Production of raw-starch digesting amyloglucosidase by Aspergillussp GP-21 in solid state fermentation

Aspergillus sp GP-21 produced a raw-starch digesting amyloglucosidase which showed optimum activity at 65°C and pH 5.0–5.5. At 50°C the enzyme converted about 40% of raw corn starch to glucose within 48 h. Enzyme production was studied in solid state fermentation using wheat bran. Productivity was affected by the level of moisture, incubation temperature and the presence or absence of supplements. Maximum enzyme production was observed at a moisture level of 75% and at 30°C. Enzyme production was stimulated by supplementing wheat bran with 0.25% proteose peptone, 1% trace mineral solution, 0.01% CaCl₂ and 0.01% MgSO₄. Received 27 October 1998/ Accepted in revised form 15 March 1999     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Purification and characterization of a novel extracellular halophilic and organic solvent-tolerant amylopullulanase from the haloarchaeon, Halorubrum sp. strain Ha25

A halophilic archaeon, Halorubrum sp. strain Ha25, produced extracellular halophilic organic solvent-tolerant amylopullulanase. The maximum enzyme production was at high salt concentration, 3–4 M NaCl. Optimum pH and temperature for enzyme production were 7.0 and 40 °C, respectively. Molecular mass of purified enzyme was estimated to be about 140 kDa by SDS–PAGE. This enzyme was active on pullulan and starch as substrates. The apparent K _m for the enzyme activity on pullulan was 4 mg/ml and for soluble starch was 1.8 mg/ml. Optimum temperature for amylolytic and pullulytic activities was 50 °C. Optimum pH for amylolytic activity was 7 and for pullulytic activity was 7.5. This enzyme was active over a wide range of concentrations (0–4.5 M) of NaCl. The effect of organic solvents on the enzyme activities showed that this enzyme was more stable in the presence of non-polar organic solvents than polar solvents. This study is the first report on amylopullulanase production in halophilic bacteria and archaea.     

A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning,heterologous expression and biochemical characterization

A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25 %) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39 % activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93 % activity even at 4 M NaCl. The catalytic efficiency (k _cat/K _m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg^?1 s^?1 (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl₂. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.     

Psychrophilic α-amylase from Aeromonas veronii NS07 isolated from farm soils

Among 120 isolates examined in this study, three isolates were selected for amylase production on starch agar plates following incubation at 10 °C. Identification by 16SrRNA on selected bacterium disclosed the highest similarity for protean regions of this gene as Aeromonas veronii NS07. A 63 kDa psychrophilic amylase enzyme from NS07 strain was purified by two-steps chromatography. The enzyme had the highest specific activity at pH 4 and was active at the range of temperatures from 0 to 50 °C, although the optimum temperature for enzyme activity was found at 10 °C. Analysis of the N-terminal amino acid sequencing disclosed 20 amino acids from purified amylase which had no similarity with other known α-amylases, indicating that the presented enzyme was novel. Amylase activity was enhanced in relation to optimum activity with the presence of sodium sulphate (161%), MnCl₂ (298%), CaCl₂ (175%), FeCl₂ (182%), MgCl₂ (237%), ZnCl₂ (169%), NiCl₂ (139%), NaCl (158%), each at 5 mM, while EDTA, phenylmethane sulphonylfluoride (PMSF) (3 mM), urea (8 M) and SDS (1%) inhibited the enzyme up to 5%, 2%, 80% and 18%, respectively. NS07 strain seems to be suitable as biocatalyst for practical use in liquefaction of starch at low temperatures, detergent and textile industries.     

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K _cat). The enzyme was also calcium dependent and with 2 mM Ca⁺², the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca⁺², respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.     

Simultaneous saccharification and ethanol fermentation of sago starch using immobilized Zymomonas mobilis

Simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and immobilized Zymomonas mobilis ZM4 on sodium alginate was studied. The immobilized Zymomonas cells were more thermo-stable than free Zymomonas cells in this system. The optimum temperature in the SSF system was 40°C, and 0.5% (v/w) AMG concentration was adopted for the economical operation of the system. The final ethanol concentration obtained was 68.3 g/l and the ethanol yield, Y_p/s, was 0.49 g/g (96% of the theoretical yield). After 6 cycles of reuse at 40°C with 15% sago starch hydrolysate, the immobilized Z. mobilis retained about 50% of its ethanol fermenting ability.     

Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense

An extracellular haloalkaliphilic thermostable α-amylase producing archaeon was isolated from the saltwater Lake Urmia and identified as Halorubrum xinjiangense on the basis of morphological, biochemical, and molecular properties. The enzyme was purified to an electrophoretically homogenous state by 80 % cold ethanol precipitation, followed by affinity chromatography. The concentrated pure amylase was eluted as a single peak on fast protein liquid chromatography. The molecular mass of the purified enzyme was about 60 kDa, with a pI value of 4.5. Maximum amylase activity was at 4 M NaCl or 4.5 M KCl, 70 °C, and pH 8.5. The K _m and V _max of the enzyme were determined as 3.8 mg ml^?1 and 12.4 U mg^?1, respectively. The pure amylase was stable in the presence of SDS, detergents, and organic solvents. In addition, the enzyme (20 U) hydrolyzed 69 % of the wheat starch after a 2-h incubation at 70 °C in an aqueous/hexadecane two-phase system.     

Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A

An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl₂ or MgCl₂. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl₂, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.     

Purification and characterization of α-amylase fromAspergillus flavus

Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an α-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate— polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5±2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55°C and it was stable for 1 h up to 50°C. TheK _m andV for gelatinized tapioca starch were 0.5 g/L and 108.67 μmol reducing sugars per mg protein per min, respectively.     

Ethanol production from raw starch by simultaneous fermentation using Schizosaccharomyces pombe and a raw starch saccharifying enzyme from Corticium rolfsii

Alcoholic fermentation from raw corn starch using Schizosaccharomyces pombe AHU 3179 and a raw starch saccharifying enzyme (RSSE) from Corticium rolfsii AHU 9627 was investigated. The optimum ethanol production was achieved at pH 3.5, 27°C and under the yeast cell concentration of 2.7 × 10⁹ cells/ml. Addition of RSSE 5 units (as glucoamylase)/g raw corn starch was found sufficient. Under these optimum conditions, 18.5% (v/v, at 15°C) ethanol was obtained from 30% raw corn starch (30.8% as glucose) after incubation for 48 h.     

Co-encapsulation of amyloglucosidase with starch and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> as basis for a long-lasting CO<Subscript>2</Subscript> release

CO₂ is known as a major attractant for many arthropod pests which can be exploited for pest control within novel attract-and-kill strategies. This study reports on the development of a slow-release system for CO₂ based on calcium alginate beads containing granular corn starch, amyloglucosidase and Saccharomyces cerevisiae. Our aim was to evaluate the conditions which influence the CO₂ release and to clarify the biochemical reactions taking place within the beads. The amyloglucosidase was immobilized with a high encapsulation efficiency of 87% in Ca-alginate beads supplemented with corn starch and S. cerevisiae biomass. The CO₂ release from the beads was shown to be significantly affected by the concentration of amyloglucosidase and corn starch within the beads as well as by the incubation temperature. Beads prepared with 0.1 amyloglucosidase units/g matrix solution led to a long-lasting CO₂ emission at temperatures between 6 and 25?°C. Starch degradation data correlated well with the CO₂ release from beads during incubation and scanning electron microscopy micrographs visualized the degradation of corn starch granules by the co-encapsulated amyloglucosidase. By implementing MALDI-ToF mass spectrometry imaging for the analysis of Ca-alginate beads, we verified that the encapsulated amyloglucosidase converts starch into glucose which is immediately consumed by S. cerevisiae cells. When applied into the soil, the beads increased the CO₂ concentration in soil significantly. Finally, we demonstrated that dried beads showed a CO₂ production in soil comparable to the moist beads. The long-lasting CO₂-releasing beads will pave the way towards novel attract-and-kill strategies in pest control.     

Characterization of a membrane bound β-glucosidase responsible for the activation of oat leaf saponins

Oat leaves contain a β-glucosidase (= avenacosidase) specific for the cleavage of the C-26 bound glucose moiety of the oat saponins avenacosides A and B. This transformation activates the fungitoxicities of the avenacosides. Evidence is presented that this enzyme is bound to the tonoplast membrane. The solubilized enzyme showed a pH optimum of 6.0–7.0, a temperature optimum around 40°, a molecular weight of 68 000±3000 and a K_m of 183 (±16) μM. The enzyme is inhibited by Hg²⁺ (10^-2 M) but not by Cu²⁺ (10^-2 M).     

Isomerization of glucose to fructose: production and some properties of glucose isomerase from Streptomyces sp. strain C7

Nine strains of actinomycetes isolated from Iraqi soils were investigated for glucose isomerase production. Only one strain, Streptomyces sp., C₇, was active. The maximum conversion ratio of the enzyme for the cells grown in d-xylose medium after 24 h incubation at 70°C and pH 6.9, was 64 and 48% for crude extract and cell-bound enzyme, respectively. The optimum pH value and temperatures for both enzymes were 8.0 and 70°C.     

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.     

Amylolytic enzymes ofEndomycopsis capsularis

Endomycopsis capsularis 311/1 is a good producer of extracellular α-glucosidases. An enzyme exhibiting maltase and transglucosidase activities is released at the beginning of the logarithmic phase of growth. The enzyme is able to produce isomaltose and panose. Starch-hydrolysing enzymes are excreted into the medium at the end of the logarithmic phase of growth: an enzyme of α-amylase type is produced in the presence of calcium carbonate and pH about 6.0, whereas an enzyme of the amyloglucosidase type is formed in the absence of calcium carbonate and pH about 4.0. The enzyme production is identical in media containing different carbon sources, glucose, maltose or soluble starch.     

Biochemical characterization of a raw starch degrading <Emphasis Type="Italic">α</Emphasis>-amylase from the Indonesian marine bacterium <Emphasis Type="Italic">Bacillus</Emphasis> sp. ALSHL3

An Indonesian marine bacterial isolate, which belongs to genus of Bacillus sp. based on 16S rDNA analysis and was identified as Bacillus filicolonicus according to its morphology and physiology, produced a raw starch degrading α-amylase. The partially purified α-amylase using a maize starch affinity method exhibited an optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme retained 72% of its activity in the presence of 1.5 M NaCl. Scanning electron micrographs showed that the α-amylase was capable of degrading starch granules of rice and maize. This α-amylase from Bacillus sp. ALSHL3 was classified as a saccharifying enzyme since its major final degradation product was glucose, maltose, and maltotriose.     

Production,Purification, and Characterization of α-Amylase from Solventogenic Clostridium sp. BOH3

A solventogenic strain of Clostridium sp. BOH3 produces extracellular α-amylase (7.15 U/mg protein) in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate (1 % w/v) and a small amount of starch (0.1 % w/v), which is essential for the expression of α-amylase. In the presence of α-amylase, BOH3 utilizes starch directly without any pretreatment and produces butanol almost equivalent (～90 %) to the production of butanol from glucose. α-Amylase can be purified from culture supernatant by using one-step weak anion exchange chromatography with a yield of 43 %. In peptide fingerprinting analysis, this enzyme shows homology with α-amylase produced by Clostridium acetobutylicum ATCC824. However, the molecular weight is 54 kDa, which is smaller than α-amylase of ATCC824 (84 kDa). This enzyme has optimum temperature at 45–50 °C and optimum pH at 4.5–5.5. Under this condition, the enzyme activity is 91.32 U/mg protein, and its K _m and V _max values are 1.71?±?0.02 mg/ml and 96.13?±?0.15 μmol/min/mg protein, respectively. Activity of this α-amylase can be enhanced (>1.5 times) by addition of Ca²⁺ and Co²⁺ and its activity can be maintained at an acidic pH (pH 3–5) for about 24 h. These unique characteristics suggest that this enzyme can be used for saccharification of starch for production of biofuel in one pot.     

Isolation and characterization of a protease-producing novel haloalkaliphilic bacterium <Emphasis Type="Italic">Halobiforma</Emphasis> sp. strain BNMIITR from Sambhar lake in Rajasthan,India

A novel haloalkaliphilic bacterium designated as strain BNMIITR was isolated from a soil sample collected from Sambhar lake, Rajasthan, in northern India. Colonies of the isolated strain were dark orange and comprised Gram-negative bacilli; there was a slight pleomorphism towards the stationary phase of growth. Experiments revealed that the isolate can grow in the range of 2–5 M NaCl, pH 6–11 and 18–55 °C, with optimum growth observed at 3 M NaCl, pH 8–8.5 and 45 °C. No growth was observed in culture medium without NaCl. The isolate showed no requirement for magnesium sulphate heptahydrate (MgSO₄ ^.7H₂O) for growth. Major cellular fatty acids were C _14:0, C _15:0 iso, C _15:0 anteiso, C _16:0, C _17:0 iso, C _17:0 anteiso and C _20:2 w6, 9c. The result of 16S rRNA gene sequence analysis showed 98 % sequence similarity with Halobiforma lacisalsi and Hbf. haloterrestris. Halobiforma sp. strain BNMIITR showed resistance towards several antibiotics and produced an extracellular alkaline protease. The crude enzyme was found to be active in broad range of alkaline pH and temperature (30–80 °C).     

Preparation of Triglyceride Hydroperoxides

Corticium rolfsii AHU 9627, which we isolated from a tomato stem, is one of the most promising producers of a raw starch saccharifying enzyme. The effects of the cultural conditions and medium components on the enzyme production were investigated. The enzyme production was improved by increasing both the concentrations of carbon sources and organic nutrients in the medium. Under the optimum cultural conditions, the enzyme activity of the culture supernatant against raw starch reached a maximum after 8-days incubation at 27°C and the activity reached 80 units per ml (when determined at 40°C and pH 4.0). The optimal pH and temperature for the enzyme reaction were 4.0 and 65°C, respectively. The saccharifying reaction was scarcely inhibited even with a high substrate concentration, and raw starch was rapidly hydrolyzed into glucose.