cultures may be analyzed in less than 20 min without separation of cells from media. Liquid chromatography was also used to detect dipicolinic acid in more complex substrates, e.g., guinea pig feces containing Metabacterium polyspora spores and canned food. Dipicolinic acid could be detected in unspoiled canned salmon containing <10⁶ added Bacillus cereus spores per g.     

Physicochemical Characterization of NPC 1161C,A Novel Antimalarial 8-Aminoquinoline,in Solution and Solid State

NPC 1161C is a novel antimalarial drug of interest because of its superior curative and prophylactic activity, and favorable toxicity profile against in vivo and in vitro models of malaria, pneumocystis carinii pneumonia, and leishmaniasis. The preformulation studies performed included determination of pK_as, aqueous and pH solubility, cosolvent solubility, log P, pH stability, thermal analysis, and preliminary hygroscopicity studies. The mean pK_a1, pK_a2, and pK_a3 were determined to be 10.12, 4.07, and 1.88, respectively. The aqueous solubility was found to be 2.4 × 10⁻⁴ M having a saturated solution pH of 4.3–5.0 and a low intrinsic solubility of 1.6 × 10⁻⁶ M. A mathematical model of the pH-solubility profile was derived from pH 2.2 to 8.0. An exponential decrease in solubility was observed with increasing pH. The excess solid phase in equilibrium with the solution in aqueous buffers was determined to be the free-base form of the drug. A significant increase in solubility was observed with all the cosolvents studied, in both unbuffered and buffered systems. Mean log P of the salt and the free base were estimated to be 2.18 and 3.70, respectively. The compound had poor stability at pH 7.0 at 37°C, with a t ₉₀ of 3.58 days. Thermal analysis of the drug using DSC and TGA revealed that the drug is present as a semi-crystalline powder, which transformed into the amorphous state after melting. The drug was also found to sublime at higher temperatures. Determination of physicochemical properties of NPC 1161C provided useful information for the development of a dosage form and preclinical evaluation.     

Studies on some aspects of peroxidase from submaxillary gland

A peroxidase has been purified 25- to 30-fold over crude homogenate from goat submaxillary gland, which shows a single band of protein on polyacrylamide gel electrophoresis at four different pH values (4.6–10.0). A molecular weight (M_r) of approximately 2 × 10⁴ per heme binding site has been found. The molecular weight of the enzyme determined by Sephadex-gel filtration method, appeared to be 4 × 10⁴. The sedimentation pattern of the purified enzyme shows a symmetrical peak, although there was evidence of some small heterogenous material near the meniscus. The sedimentation coefficient of the enzyme (s^o 20^wat 0.4% of the enzyme concentration) was found to be 4.18, which indicates the molecular weight of the enzyme to be approximately 6 × 10⁴.     

Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp.

Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic Bacillus sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C in pH 8 and 9 buffers.     

Leukocyte beta-glucosidase in homozygotes and heterozygotes for Gaucher disease

Human leukocytes contain at least two isozymes of 4-methylumbelliferyl-β-glucosidase acting optimally at pH 4.0 and 4.8; in Gaucher disease, only the former is deficient. Brief exposure of the leukocyte homogenate to pH 4.0 at room temperature results in irreversible inactivation of the pH 4.8 activity, while the activity at pH 4.0 remains unaffected. The more acidic isozyme is stimulated four- to fivefold by 0.2% sodium taurodeoxycholate (TDC) with a shift in the pH optimum to 5.0. The less acidic isozyme is completely suppressed in the presence of this detergent. Both leukocyte isozymes appear to be membrane-bound since gel filtration of Sephadex G-200 produces only one peak of activity located at the void volume, unlike in liver and kidney where a second peak also can be demonstrated. Heat inactivation analysis indicated that in controls, assayed in the absence of detergent, pH 4.0 activity is more thermostable than pH 4.8 activity. However, in Gaucher disease, the residual β-glucosidase at pH 4.0 is just as thermolabile as the unaffected pH 4.8 activity. Heat inactivation of the enzyme in the presence of TDC resulted in rapid loss of activity, suggesting a direct effect of the bile salt on the configuration of the enzyme decreasing its thermal stability. In the absence of detergent, acid β-glucosidase shows two K_m's, one at 3.2 mM and another at 0.9 mM. In the presence of detergent, only the higher K_m at 3.3 mM is obtained. In patients with Gaucher disease and in obligate carriers, the K_m remains essentially unaffected while the V_max shows the expected deficiency.

A reliable and reproducible selective assay technique has been developed for the diagnosis of Gaucher disease homozygotes and obligate heterozygotes and for the carrier screening of individuals at risk for this inherited disorder. The efficacy of this technique has been demonstrated by studying the activity in 42 controls, 26 patients, 32 obligate heterozygotes, and 23 healthy relatives of patients with Gaucher disease.

     

Adaptation of Escherichia coli O157:H7 to pH Alters Membrane Lipid Composition, Verotoxin Secretion, and Resistance to Simulated Gastric Fluid Acid

The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1ω7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10⁸ CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.     

Stability of 2'-deoxyxanthosine in DNA

The deamination of nucleobases in DNA occurs by a variety of mechanisms and results in the formation of hypoxanthine from adenine, uracil from cytosine, and xanthine and oxanine from guanine. 2′-Deoxyxanthosine (dX) has been assumed to be an unstable lesion in cells, yet no study has been performed under biological conditions. We now report that dX is a relatively stable lesion at pH 7, 37°C and 110 mM ionic strength, with a half-life (t_1/2) of 2.4 years in double-stranded DNA. The stability of dX as a 2′-deoxynucleoside (t_1/2 = 3.7 min at pH 2; 1104 h at pH 6) was increased substantially upon incorporation into a single-stranded oligodeoxynucleotide, in which the half-life of dX at different pH values was found to range from 7.7 h at pH 2 to 17 700 h at pH 7. Incorporation of dX into a double-stranded oligodeoxynucleotide resulted in a statistically insignificant increase in the half-life to 20 900 h at pH 7. Data for the pH dependence of the stability of dX in single-stranded DNA were used to determine the rate constants for the acid-catalyzed (2.6 × 10^–5 s^–1) and pH-independent (1.4 × 10^–8 s^–1) depurination reactions for dX as well as the dissociation constant for the N⁷ position of dX (6.1 × 10^–4 M). We conclude that dX is a relatively stable lesion that could play a role in deamination-induced mutagenesis.     

Affinity Immobilization of Escherichia coli: Catalysis by Intact and Permeable Cells Bound to Starch

The binding of viable Escherichia coli cells to an immobilized ligand of a surface receptor for maltodextrins has recently been demonstrated (T. Ferenci and K. S. Lee, J. Mol. Biol. 160:431-444, 1982). The interaction of bacteria and ligand immobilized in a chromatographic column was investigated over a wide range of applied cell densities, temperatures, eluant pH values, osmotic concentrations, and flow rates. Over 95% retention of bacteria applied to starch-Sepharose was found at cell densities up to 10⁹ per ml of matrix, between pH 5.5 and 8.0, between 8 and 55°C, in the presence of 0 to 0.5 M NaCl, and at elution flow rates up to 37 column volumes per h. The catalytic capability and stability of affinity-immobilized cells was demonstrated with the cytoplasmic β-galactosidase activity of starch-bound cells. Intact immobilized bacteria exhibited slowly increasing β-galactosidase activity over several days with a plateau after 6 days. Bacteria made permeable by treatment with toluene were also bound to starch-Sepharose but showed maximum β-galactosidase activity within 1 day and exhibited no loss of enzyme activity in 8 days of continuous elution at ambient temperatures.     

Cloning,expression of b-1,3-1,4 glucanase from Bacillus subtilis SU40 and the effect of calcium ion on the stability of recombinant enzyme: in vitro and in silico analysis

A new glucanolytic bacterial strain, SU40 was isolated, and identified as Bacillus subtilis on the basis of 16S rRNA sequence homology and phylogenetic tree analysis. The gene encoding β-1,3-1,4-glucanase was delineated, cloned into pET 28a+ vector and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme was about 24 kDa. The enzyme exhibited maximum activity (36.84 U/ml) at 60°C, pH 8.0 and maintained 54% activity at 80°C after incubation for 60 min. The enzyme showed activity against β-glucan, lichenan, and xylan. Amino acid sequence shared a conserved motif EIDIEF. The predicted three-dimensional homology model of the enzyme showed the presence of catalytic residues Glu105, Glu109 and Asp107, single disulphide bridge between Cys32 and Cys61 and three calcium binding site residues Pro9, Gly45 and Asp207. Presence of calcium ion improves the thermal stability of SU40 β-1,3-1,4-glucanase. Molecular dynamics simulation studies revealed that the absence of calcium ion fluctuate the active site residues which are responsible for thermostability. The high catalytic activity and its stability to temperature, pH and metal ions indicated that the enzyme β-1,3-1,4-glucanase by B. subtilis SU40 is a good candidate for biotechnological applications.