Enzymatic properties of recombinant kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus ATCC43494

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP ≧ 4 than KP from Thermoanaerobacter brockii ATCC35047.     

A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1,6-glucosidases

Summary A p-nitrophenyl-α-d-glucopyranoside-hydrolysing oligo-1,6-glucosidase (dextrin 6-α-d-glucanohydrolase, EC 3.2.1.10) of Bacillus sp. KP 1071 capable of growing at 30°–66°C was purified to homogeneity. The molecular weight was estimated to be 62,000. The amino-terminal amino acid was methionine. The enzyme shared its antigenic groups in part with its homologous counterpart from Bacillus thermoglucosidasius KP 1006 (obligate thermophile), but did not at all with any one of oligo-1,6-glucosidases from Bacillus cereus ATCC 7064 (mesophile), Bacillus coagulans ATCC 7050 (facultative thermophile) and Bacillus flavocaldarius KP 1288 (extreme thermophile). A comparison of amino acid composition showed that the proline content increased greatly in a linearity with the rise in thermostability in the order, mesophile → facultative thermophile → KP 1071 → obligate thermophile → extreme thermophile enzymes. Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Kyoto, April 3, 1986     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between elongation growth and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000g pelletable (2KP) fraction from lettuce (Lactuca sativa L., cv. Arctic) hypocotyl sections has been examined. Sections were loaded with incremental amounts of GA₁ under conditions where growth was arrested (5° C) or permitted (30° C) and, after 16 h, all were transferred to a GA-free medium at 30° C. Growth and 2KP radioactivity were measured at this point and after a further 24 h in the chase medium. Uptake was reduced by 80% at 5° C, as compared to 30° C, but 2KP labelling and protein synthesis were only reduced by half. The growth rate of the 5° C pretreated sections during the chase period was comparable to that observed during the pulse in the 30° C material but the dose/response relationship was flatter. Low temperature sections incorporated a much higher percentage of GA₁ uptake into the 2KP fraction (27% at maximum) but the absolute levels of labelling at this temperature were lower than those measured at 30° C. The data are interpreted as showing that 2KP labelling is not a consequence of growth. It must either precede response or be an unconnected concurrent process.     

Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800

The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN₃ and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.     

Purification and Properties of Neutral-cyclodextrin Glycosyl-transferase of an Alkalophilic Bacillus sp.

Neutral-cyclodextrin glycosyltransferase (EC 3.2.1.19) of alkalophilic Bacillus sp. (ATCC 21783) was purified by starch adsorption, DEAE-cellulose chromatography and Sephadex G–150 gel filtration chromatography followed by preparative polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was 85,000-88,000 by SDS-disc gel electrophoresis. The enzyme was most active at pH 7 and 50°C, and stable up to 60°C at pH 7 and in the range of pH 6~8 at 60°C by 30 min incubation. The apparent V_max and Km values for α- and β-cyclodextrin at a constant concentration of sucrose were 417, 70 µmoles glucose/min · mg protein and 10, 0.83 nm, respectively. About 85~90% of amylose, 75~80% of potato starch, 65~70% of amylopectin, 55~60% of glycogen, 45~50% of amylopectin β-limit dextrin, 20~25% of maltotriose and 10~15% of maltose were converted to cyclodextrins with 0.5~1% (w/v) of each substrate.

Schardinger β-dextrin was preferentially produced from starch, and α- or γ-dextrin was gradually formed after prolonged incubation. After 20 min incubation, about 0.4, 14 and 2.5% of α-, β- and γ-dextrin were formed from starch, respectively.     

Polynucleotide Phosphorylase from Acromobacter sp. KR 170-4

Eighty-five strains of bacteria were screened for selection of microorganisms suitable for industrial production of polynucleotides. Among these bacteria, Achromobacter sp. KR 170-4 (ATCC 21942) was found to be rich in polynucleotide Phosphorylase (PNPase) in its “salt-shockate” as compared with the other strains tested. PNPase was purified about 50-fold from the “salt-shockate” of Achromobacter sp. KR 170-4, and properties of the enzyme were elucidated. Optimal pH for reaction was 10.1. Stable pH range at 37°C was between pH 6.5 and 10.5. Optimal temperatures were 46°C for polymerization of ADP or IDP, and 43°C for CDP or UDP. The enzyme was stable below 55°C at pH 9.2. The enzyme required Mn²⁺ rather than Mg²⁺ unlike the other PNPases reported. Optimal concentration of Mn²⁺ was 6 mM.     

Laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613

Conditions for the laboratory-scale production of acetoin plus diacetyl by Enterobacter Cloacae ATCC 27613 were studied. Thirty-five g acetoin plus diacetyl/50 g sucrose were obtained when fermentation was carried out in 2. 5 liter medium containing 12.5 g peptone and 12. 5 g yeast extract, at pH 7.0, in a 5 liter conical flask on a shaker (240rpm) at 28–30°C for 48 hr. Recovery of pure diacetyl was 85% of the total plus diacetyl.     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

Survival of acid-adapted and non-adaptedStaphylococcus aureus in various food samples

Resistance ofStaphylococcus aureus to acid pH was studied.Staphylococcus aureus ATCC 6538 was acid-adapted at pH 5.0 in tryptic soy broth (TSB) for 4 h. Commercial products, mayonnaise pH 3.57, rape pH 3.72, fatty yogurt pH 4.01, were purchased from a local supermarket, and kisir köfte pH 4.9 samples were prepared by us. All of the samples were inoculated with acid-adapted or non-adapted cells ofS. aureus. In un-inoculated mayonnaise, rape, fatty yogurt, and kisir köfteS. aureus was not detected. The viable population of S. aureus in mayonnaise declined quickly when stored at 4 or 25 °C. After 48 h of storage, no viable cells were recovered from mayonnaise inoculated with acid-adapted or non-adapted ATCC 6538 at 25 °C. Acid-adapted cells were recovered in greater numbers than non-adapted cells during storage at 4 or 25 °C. After 24 h of storage, no viable cells were recovered from rape and yogurt inoculated with acid-adapted and non-adapted ATCC 6538 at 4 and 25 °C. Acid-adaptedS. aureus survived in kisir köfte during 48 h. After 72 h of storage, no viable cells were recovered from kisir köfte inoculated with acid-adapted and non-adapted ATCC 6538 at 25 °C and 4 °C.     

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate‐SDS‐PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6–8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl‐methyl sulfonyl‐fluoride and N‐α‐Tosyl‐L ‐lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin‐like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin‐like serine proteases occur during the developmental cycle of A. aegypti. © 2011 Wiley Periodicals, Inc.     

Studies on Tannin Acyl Hydrolase of Microorganisms

Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.

The molecular weight of the enzyme estimated by gel filtration method was about 200,000.

The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.

Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water.     

Cellular Lipid Fatty Acid Pattern Heterogeneity Between Reference and Recent Food Isolates of Listeria Monocytogenes as a Response to Cold Stress

Cells of four reference strains (Scott A, LO 28, CNL 895807 and ATCC 19115) and of five recent food isolates (A00M011, A00M018, A00M087, A00M092 and A00M123) of Listeria monocytogenes were grown until late exponential phase in Brain Heart Broth at two different temperatures (37 °C and 4 °C). Our results show that significant differences exist between the cellular lipid fatty acid profile of reference and recent food isolates. Like the reference strains, and in keeping with previous reports on the cellular lipid fatty acid profile of L. monocytogenes, the recent food isolates were characterised by the presence of ai15:0, i15:0 and ai17:0. In addition, the fatty acid ai13:0 was observed in all of the recent food isolates grown at 4 °C, whereas only two reference strains, Scott A and LO 28, showed ai13:0 in their cellular lipid fatty acid profile at 4 °C. When grown at 4 °C, the recent food isolates showed a mean aiC₁₅/aiC₁₇ ratio of 66, while reference strains were characterised by significantly lower ratios, ranging between 4.3 (ATCC 19115) and 28.9 (Scott A). These results showed that all of the recent food isolates, Scott A and LO28 strains use chain length and anteiso-branching (ai15:0) as their major response to cold temperature adaptation. However, the cold adaptation response of reference strains CNL 895807 and ATCC 19115 appears to be different.     

Phospholipid metabolism in Mycobacterium smegmatis ATCC 607 grown at 37° C and 27° C

The rate of synthesis and degradation of phospholipids in Mycobacterium smegmatis ATCC 607, grown at 27° C and 37° C was studied by incorporation of ³²P into phospholipids and chase of radioactivity of the pulse-labelled phospholipids. A relatively low rate of synthesis and degradation of phospholipids in cells growth at 27° C was observed as compared to those grown at 37° C. Phosphatidylethanolamine (PE) had the maximum turnover at 37° C. However, at 27° C, cardiolipin (CL) showed a turnover rate higher than PE. Phosphatidylinositol mannosides (PIMs) were metabolically more active at 37° C than at 27° C. The differences in metabolic activity of the phospholipids at the two temperatures have been discussed.     

The lack of identity between Bacillus stearothermophilus and Bacillus thermoglucosidasius in serological patterns of exo-oligo-1,6-glucosidase and exo-α-1,4-glucosidase

Summary Among 16 Bacillus stearothermophilus strains, 11 strains (ATCC 7953, ATCC 10149, ATCC 12976, ATCC 12978, ATCC 12980, ATCC 15951, ATCC 21365, IAM 11001, IAM 11004, IAM 11062 and IFO 12550) produced a protein reactable on double immuno-diffusion with the antiserum against Bacillus thermoglucosidasius KP 1006 (DSM 2542) exo-oligo-1,6-glucosidase (dextrin 6-glucanohydrolase, EC.3.2.1.10). However, these antigens in part shared their antigenic determinants. In addition to an exo-oligo-1,6-glucosidase, 6 B. thermoglucosidasius strains [KP 1006, KP 1012, KP 1013, KP 1014, KP 1019 and KP 1022 (DSM 2543)] formed a protein cross-reacted with the antiserum against B. stearothermophilus ATCC 12016 exo--1,4-glucosidase (-d-glucoside glucohydrolase, EC.3.2.1.20). These two antigens showed, however, a partial coincidence in their antigenic determinant groups. Of 16 B. stearothermophilus strains, 3 strains (ATCC 8005, ATCC 12016 and ATCC 15952) produced a protein immunologically compatible with the -1,4-glucosidase, while 4 strains (ATCC 12979, ATCC 12980, ATCC 15951 and IAM 11001) made the other protein which showed certain differences partly from this enzyme in its antigenic groups. No protein precipitated with the anti--1,4-glucosidase occurred in the remaining 9 B. stearothermophilus strains (ATCC 7953, ATCC 10149, ATCC 12976, ATCC 12977, ATCC 12978, ATCC 21365, IAM 11004, IAM 11062 and IFO 12550). These data indicate no serological identity between two thermophilic Bacillus species in their glucosidase patterns.     

Purification,Properties and Recognition Sequence of Site-specific Restriction Endonuclease from Gluconobacter cerinus IFO 3285

A type II restriction endonuclease, designated as GceGLI, was purified from cells of Gluconobacter cerinus IFO 3285. The purified enzyme was found to be homogeneous on Polyacrylamide gel disc electrophoresis. The enzyme worked best at 37°C and pH 7.5 and required 7 mM MgCl₂ and 100 mM NaCl. The purified enzyme was stable when preincubated over a pH range of 7.5 to 9.5 for 12 hr at 4°C and a temperature range of 37 to 40°C for 5 min at pH 7.5. The enzyme was shown to cleave λ φX174 RF, SV40, pBR322, M13 mp7 RF and Ad2 DNAs at 4, 1,0, 0, 0 and 25 or more sites, respectively, and to recognize the DNA sequence of 5′-C-C-G-C-G-G-3′ and to cut between C and G on the right side of the sequence, being an isoschizomer of SacII of Streptomyces achromogenes ATCC 12767.     

Application of a microtitre reader system to the screening of inulinase nulinase-producing yeasts

 Fourteen strains of yeast from genera Kluyveromyces, Candida, Debaryomyces and Schizosaccharomyces were investigated for inulinase production. In the first stage, the microtitre reader system SLT was used for the determination of enzyme activity and the evaluation of cellular growth. Different culture conditions were tested and four strains of Kluyveromyces were selected on the basis of enzyme activity and growth capacity at low pH and high temperature: K. marxianus CBS 6397, DSM 70792, ATCC 36907 and IZ 619. These strains were tested in greater volume using pH 4.0, 45^°C and inulin (10 g/l) as selection conditions. On the basis of results obtained, the strain K. marxianus ATCC 36907 was selected for inulinase production. Enzyme stability at low pH (4.0) as well as high temperature (50^°C) for 10, 30 and 60 min was also evaluated, but no significant difference in enzyme activity was observed. It could be demonstrated that the microtitre reader system is an excellent method for the screening of microorganisms. Received: 31 May 1995/Received revision: 20 September 1995/Accepted: 29 September 1995     

Purification and characterization of the intracellular β-glucosidase from Aureobasidium sp ATCC 20524

β-Glucosidase hydrolyzing cellobiose was extracted from Aureobasidium sp ATCC 20524 and purified to homogeneity. The molecular mass was estimated to be about 331 kDa. The enzyme contained 26.5% (w/w) carbohydrate. The optimum pH and temperature for the enzyme reaction were pH 4 and 80°C, respectively. The enzyme was stable at a wide range of pH, 2.2–9.8, after 3 h and at 75°C for 15 min. The kinetic parameters were determined. The enzyme was relatively stable against typical organic enzyme inhibitors. The enzyme also hydrolyzed gentiobiose, p-nitrophenyl-β-glucoside and salicin. Received 05 November 1998/ Accepted in revised form 14 February 1999     

Induction of an Increase in Nerve Growth Factor Content in the Cell-conditioned Medium of Mouse L-M Cells by Basic Peptides

Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80–82% at 30–40°C. The activity was inhibited by Cu²⁺ , Hg²⁺, Zn²⁺, and Tris.     

Characterization of a thermostable xylanase from an alkaliphilic Bacillus sp.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity.