Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties

Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO₄.7H₂O, 0.05% KCl, 0.2% K₂HPO₄ and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme retained more than 90% of its activity. Enzyme activity was inhibited by Hg²⁺ and K⁺, whereas Ca²⁺ and Mn²⁺ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Development of fermentation process for PLA-degrading enzyme production by a new thermophilic Actinomadura sp. T16-1

The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-degrading Actinomadura sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at 50°C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2 % KH₂PO₄, and 0.02% MgSO₄ · 7H₂O. The optimal concentration of PLA film and gelatin obtained by response surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238% (w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter.     

Biochemical studies of citric acid production and accumulation by Aspergillus niger mutants

The biochemical rationale for the inhibition of citric acid fermentation by Aspergillus niger in the presence of Mn²⁺ ions has been investigated using high citric acid-yielding, Mn²⁺ ion-sensitive as well as Mn²⁺ ion-tolerant mutant strains of A. niger. In the presence of Mn²⁺ (1.5 mg/l), citric acid production by the Mn²⁺ ion-sensitive strain (KCU 520) was reduced by about 75% with no apparent effect on citric acid yield by the Mn²⁺ ion-tolerant mutant strain (GS-III) of A. niger. The significantly increased level of the Mn²⁺ ion-requiring NADP⁺-isocitrate dehydrogenase activity in KCU 520 cells and the lack of effect on the activity level of the enzyme in GS-III mutant cells by Mn²⁺ ions during fermentation seem to be responsible for the Mn²⁺ ion inhibition of citric acid production by the KCU 520 strain and the high citric acid yield by the mutant strain GS-III of A. niger even in the presence of Mn²⁺.     

Estimation of coefficient of coancestry using molecular markers in maize

Summary The coefficient of coancestry (f_AB) between individuals A and B is the classical measure of genetic relationship. f_AB is determined from pedigree records and is the probability that random alleles at the same locus in A and B are copies of the same ancestral allele or identical by descent (ibd). Recently, the proportion of molecular marker variants shared between A and B (S_AB) has been used to measure genetic relationship. But S_AB is an upwardly-biased estimator of f_AB, especially between distantly-related lines. f_AB, S_AB, and adjusted (to remove bias) estimates of molecular marker similarity (f _AB ^M ) were compared. RFLP banding patterns at 46 probe-restriction enzyme combinations were obtained for 23 maize inbred lines derived from the Iowa Stiff Stalk Synthetic (BSSS) maize (Zea mays L.) population, and for 4 non-BSSS lines. f _AB ^M was estimated as , where _A (or _B) was the average proportion of RFLP variants shared between inbred A (or inbred B) and the non-BSSS lines. The average f_AB among 253 pairwise combinations of BSSS lines was 0.212, whereas the average S_AB was 0.397. The average f _AB ^M was 0.162, indicating that the upward bias in S_AB was effectively removed. S_AB and f_AB were significantly different ( = 0.05) in 76.3% of the comparisons, whereas 24.9% of the f _AB ^M values differed significantly from f_AB. The latter result suggests that selection and/or drift were present during inbred line development and that f_AB may not be an accurate measure of the true proportion of ibd alleles between two lines. Cluster analyses based on S _AB ^M and f _AB ^M grouped lines according to pedigree, although several exceptions were noted. The presence of shared molecular marker variants between unrelated lines must be considered when setting S_AB-based minimum distances for varietal protection. Under simplified conditions, more than 250 molecular marker loci are necessary to obtain sufficiently precise estimates of coefficient of coancestry using molecular markers.A contribution from Limagrain Genetics, a Group Limagrain company     

Microbial and enzymatic improvement of anaerobic digestion of waste biomass

Metabolic activities of different microorganisms (Bacillus subtilis, B. licheniformis and Aspergillus niger) and hydrolytic enzymes (concentrations: 1 to 200 mg enzyme solids g^–1 feed) were studied individually and in combinations with respect to H₂ and methane production from damaged wheat grains. Bacillus subtilis, B. licheniformis and pre-existing hydrogen producers (control) produced 45 to 64 l H₂ kg^–1 total solids and subsequently, with the help of added methanogens, 155 to 220 l methane kg^–1 total solids could be produced. H₂ production from damaged wheat grains could be decreased to 28% or enhanced up to 152% with respect to control, by employing various microbial and enzymatic treatments. Similarly, it has been made possible to vary methane production capacities from as low as 17% to as high as 110% with respect to control.     

Purification and characterization of chitinase from Alcaligenes xylosoxydans

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecularmass of chitinase was estimated to be 45 kDa and44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 °C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l^–1. Cu²⁺ and Na⁺ at 5 mM inhibited chitinase activity to 25% while Ca²⁺, Mg²⁺ and Ba²⁺ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.     

Effect of different cultural conditions for phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> CFR 335 in submerged and solid-state fermentations

The present article deals with the studies on the effect of media ingredients, such as carbon, nitrogen, inorganic phosphates, surfactants, and metal salts, on phytase enzyme production by Aspergillus niger CFR 335 in submerged (SmF) and solid-state fermentations (SSF). The results obtained showed a 1.5-fold higher enzyme yield in the presence of sucrose in both SmF and SSF, while peptone was found to be a favorable nitrogen source for SmF. Sodium dihydrogen phosphate (NaH₂PO₄) favored 34% higher enzyme yield than the control, which was followed by 19% higher activity in potassium dihydrogen phosphate (KH₂PO₄) in SSF at 0.015% w/v. The addition of Tween-20 in SmF showed a maximum yield of 12.6 U/mL while, SDS suppressed the growth of the fungus. None of the surfactants favored the enzyme yield in SSF. Calcium chloride (CaCl₂) was extensively efficient in stimulating more than 55% higher phytase production in SmF at 0.01% v/v. In SSF, none of the metal salts stimulated phytase production.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

Thermostable Alkaline Protease from a Novel Marine Haloalkalophilic Bacillus Clausii Isolate

An oxidative and SDS-stable alkaline protease secreted by a marine haloalkalophilic Bacillus clausii isolated from the tidal mud flats of the Korean Yellow Sea near Inchon City was investigated in batch fermentation in shake flasks and in a bioreactor under a range of conditions. The isolate produced maximum protease yields (15,000 U ml⁻¹) under submerged fermentation conditions at 42 °C for 40 h with an aeration of 1.5 v/v/min and agitation of 400 rev/min in a formulated soybean—casein medium (pH 9.6) containing (w/v): soybean meal (2%), casein (1%), corn starch (0.5%), NH₄Cl (0.05%), NaCl (0.05%), KH₂PO₄(0.04%), K₂HPO₄(0.03%), MgSO₄(0.02%), yeast extract (0.01%) and Na₂CO₃(0.6%). The optimal pH and temperature of activity of the partially purified enzyme were 11.5 and 80 °C, respectively. The alkaline protease showed extreme stability towards SDS and oxidizing agents, retaining its activity above 96 and 75% on treatment for 72 h with 5% SDS and 5% H₂O₂, respectively. The inhibition profile exhibited by phenylmethanesulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases.     

Optimization of Process Parameters for the Production of Inulinase from a Newly Isolated Aspergillus niger AUP19

Summary Fifty strains were isolated from different soil samples on synthetic medium containing inulin as a sole carbon source for the production of extracellular inulinase. Of them, five isolates showed high inulinase activity and one of them was selected for identification and medium optimization studies. The isolate was identified as Aspergillus niger. Various physical and chemical parameters were optimized for inulinase production. Maximum productivity of inulinase (176 U ml⁻¹) was achieved by employing medium containing 5% (w/v) inulin, galactose as additional carbon source, corn steep liquor and (NH₄)H₂PO₄ as nitrogen sources, incubation period of 72 h, incubation temperature of 28 °C, pH 6.5, inoculum load at 10% (v/v) level and medium volume to flask volume ratio of 1:20 (v/v) with indented flasks.     

Optimization of medium for extracellular nuclease formation from Rhizopus stolonifer

A strain of Rhizopus stolonifer produced a high activity of extracellular DNAase when grown on YPG (yeast extract peptone glucose) medium. The source of peptone had a marked effect on the enzyme yield and only one peptone (i.e. from Sarabhai M. Chemicals Ltd, India) supported enzyme production. Maximum enzyme activity (88 U/ml) was obtained after 4 days' growth under submerged conditions in YPG medium containing 100 M Mn²⁺, Co²⁺ or Mg²⁺, and glucose as the sole carbon source. The unpurified enzyme was optimally active at pH 7.5 and 45°C. It had a higher activity with sonicated and heat-denatured DNA than native DNA.     

Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.     

Optimization of an ethanol production medium in very high gravity fermentation

Concentrations of Mg²⁺, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg²⁺ and peptone were identified as the critical factors: 50 mM Mg²⁺ and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.     

Studies onAspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grownAspergillus niger was increased 3–5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH ₄ ⁺ , and further, the enzyme is repressed by increasing concentrations of NH ₄ ⁺ . In contrast to other micro-organisms, theAspergillus niger enzyme was neither specifically inactivated by NH ₄ ⁺ or L-glutamine nor regulated by covalent modification. Glutamine synthetase fromAspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn²⁺ or Mg²⁺-dependent synthetase and Mn²⁺-dependent transferase activity. Aspergillusniger glutamine synthetase was completely inactivated by two mol of phenyl-glyoxal and one mol of N-ethylmaleimide with second order rate constants of 3.8 M^-1 min^-1 and 760 M^-1 min^-1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH ₄ ⁺ , Mn²⁺ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.     

H2 production from maltose derived from starch using the visible light, photosensitization of Mg chlorophyll-a from Spirulina

A photoinduced H₂ production system, coupling d-maltose degradation by glucoamylase and glucose dehydrogenase (GDH) and H₂ production with colloidal platinum as a catalyst using the visible light-induced photosensitization of Mg chlorophyll-a (Mg Chl-a), has been developed. H₂ production was continuous when the reaction mixture containing d-maltose, glucoamylase, GDH, NAD⁺, Mg Chl-a, Methyl Viologen (MV²⁺, an electron relay reagent) and colloidal platinum was irradiated by visible light. The amount of H₂ production was estimated to be 5 ± 0.5 mol after 4 h irradiation. The yield of the conversion of d-maltose to H₂ in this system was ca. 1.8 %.     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.