Physiological adsorption of Sulfolobus acidocaldarius on coal surfaces

Summary The adsorption of Sulfolobus acidocaldarius on bituminous coal surfaces and the respiration rate during adsorption at 70° C were enhanced at pH 1.0–2.0, in comparison with those at pH 3.0–5.0. The maximum number of bacterial cells adsorbed per unit area of coal attained a maximum (1.4 × 10¹¹ cells/m²) at pH 2.0. The rate of desulphurization at pH 2.2–2.5 was higher than at other pHs tested. Micrographs of S. acidocaldarius obtained by TEM and SEM indicated that the cells were adsorbed to the coal surfaces by extracellular slime. Specific inhibitors of membrane-bound ATPase (NaF, 20 mm) and respiration (NaN₃, 1 mm; KCN, 1 mm) had pronounced effects on suppressing adsorption. The amount of S. acidocaldarius adsorbed decreased when the coal particles were leached in advance with 2.0 m HNO₃. These facts lead to the conclusion that the adsorption of S. acidocaldarius on coal surfaces requires physiological activity relatd to respiration or energy conversion. Offprint requests to: V. B. Vitaya     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.     

Evidence for the presence of membrane-bound forms of acid protease in Aspergillus oryzae.

While approximately 85% of the cell-bound acid protease of $\text{Aspergillus oryzae}$ were recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be present tightly associated with the membranes. Two forms of membrane-bound enzyme, which were solubilized with Triton X-100, were similar to the external acid protease found in culture medium in that they had an optimum pH at 3.2, activated trypsinogen at pH 3 and lost their activity upon treatment with 5.1 mM sodium dodecylsulfonate. However, they differed in their hydrophobic properties (i.e. aggregation in the absence of Triton X-100 and activation by the detergent) from both the cell-bound, soluble form and the one excreted into culture medium.     

Purification and characterization of highly thermostable α-amylase from thermophilic <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis>

In this study, the production of extracellular thermostable α-amylase by newly isolated thermophilic Alicyclobacillus acidocaldarius was detected on LB agar plates containing 1.0% soluble potato starch and incubated at 60°C. This extracellular α-amylase was purified to homogeneity by ammonium sulphate precipitation followed by Sephadex and ion-exchange chromatography. The α-amylase was purified to 8.138 fold homogeneity with a final recovery of 58% and a specific activity of 3,239 U/mg proteins. The purified α-amylase appeared as a single protein band on SDS-PAGE with a molecular mass of 94.5 kDa. Non-denaturing PAGE analysis showed one major band associated with enzyme activity, indicating the absence of isoenzymes. A TLC analysis showed maltose as major end product of the enzyme. The optimum assay temperature and pH for enzyme activity were 60°C and 6.0 respectively; however, the enzyme activity was stable over a wide range of pH and temperatures. The α-amylase retained its activity in the presence of the denaturing agents — SDS, Triton X-100, Tween-20, Tween-80, and was significantly inhibited by EDTA and urea. Calcium ions increased the enzyme activity, while Hg²⁺, Zn²⁺, and Co²⁺ had inhibitory effects. The K _m and V _max values were found to be 2.9 mg/mL and 7936 U/mL respectively.     

Partial Purification and Characterization of Thermostable Acid Phosphatase from Thermoacidophilic Archaeon Sulfolobus acidocaldarius

Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70°C, respectively. The apparent K _m value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75°C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein. Received: 8 July 1999 / Accepted: 9 August 1999     

Heterologous expression of a gene encoding a thermostable β-galactosidase from <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis>

A genomic DNA library screen yielded the nucleotide sequence of a 12 kb fragment containing a gene (2067 bp) coding a thermostable β-galactosidase from Alicyclobacillus acidocaldarius ATCC 27009. The β-galactosidase gene was expressed in Pichia pastoris, and up to 90 mg recombinant β-galactosidase/l accumulated in shake flask cultures. Using o-nitrophenyl-β-d-galactopyranoside as a substrate, the optimum pH and temperature of the purified recombinant β-galactosidase were 5.8–6.0 and 70°C, respectively. The enzyme retained 90% of its activity when heated at 70°C for 30 min. Approximately 48% of lactose in milk was hydrolyzed following treatment with the recombinant enzyme over 60 min at 65°C.     

Factors Affecting the Activity and Stability of Alkaline Phosphatase in a Marine Pseudomonad

Conditions optimum for the assay of alkaline phosphatase of marine pseudomonad B-16 (ATCC 19855) and for maintaining the activity of the enzyme have been determined. The pH for optimal activity of the cell-bound enzyme was 9.0, whereas that for the enzyme after its release from the cells exceeded 9.4. Release was effected by first washing the cells in 0.5 M NaCl and then suspending them in 0.5 M sucrose. In the absence of salts, the activity of the cell-bound enzyme decreased rapidly at 25 C and less rapidly at 4 C. This loss of activity could be arrested but not restored by adding Mg(2+). In the presence of Na(+), activity of the cell-bound enzyme dropped to about 50% of that prevailing initially, but in this case adding Mg(2+) restored enzyme activity completely. The activity of the enzyme after its release from the cells into 0.5 M sucrose was approximately 50% of that of the equivalent amount of enzyme in the original cells. This activity was relatively stable at both 25 and 4 C. Adding Mg(2+) to the released enzyme restored its activity to that of the cell-bound form. The synthesis of alkaline phosphatase by the cells was not affected by adding 50 mM inorganic phosphate to the growth medium. The K(m) of the released enzyme for p-nitrophenyl phosphate was found to be 6.1 x 10(-5) M.     

Effect of lipids and detergents on the production and solubilization of extracellular lipase inYarrowia lipolytica

Triglycerides, oleic acid but not fatty acid-containing, nonionic detergents (Spans, Tweens) were able to stimulate the synthesis of cell-bound and soluble lipase ofYarrowia lipolytica grown in a complex medium containing citrate and urea. The optimal concentration of olive oil for induction was 0.5% (W/V). The combined effect of a high ionic strength (0.75 mol/L KCl) and of digitonin (2 mmol/L) at pH 7.6 resulted in solubilization of 80% of the cell-bound lipase and a significant activation of the enzyme. Comparison of twoY. lipolytica strains showed the effects of Mg²⁺ and Fe³⁺ concentrations in the medium on the synthesis of the enzyme to be strongly strain-dependent.     

Purification and Properties of Acid β-d-Galactosidase from Corticium rolfsii

An acid β-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SP-Sephadex C-50 chromatography. The maximum activity of the enzyme towards p-nitrophenyl β-D-galactopyranoside was found to be at pH 2.0 to 2.5 and the enzyme was fairly active at pH 1.5 to l.8. The enzyme was quite stable over a pH range 2.0 to 8.0 at 2°C for 72 hr. The enzymic activity was clearly inhibited by Hg²⁺. Km value was determined to be 3.84 × 10^?4 m, and V_max was calculated to be 6.9 μ moles per min per mg for p-nitrophenyl β-d-galactopyranoside. Contrary to high activity on the synthetic galactoside, reaction velocity was small when the enzyme acted on lactose.     

1 '-(6-Hydroxyoctanoyl) nornicotine and 1 '-(7-Hydroxyoctanoyl) nornico-tine,Two New Alkaloids from Japanese Domestic Tobacco

A Streptomyces-pepsin inhibitor (S-PI or pepstatin Ac)-insensitive carboxyl proteinase was found in a still culture filtrate of Ganoderma lucidum (Mannen-take). The new carboxyl proteinase was purified, and about 15 mg of the purified enzyme was obtained from 15 liters of culture filtrate, with 13% recovery. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis.

The enzyme was most active at pH 3.2 toward hemoglobin, and at pH 2.0 toward casein, and stable only in the narrow pH range of 3.5 to 5.2 even under mild treatment (37°C for 3hr). The molecular weight and isoelectric point were 36,000 and pH 5.3, respectively. The enzyme did not contain methionine.

The enzyme was characterized by the following points: (1) the proteolytic activity was not inhibited by carboxyl proteinase inhibitors such as S-PI, DAN, and EPNP; (2) the enzyme was very unstable; (3) the caseinolytic activity was very low compared with the hydrolysis of hemoglobin (about 15%); (4) the enzyme split preferentially the Phe(24)–Phe(25) bond of oxidized insulin B-chain at the rate of 50% for total hydrolysis. These characteristics were compared with the carboxyl proteinases isolated from Scytalidium lignicolum and Lentinus edodes, which were reported to be SPI- and DAN-insensitive carboxyl proteinases.     

Characterization and partial purification of squalene-hopene cyclase from Bacillus acidocaldarius

A membrane-bound enzyme activity from Bacillus acidocaldarius converted squalene to two pentacyclic triterpenes, hop-22(29)-ene and hopan-22-ol. The products were formed in a constant molar ratio of hopene:hopanol, 5:1, probably through parallel, and not successive, reactions. The conversion was independent of oxygen, in contrast to the biosynthesis of sterols from epoxysqualene in eukaryotes. The squalene-hopene cyclase was pufified 270-fold by extraction from B. acidocaldarius membranes at low concentrations of Triton X-100 followed by DEAE-cellulose chromatography. The enzyme showed optimal rates of squalene conversion at pH 6 and 60°C, corresponding to the intracellular pH and the optimal growth temperature of the bacterium. The apparent K_m for squalene is 3 μM. Effective inhibitors of the enzyme were some sulfhydryl reagents and the histidyl reagent diethyl pyrocarbonate. The squalene-hopene cyclase, like several eukaryotic epoxysqualene cyclases, was strongly inhibited by AMO 1618 and by high ionic strength. On the basis of these and other similarities a phylogenitic relationship between the dey enzyme of steroid and hopanoid biosynthesis was envisaged.     

Taxonomic Characteristics of a Thermophilic Acidophilic Strain of Bacillus Producing Thermophilic Acidophilic Amylase and Thermostable Xylanase

Taxonomic characteristics of a strain of thermophilic acidophilic bacillus, Bacillus sp. 11-1S, which had the ability to produce thermophilic acidophilic amylase and thermostable xylanase were examined. Cells of the organism were aerobic, heterotrophic, Gram-positive, spore-forming rods. It grew at temperatures between 45 and 70°C (optimum 65°C) in media of pHs ranging from 2.0 to 5.0 (optimum 3.5 ~ 4.0). Physiological and biochemical characteristics were identical with those of Bacillus acidocaldarius, and % GC of DNA (59%) was close to that of the latter (61 ~ 62%). From these results it was concluded that the organism belongs to B. acidocaldarius Darland and Brock.     

Effects of various inhibitors on β-galactosidase purified from the thermoacidophilic <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis> subsp. <Emphasis Type="Italic">Rittmannii</Emphasis> isolated from Antarctica

β-Galactosidase purified from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica is a member of the GH42 family. The enzyme was not effected by various concentrations of its reaction product glucose, but was greatly inhibited by the other reaction product galactose using both substrates, ONPG and lactose. Linewever-Burk plot analysis derived from both ONPG and lactose hydrolysis results showed that galactose is a mixed-type inhibitor of the purified β-galactosidase. The enzyme was slightly activated by Mg²⁺ (13% at 20 mM), while inhibited at higher concentrations of Ca⁺² (33% at 10 mM), Zn⁺² (86% at 8 mM) and Cu⁺² (87% at 4 mM). The enzyme activity was not significantly altered by the metal ion chelators EDTA and 1,10-phenanthroline up to 20 mM, indicating that this enzyme is not a metalloenzyme. 2-Mercaptoethanol and DTT were found to enhance β-galactosidase activity, while p-chloromercuribenzoic acid (PCMB) completely inhibited enzymatic activity (97% at 1 mM; 99.7% at 2 mM), indicating at least one essential Cys residue modified by the reagents in the active site of β-galactosidase. Iodoacetamide and Nethylmaleimide had little effect on the β-galactosidase. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme strongly (19.8% at 1 mM; 71.9% at 10 mM), also showing the participation of serine for enzyme activity.     

Continuous production of fructose-syrups from inulin by immobilized inulinase from recombinantSaccharomyces cerevisiae

Recombinant exoinulinase was partially purified from the culture supernatant ofS. cerevisiae by (NH₄)₂SO₄ precipitation and PEG treatment. The purified inulinase was immobilized onto Amino-cellulofine with glutaraldehyde as a cross-linking agent. Immobilization yield based on the enzyme activity was about 15%. Optimal pH and temperature of immobilized enzyme were found to be 5.0 and 60°C, respectively. The enzyme activity was stably maintained in the pH ranges of 4.5 to 6.0 at 60°C. 100% of enzyme activity was observed even after incubation for 24 hr at 60°C. In the operation of a packed-bed reactor containing 412 U inulinase, dahalia inulin of 7.5%(w/v) concentration was completely hydrolyzed at flow rate of 2.0 mL/min at 60°C, resulting in a volumetric productivity of 693 g-reducing sugars/L/h. Under the reaction conditions of 1.0 mL/min flow rate with 2.5% inulin at 60°C, the reactor was successfully operated over 30 days without loss of inulinase activity.     

Penicillinase (β-lactamase) formation by blue-green algae

-Lactamase (penicillinase) activity was found in a number of strains of blue-green algae. In some cases, this enzyme permitted algae to overcome the inhibitory effects of penicillin. Production and localization of -lactamase were studied in a unicellular species, Coccochloris elabens (strain 7003), and in a filamentous, nitrogen-fixing Anabaena species (strain 7120). When cells were grown in a neutral medium with NaNO₃ as N source, the pH rose during growth; at a pH of about 10, most of the enzyme was extracellular and all the cell-bound enzyme was expressed equally well in intact or disrupted cells. If the pH was kept near neutrality during growth by gassing with CO₂ in N₂ or by growth under conditions of N₂ fixation, the enzyme remained cell-bound and cryptic for most of the growth phase, being measurable only after cells were disrupted. The enzymes from strains 7003 and 7120 had greater activity on benzyl penicillin and other penicillins than on cephalo-sporins. Some differences were observed in the substrate profiles of penicillinases from the two strains against different penicillins.A preliminary account of this work was presented at the 1974 meetings of the American Society for Microbiology in Chicago (Abstracts of Meetings, M37)     

Purification of lipase from Geotrichum candidum: conditions optimized for enzyme production using Box–Behnken design

The fungus Geotrichum candidum was selected from isolates of oil-mill waste as a potent lipase producer. Factors affecting lipase production by the fungus G. candidum in yeast-extract-peptone medium have been optimized by using a Box–Behnken design with seven variables to identify the significant correlation between effects of these variables in the production of the enzyme lipase. The experimental values were found to be in accordance with the predicted values, the correlation coefficient is 0.9957. It was observed that the variables days (6), pH (7.0), temperature (30 °C), carbon (1.25%), nitrogen (2.0%), Tween (1.0%) and salt concentrations (0.5 mM) were the optimum conditions for maximum lipase production (87.7 LU/ml). The enzyme was purified to homogeneity with an apparent molecular mass of 32 kDa by SDS-PAGE. The optimum pH at 40 °C was 7.0 and the optimum temperature at pH 7.0 was 40 °C. The enzyme was stable within a pH range of 6.5 to 8.5 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, and EDTA. However, the presence of Ca²⁺ and Ba²⁺ ions enhanced the activity of the enzyme.     

Simultaneous production of glucose oxidase and catalase by Alternaria alternata

Summary A number of factors affecting simultaneous production of cell-bound glucose oxidase and catalase by the fungus Alternaria alternata have been investigated. Consecutive optimization of the type and concentration of nitrogen and carbon source, the initial pH and growth temperature resulted in a simultaneous increase in glucose oxidase and catalase by 780% and 68% respectively. Two second-order equations, describing the combined effect of pH and temperature on the activity of each enzyme, revealed that glucose oxidase had its optima at pH 7.9 and 32.3°C and catalase at pH 8.5 and 18.1°C. Under certain growth conditions, yields as high as 23.5 and 18,100 units/g carbon source for glucose oxidase and catalase, respectively, were simultaneously obtained.Offprint requests to: B. J. Macris     

Surface-Bound Nuclease of Staphylococcus aureus: Localization of the Enzyme

The cellular localization of staphylococcus nuclease, previously known as an exoenzyme, was investigated, and the following results were obtained. (i) When Staphylococcus aureus cells were converted to protoplasts by cell wall lytic enzyme L-11 (a bacteriolytic enzyme purified from Flavobacterium sp. which specifically hydrolyzes amide and peptide linkages of murein layers), over 80% of the cell-bound nuclease was released into the surrounding sucrose medium. (ii) The cell-bound nuclease was associated with the cell-wall membrane fraction of mechanically disrupted cells. (iii) The nuclease activity of cell-wall membrane fractions from cells during early and late stages of protoplast formation were compared. Less activity was found in the late stage. These results suggest that nuclease may be located at or near the surface of the cells. The distribution of cell-bound nuclease in the cell-wall membrane fraction varied with the growth conditions of S. aureus. The activity of alkaline phosphatase, another surface enzyme, was also investigated. Less of this enzyme than nuclease was released when the cells were converted to protoplasts.     

Purification and properties of an extracellular β-glucosidase from the cellulolytic thermophile Clostridium stercorarium

Summary Clostridium stercorarium cultures grown on cellobiose contain both an extracellular and a cell-bound -glucosidase activity. A substantial portion of the cell-bound enzyme could be extracted by osmotic shock, suggesting a periplasmic localization. The -glucosidase present in culture supernatants was purified to homogeneity. It was found to be identical in all aspects tested with the cell-bound -glucosidase. The enzyme exists as a monomer with an apparent molecular weight of 85.000 (SDS-PAGE) and a pI of 4.8. It shows optimal activity as pH 5.5 and 65° C. Thiol groups are essential for enzyme activity. In the presence of reducing agents and divalent cations the half-life of the purified enzyme was more than 5 h at 60°C. The enzyme hydrolyses at different rates a wide range of substrates including aryl--glucosides, cellobiose, and disordered cellulose. K _m values were determined as 0.8 mM for p-nitrophenyl--glucoside (PNPG) and 33 mM for cellobiose. The cellular localization and the substrate specificity pattern are consistent with a dual role of the C. stercorarium -glucosidase in cellulose saccharification: (1) Cleavage of cellobiose formed by exoglucanase and (2) degradation of cellodextrins produced by endoglucanase action.     

Amylolytic lactic acid bacteria in fish silage

An ∝aL-amylase activity has been observed in lactic acid bacteria occurring initially in fermented fish silage. The organisms belong to the genus Leuconostoc . The main fraction of the amylolytic enzyme produced by one of the isolated bacteria is cell-bound and is released into the medium at a late stage of growth. Treating cells with ultrasound or Triton X-100 increases enzyme activity in the culture filtrate. The pH range for enzyme activity is 5.0–7.0, with an optimum at pH 6.0. The enzyme is extremely labile at pH 8.0 and is inactivated at temperatures above 50°C at pH 5.8. Two enzyme fractions were found by isoelectric focusing, the main one at pH 5.00 and another at pH 4.5. Chromatography on DEAE cellulose gave two active peaks.