Identification and characterization of a new naringinase-producing strain, Williopsis californica Jmudeb007

A new naringinase-producing strain, Jmudeb007 was indentified by means of morphological observation, gene homogeneous analysis and physiological and biochemical test. Its characteristics in expressing naringinase were investigated by batch culture in 7 L fermentors. Jmudeb007 grown white, circular, convex, and smooth edged colonies, and single, oval-shaped and nucleus contained cells. Its gene sequences of 26S rDNA D1/D2 region and 5.8S rDNA-ITS region both showed homogeneities at 99% to Williopsis californica. It appeared positive in glucose fermentation test, negative in urease test and diazo blue B (DBB) test. Strain Jmudeb007 was identified to W. californica. Cultivation of Jmudeb007 with three media containing different amount of glucose showed it was capable of expressing naringinase which hydrolyze naringin to naringenin. The naringinase synthesis of Jmudeb007 was regulated by glucose which depended on the concentration. Jumdeb007 could express naringinase in medium containing glucose no more than 4 g/L. The present work provides a new source of naringinase, W. californica Jmudeb007, which is non-pathogenic, convenient to conduct breeding operation, easy to develop fermentation process. It is the first report about yeast that can produce naringinase, which help to explore naringinase and other glycosidase from yeast.     

STATISTICAL MEDIA OPTIMIZATION FOR THE BIOMASS PRODUCTION OF POSTHARVEST BIOCONTROL YEAST Rhodosporidium paludigenum

A cane molasses-based medium for the biomass production of biocontrol agent Rhodosporidium paludigenum was statistically optimized. Molasses concentration (after pretreatment), yeast extract, and initial pH were identified by the Plackett–Burman design to show significant influence on the biomass production. The three factors were further optimized by central composite design and response-surface methodology. The statistical analysis indicated the optimum values of the variables were 89.98 g/L for cane molasses, 2.35 g/L for yeast extract and an initial pH of 8.48. The biomass yield at the optimal culture achieved 15.89 g/L in flask fermentation, which was 2.1 times higher than that at the initial NYDB medium. In a 10-L fermenter, 18.97 g/L of biomass was obtained after 36 hr of cultivation. Moreover, the biocontrol efficacy of the yeast was investigated after culture optimization. The results showed the yeast harvested in the optimal medium maintained its initial biocontrol properties by reducing the percentage of decayed apples to below 20%.     

STATISTICAL APPROACH FOR THE ENHANCED PRODUCTION OF COLD-ACTIVE β-GALACTOSIDASE FROM Thalassospira frigidphilosprofundus: A NOVEL MARINE PSYCHROPHILE FROM DEEP WATERS OF BAY OF BENGAL

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active β-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for β-galactosidase production. Under optimal conditions, the production of β-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. β-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active β-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active β-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.     

Solid‐state fermentation for the production of debittering enzyme naringinase using Aspergillus niger MTCC 1344

Aspergillus niger produced high levels of naringinase using easily available, inexpensive industrial waste residues such as rice bran, wheat bran, sugar cane bagasse, citrus peel, and press mud in solid‐state fermentation (SSF). Among these, rice bran was found to be the best substrate. Naringinase production was highest after 96 h of incubation at 27°C and at a substrate‐to‐moisture ratio of 1:1 w/v. Supplementation of the medium with 10% naringin caused maximum induction. An inoculum age of 72 h and an inoculum level of 15% resulted in maximum production of naringinase. Enzyme production was stimulated by the addition of nutrients such as naringin and peptone. Thus, A. niger produced a very high level of naringinase within a short time in solid‐state fermentation using inexpensive agro‐residues, a level that is much higher than reported for any other microbes.     

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04?mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14?mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778?U/L at 34?hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164?U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.     

Comparing submerged and solid-state fermentation of agro-industrial residues for the production and characterization of lipase by Trichoderma harzianum

Lipase production by Trichoderma harzianum was evaluated in submerged fermentation (SF) and solid-state fermentation (SSF) using a variety of agro-industrial residues. Cultures in SF showed the highest activity (1.4 U/mL) in medium containing 0.5 % (w/v) yeast extract, 1 % (v/v) olive oil and 2.5 C:N ratio. This paper is the first to report lipase production by T. harzianum in SSF. A 1:2 mixture of castor oil cake and sugarcane bagasse supplemented with 1 % (v/w) olive oil showed the best results among the cultures in SSF (4 U/g ds). Lipolytic activity was stable in a slightly acidic to neutral pH, maintaining 50 % activity after 30 min at 50 °C. Eighty percent of the activity remained after 1 h in 25 % (v/v) methanol, ethanol, isopropanol or acetone. Activity was observed with vegetable oils (olive, soybean, corn and sunflower) and long-chain triacylglycerols (triolein), confirming the presence of a true lipase. The results of this study are promising because they demonstrate an enzyme with interesting properties for application in catalysis produced by fermentation at low cost.     

Controlling filamentous fungi morphology with microparticles to enhanced β-mannanase production

β-mannanase was produced mainly by Aspergillus species and can degrade the β-1,4-mannose linkages of galactomannans. This study was undertaken to enhance mannanase production using talcum and aluminum oxide as the microparticles, which control cell morphology of recombinant Aspergillus sojae in glucose and carob extract medium. Both microparticles improved fungal growth in glucose and carob pod extract medium. Aluminum oxide (1 g/L) was the best agent for glucose medium which resulted in 514.0 U/ml. However, the highest mannanase activity was found as 568.7 U/ml with 5 g/L of talcum in carob extract medium. Increase in microparticle concentration resulted in decreasing the pellet size diameter. Furthermore, more than 10 g/L of talcum addition changed the filamentous fungi growth type from pellet to pellet/mycelium mixture. Results showed that right type and concentration of microparticle in fermentation media improved the mannanase activity and production rate by controlling the growth morphology.     

Influence of carbon and nitrogen sources on lipase production by a newly isolated Candida viswanathii strain

Microorganisms can produce lipases with different biochemical characteristics making necessary the screening of new lipase-producing strains for different industrial applications. In this study, 90 microbial strains were screened as potential lipase producers using a sensitive agar plate method with a suitable medium supplemented with Tween 20 and also a liquid culture supplemented with olive oil. The highest cell growth and lipase production for Candida viswanathii were observed in triolein and oleic acid when used as the only pure carbon source. Renewable low-cost triacylglycerols supported the best cell growth, and olive oil was found to be the best inducer for lipase production (19.50 g/L and 58.50 U). The selected conditions for enzyme production were found with yeast extract as nitrogen source and 1.5 % (w/v) olive oil (85.70 U) that resulted in a good cell growth yield (Y_X/S?=?1.234 g/g) and lipase productivity (1.204 U/h) after 72 h of shake-flask cultivation. C. viswanathii lipase presented high hydrolytic activity on esters bonds of triacylglycerols of long-chain, and this strain can be considered an important candidate for future applications in chemical industries.     

OPTIMIZATION OF FERMENTATION MEDIUM FOR ACETOIN PRODUCTION BY Bacillus subtilis SF4-3 USING STATISTICAL METHODS

To improve the acetoin-producing ability of Bacillus subtilis SF4-3, isolated from “natto,” a Japanese traditional food, the fermentation medium was optimized in shake-flask fermentation by statistically designed methods. Based on results of the single-factor experiment, orthogonal experiment, and Plackett–Burman design, yeast extract, corn steep liquor, and urea were identified as showing significant influence on the acetoin production. Subsequently, the optimum combination of the three factors was investigated by the Box–Behnken design (BBD) of response surface methodology (RSM) in order to further enhance the acetoin production. The maximum acetoin yield of 45.4 g/L was predicted when the concentrations of yeast extract, corn steep liquor, and urea were 8.5 g/L, 14.6 g/L, and 3.8 g/L, respectively. The results were further confirmed in triplicate experiments using the optimized medium (glucose 160 g/L, yeast extract 8.5 g/L, corn steep liquor 14.6 g/L, urea 3.8 g/L, manganese sulfate 0.05 g/L, ferrous sulfate 0.05 g/L), and an acetoin yield of 46.2 g/L was obtained in the validation experiment, which was in agreement with the prediction. After the optimization of medium components, an increase of 36.28% in acetoin production was achieved in comparison to that at the initial medium levels.     

Enhancing Endo-nitrilase production by a newly isolated Arthrobacter nitroguajacolicus ZJUTB06-99 through optimization of culture medium

The medium components of nitrilase production by Arthrobacter nitroguajacolicus ZJUTB06-99 were optimized in this study. Effects of factors such as carbon sources, nitrogen sources, and inducers on nitrilase production were investigated. Glucose, yeast extract, and ε-caprolactam were chosen as the suitable components. Moreover, experiments were carried out to fix the concentration of three factors for the zero coded level of variables in the subsequent optimization. Response surface methodology (RSM) and central composite design (CCD) were employed for further optimization. A quadratic model was found to fit the nitrilase activity and the variables. The results revealed that the optimized medium contained (%, w/v) 2.80, glucose; 0.57, yeast extract; and 0.42, ε-caprolactam. Validation experiments were carried out under the optimized conditions and nitrilase activity of 107.49 U/L was close to the predicted activity 110.82 U/L. After optimization, the nitrilase activity attained 2.86 fold of activity compared to the unoptimized conditions and the conversion of acrylonitrile was significantly improved. The strain growth curve and nitrilase activity alteration in the course of culture were tested. The cells were suitably harvested after cultured for 72∼78 h.     

Production and partial purification of naringinase by Penicillium decumbens PTCC 5248

Penicillium decumbens PTCC 5248 produced naringinase when grown in a medium contained naringin as a source of carbon. Rhamnose also induced production of naringinase. Prunin disappeared as the time of enzymatic reaction increased. On fractionation with isopropanol 24-fold purification was achieved. Optimum pH and temperature for naringinase activity were determined to be 4.5 and 55 °C respectively. The K_m value of the enzyme with respect to naringin was found to be 1.7 mM. Citric acid, glucose, Ca²⁺, Mg²⁺, Zn²⁺ all inhibited naringinase activity.     

Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods

Polysaccharides and ganoderic acids (GAs) are the major bioactive constituents of Ganoderma species. However, the commercialization of their production was limited by low yield in the submerged culture of Ganoderma despite improvement made in recent years. In this work, twelve Ganoderma strains were screened to efficiently produce polysaccharides and GAs, and Ganoderma lucidum 5.26 (GL 5.26) that had been never reported in fermentation process was found to be most efficient among the tested stains. Then, the fermentation medium was optimized for GL 5.26 by statistical method. Firstly, glucose and yeast extract were found to be the optimum carbon source and nitrogen source according to the single-factor tests. Ferric sulfate was found to have significant effect on GL 5.26 biomass production according to the results of Plackett–Burman design. The concentrations of glucose, yeast extract and ferric sulfate were further optimized by response surface methodology. The optimum medium composition was 55 g/L of glucose, 14 g/L of yeast extract, 0.3 g/L of ferric acid, with other medium components unchanged. The optimized medium was testified in the 10-L bioreactor, and the production of biomass, IPS, total GAs and GA-T enhanced by 85, 27, 49 and 93 %, respectively, compared to the initial medium. The fermentation process was scaled up to 300-L bioreactor; it showed good IPS (3.6 g/L) and GAs (670 mg/L) production. The biomass was 23.9 g/L in 300-L bioreactor, which was the highest biomass production in pilot scale. According to this study, the strain GL 5.26 showed good fermentation property by optimizing the medium. It might be a candidate industrial strain by further process optimization and scale-up study.     

Production,Purification, and Antibiofilm Activity of a Novel Exopolysaccharide from Arthrobacter sp. B4

A novel exopolysaccharide (EPS), namely, B4-EPS, is produced by Arthrobacter sp. B4. Response surface methodology (RSM) was employed to optimize the fermentation medium for increasing B4-EPS production. Based on Plackett–Burman design (PBD), glucose, yeast extract, and KH₂PO₄ were selected as significant variables, which were further optimized by a central composite design (CCD). According to response surface and canonical analysis, the optimal medium was composed of 16.94 g/L glucose, 2.33 g/L yeast extract, and 5.32 g/L KH₂PO₄. Under this condition, the maximum yield of B4-EPS reached about 8.54 g/L after 72 hr of batch fermentation, which was pretty close to the predicted value (8.52 g/L). Furthermore, B4-EPS was refined by column chromatography. The main homogeneous fraction (B4-EPS1) was collected and applied to assay of antibiofilm activity. B4-EPS1 exhibited a dose-dependent inhibitory effect on biofilm formation of Pseudomonas aeruginosa PAO1 without antibacterial activity. About 86.1% of biofilm formation of P. aeruginosa PAO1 was inhibited in the presence of 50 µg/mL B4-EPS1, which was more effective than the peer published data. Moreover, B4-EPS1 could prevent biofilm formation of other strains. These data suggest B4-EPS may represent a promising strategy to combat bacterial biofilms in the future.     

Optimization,purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2°C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn²⁺ and strongly inhibited by Ba²⁺. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.     

Utilization of renewable agricultural residues for the production of extracellular halostable cellulase from newly isolated Halomonas sp. strain PS47

A newly isolated biopolymer-degrading halophilic bacterium, Halomonas sp. strain PS47, yielded higher cellulase activity (0.0076 U/ml) in mineral salt medium (MM63). Activity increased to 0.029 U/ml when carboxymethyl cellulose (0.5 % w/v) was used as carbon source and further to 0.138 U/ml when a combination of yeast extract and peptone was used as nitrogen source. Enzyme secretion was maximal during late exponential and stationary phases (0.15 U/ml, 48 h). Among different agro-residues (1 % w/v), wheat bran gave the highest activity (0.12 U/ml) at pH 7.5, 30 °C and 6 % (w/v) NaCl. The cellulase exhibited higher activity at pH 7.1 and 50 °C. The enzyme exhibited activity over a wide range of NaCl concentrations (0–4 M). Optimum activity was at 0–1 M NaCl. At 4 M NaCl, activity was reduced to 65 % of the initial value. The present investigation thus contributes to the limited information available on halostable cellulases.     

Isolation,screening, and selection of an L-glutaminase producer from soil and media optimization using a statistical approach

A culture isolated from garden soil was found to be a promising L-glutaminase producer. Biochemical identification tests and 16S rRNA sequencing identified this isolate to be Klebsiella oxytoca. Subsequently, media optimization using one-factor-at-a-time approach and response surface methodology was undertaken. A face centered central composite design was employed to investigate the interactive effects of four variables, viz. concentrations of maltose, yeast extract, beef extract, and ammonium acetate on glutaminase production. Almost all factors had significant interactive effects on glutaminase production. A medium containing (g/L): maltose, 23.31; yeast extract, 20.0; beef extract, 20.01; ammonium acetate, 10.0; mannitol, 10.0; KH₂PO₄, 0.4; Na₂SO₄, 0.4; and MgCl₂, 0.4 was optimum for glutaminase production. The applied methodology was validated using this optimized media and enzyme activity of 458.91 ± 9.49 U/L and specific activity of 0.441 ± 0.04 U/mg protein after 42 h of incubation at 33°C were obtained.     

OPTIMIZATION OF FERMENTATION CONDITIONS AND PURIFICATION OF CORDYCEPIN FROM Cordyceps militaris

The fermentation medium and conditions for the production of cordycepin were optimized in static culture using single-factor experiments, Placket–Burman design, a central composite design, and response surface methodology. Among seven variables including temperature, pH, and the concentrations of glucose, tryptone, yeast extract, KH₂PO₄, and MgSO₄ · 7H₂O, temperature and the concentrations of yeast extract and tryptone were found to be the important factors that significantly affected cordycepin production. The optimized medium consisted of yeast extract 9.00 g/L and tryptone 17.10 g/L, while the optimized culture conditions consisted of seed age 3 days, with an inoculum size of 10% and incubation temperature of 27.1°C. A maximum cordycepin yield of 7.35 g/L was achieved in a 5-L fermenter under the optimized conditions. Next, cordycepin was partially purified and determined. The resulting product showed 90.54% high-performance liquid chromatography (HPLC)–ultraviolet (UV) purity. Therefore, cordycepin was applied to a cell viability assay on SH-SY5Y cells and RM-1 cells. Cordycepin can inhibit the proliferation of RM-1 cells with IC₅₀ of 133 µmol/L, but it has no inhibitory effect on SH-SY5Y cells.

Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Inulinase production by a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the crude inulinase

Marine yeast strain 1, isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium. This marine yeast was identified as a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast worked optimally at pH 6.0 and 60°C. The optimal medium for inulinase production was seawater containing 4.0% (w/v) inulin and 0.5% (w/v) yeast extract, while the optimal cultivation conditions for inulinase production were pH 8.0, 28°C and 170 rpm. Under the optimal conditions, over 60 U ml⁻¹ of inulinase activity was produced within 48 h of fermentation in shake flasks. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase activity.     

Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.

Cholesterol oxidase production (COD) by a new isolate characterized as Streptomyces sp. was studied in different production media and fermentation conditions. Individual supplementation of 1 % maltose, lactose, sucrose, peptone, soybean meal and yeast extract enhanced COD production by 80–110 % in comparison to the basal production medium (2.4 U/ml). Supplementation of 0.05 % cholesterol (inducer) enhanced COD production by 150 %. COD was purified 14.3-fold and its molecular weight was found to be 62 kDa. V_max (21.93 μM/min mg) and substrate affinity K_m (101.3 μM) suggested high affinity of the COD for cholesterol. In presence of Ba²⁺ and Hg²⁺ the enzyme activity was inhibited while Cu²⁺ enhanced the activity nearly threefold. Relative activity of the enzyme was found maximum in triton X-100 whereas sodium dodecyl sulfate inactivated the enzyme. The enzyme activity was also inhibited by the thiol-reducing reagents like Dithiothreitol and β-mercaptoethanol. The COD showed moderate stability towards all organic solvents except acetone, benzene and chloroform. The activity increased in presence of isopropanol and ethanol. The enzyme was most active at pH 7 and 37 °C temperature. This organism is not reported to produce COD.