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.     

Optimization of culture conditions for production of cellulases and xylanases by Scytalidium thermophilum using Response Surface Methodology

Summary Scytalidium thermophilum type culture Humicola insolens MTCC 4520 isolated from composting soil was optimized for production of cellulolytic and hemicellulolytic enzymes (endoglucanase, Avicel-adsorbable endoglucanase, FPase, β-glucosidase, xylanase and mannanase) by solid-state fermentation (SSF). Initial experiments showed that culture medium containing rice straw and wheat bran (1:3) as carbon source prepared in a synthetic basal medium supported maximal enzyme production at 45 °C. Further optimization of enzyme production was carried out using Box-Behnken design of experiments to study the influence of process variables (inoculum level, (NH₄)₂SO₄ and pH) on enzyme production. The response surface plots revealed the conditions for obtaining optimal enzyme levels. The models computed for R ² value ranged between 95% and 98.7% indicating they are appropriate and can be useful to predict the effect of inoculum level, (NH₄)₂SO₄ and pH on enzyme production. Under optimized conditions 62.5 ± 0.50, 23.0 ± 0.58, 3.0 ± 0.50, 151.00 ± 8.194, 196 ± 5.033 and 4.9 ± 0.32 (units/g substrate) of endoglucanase (EG), Avicel-adsorbable endoglucanase (AAEG), FPase, β-glucosidase, xylanase and mannanase were produced, respectively. Isoelectric focusing (IEF) of the crude extract showed that S. thermophilum produced six different EG isoforms, of which the EG corresponding to pI values of 8.4, 7.9 and 6.5 showed affinity for Avicel, thereby indicating the presence of a cellulose-binding domain (CBD). Furthermore, seven isoforms of β-glucosidase and ten multiple forms of xylanase distributed over a wide range of pI were also detected.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

Biochemical and biotechnological studies on a novel purified bacillus cholesterol oxidase tolerant to solvent and thermal stress

A novel bacterial strain was isolated and identified as Bacillus pumilus, with the capability to produce cholesterol oxidase enzyme (55?kDa). The production of the enzyme was optimized via two-step statistical approach. Out of eight factors screened in Plackett–Burman, only four had significant effects on enzyme activity. The optimization process of these four variables by Box–Behnken revealed that the maximum enzyme activity (90?U/mL) was significantly obtained after 6 days of fermentation with 0.3%, 1% and 0.2% of NH₄NO₃, yeast extract and Tween 80, respectively. The purified enzyme showed optimum activity at pH 7.5 and temperature of 40?°C. The enzyme retained 100% of its activity after storage at 40?°C for 60?min. The enzyme also exhibited enhanced stability in the presence of Tween 80, methanol and isopropanol. This solvent and thermal stress tolerant enzyme, produced by B. pumilus, may provide a practical option for industrial and analytical applications.     

Thermostable lipoxygenase is a key enzyme in the conversion of linoleic acid to trihydroxy-octadecenoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Lipoxygenases (LOXs) constitute a family of lipid-peroxidizing enzymes that catalyze the oxidation of unsaturated fatty acid containing a (1Z,4Z)-pentadiene structural unit, leading to formation of conjugated (Z,E)-hydroperoxydienoic acid. LOXs are known to be widely distributed in plants and animals. Recently, several microbial LOXs were reported to be involved in the production of hydroperoxy fatty acids. Among the microorganisms that produce hydroxy fatty acids, Pseudomonas aeruginosa PR3 is known to convert linoleic acid to trihydroxy fatty acid, which suggests the involvement of a LOX enzyme. Based on these reports, we identified a novel thermostable LOX from P. aeruginosa PR3 strain. The protein was purified 34.3-fold with a recovery rate of 5.14%. The K_m and V_max values of the purified enzyme were 3.57 mM and 0.73 μmol/min//mg, respectively. Heat stability of the purified enzyme was unexpectedly high with an LD₅₀ of 90 min at 80°C, although P. aeruginosa PR3 is known as a mesophilic bacterium. Substrate specificity of the purified enzyme was restricted only to unsaturated fatty acids carrying a (1Z,4Z)-pentadiene unit.     

Extracellular β-glucosidase production by a marine Aspergillus sydowii BTMFS 55 under solid state fermentation using statistical experimental design

A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as Aspergillus sydowii BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of Aspergillus sydowii in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH₄)₂SO₄ precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ∼95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards pNPG and enzyme has a K _m and V _max of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a K _i of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of Saccharomyces cerevisiae in presence of cellulase and the purified β-glucosidase of Aspergilus sydowii BTMFS 55.     

Extracellular Production of l-Lysine α-Oxidase in Wheat Bran Culture of a Strain of Trichoderma viride

A mold strain Y244-2 capable of producing l-lysine α-oxidase, a new enzyme catalyzing the α-oxidative deamination of l-lysine, was identified as Trichoderma viride. Among strains belonging to the genus Trichoderma tested, only Trichoderma viride Y244-2 produced the enzyme in wheat bran culture. The maximum enzyme production by the mold grown on wheat bran was observed after 10 and 14 days incubation with and without NaN0₃, respectively. Addition of NaN0₃, NH₄N0₃, adenine, purine nucleosides, l-histidine, glycine or l-glutamine to wheat bran stimulated the production of the enzyme. In the liquid culture, the enzyme was produced extracellulary under the aerobic conditions, although the production was much lower than that in the wheat bran culture.     

Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

The filamentous fungus Paecylomices variotii was able to produce high levels of cell extract and extracellular invertases when grown under submerged fermentation (SbmF) and solid-state fermentation, using agroindustrial products or residues as substrates, mainly soy bran and wheat bran, at 40°C for 72 h and 96 h, respectively. Addition of glucose or fructose (≥1%; w/v) in SbmF inhibited enzyme production, while the addition of 1% (w/v) peptone as organic nitrogen source enhanced the production by 3.7-fold. However, 1% (w/v) (NH₄)₂HPO₄ inhibited enzyme production around 80%. The extracellular form was purified until electrophoretic homogeneity (10.5-fold with 33% recovery) by DEAE-Fractogel and Sephacryl S-200 chromatography. The enzyme is a monomer with molecular mass of 102 kDa estimated by SDS–PAGE with carbohydrate content of 53.6%. Optima of temperature and pH for both, extracellular and cell extract invertases, were 60°C and 4.0–4.5, respectively. Both invertases were stable for 1 h at 60°C with half-lives of 10 min at 70°C. Mg²⁺, Ba²⁺ and Mn²⁺ activated both extracellular and cell extract invertases from P. variotii. The kinetic parameters K_m and V_max for the purified extracellular enzyme corresponded to 2.5 mM and 481 U/mg prot⁻¹, respectively.     

Improved production and properties of β-glucosidase influenced by 2-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-glucose in the culture medium of <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

Increased production, secretion, and activity of β-glucosidase in the filamentous fungus Termitomyces clypeatus was achieved in presence of the glycosylation inhibitor 2-deoxy-d-glucose (0.05%, w/v) during submerged fermentation. Enzyme activity increased to 163 U/mL by adding mannose (2 mg/mL) to the medium. Such a high enzyme activity has not been achieved without mutation or genetic manipulation. The K_m and V_max of the enzyme in culture medium were determined to be 0.092 mM and 35.54 U/mg, respectively, with p-nitrophenyl β-d-glucopyranoside as substrate, confirming its high catalytic activity. The enzyme displayed optimum activity at pH 5.4 and 45°C. The enzyme was fairly stable between acidic to alkaline pH and retained about 75 ∼ 65% residual activities between pH 4 and 10.6 and demonstrated full activity at 45°C for 3 days. The enzyme was also stable in the presence of Zn²⁺ and Mg²⁺ and 80% of the residual activity was observed in the presence of Mn²⁺, Ca²⁺, K⁺, Cu²⁺, EDTA, and sodium azide. Around 70% of the activity was retained in the presence of 2 M guanidium HCl and 3 M urea, whereas the activity was 5 and 2 times higher in the presence of 4 mM beta-mercaptoethanol and 50 mM DTT, respectively. The enzyme obtained from the culture filtrate showed potential cellulose saccharifying ability which increased further when supplemented with commercial cellulase. Thus, this enzyme could be used without any additional downstream processing for commercial cellulase preparation and production of bioethanol or for other biotechnological applications.     

Effects and statistical optimization of fermentation conditions on growth and poly (vinyl alcohol)-degrading enzyme production of Streptomyces venezuelae GY1

The effects of environmental conditions, including temperature, pH and dissolved oxygen, on growth and production of polyvinyl alcohol (PVA)-degrading enzymes of the newly-isolated strain Streptomyces venezuelae GY1 were investigated. The medium composition for strain GY1 was studied first by single factorial design and then optimized using a central composite design. PVA with high saponification is better for growth of, and PVA-degrading enzyme production by S. venezuelae GY1 compared with PVA with low saponification, in contrast with the characteristics of other bacteria producing PVA-degrading enzymes. The optimal temperature and initial pH for production of PVA-degrading enzyme by strain GY1 was 30°C and 7.0, respectively. The optimal medium composition for PVA-degrading enzyme production is: 1.01 g L^?1 of PVA1799, 0.307 g L^?1 of NaNO₃ and 0.512 g L^?1 of MgSO₄?7H₂O.     

Optimization of culture conditions for glucose oxidase production by a Penicillium chrysogenum SRT 19 strain

The enzyme glucose oxidase (GOD) has been used for a variety of biotechnological applications in food and pharmaceutical industries. In this study, the optimization of extracellular GOD production was carried out in a Penicillium chrysogenum SRT 19 strain isolated from contaminated and decaying cheese samples. Maximum GOD production was attained at pH 6 and 20°C in fermentation broth after 72 h of incubation. The effects of metal ions and sugars were screened for the induction of higher GOD production. The results revealed that glucose and lactose give the highest production of enzyme (0.670 and 0.552 U/mL, respectively) as compared with other sugars (sucrose, cellulose, mannitol and fructose). Out of the seven metal ions studied, CaCO₃ (1.123 U/mL) and FeSO₄ (0.822 U/mL) act as modulators, while MgSO₄ (0.535 U/mL), CuSO₄ (0.498 U/mL), HgCl₂ (0.476 U/mL), ZnSO₄ (0.457 U/mL) and BaSO₄ (0.422 U/mL) yield lower production. The study therefore suggests that a strain of P. chrysogenum SRT 19 can be used as a new strain for GOD production.     

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH₄)₂SO₄ and soybean flour as nitrogen sources and KH₂PO₄ at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.     

Optimization of cultivation medium composition for isoamylase production

Summary The medium composition for production of isoamylase by Pseudomonas amyloderamosa JD210 was optimized using response surface methodology. The factors chosen for optimization were maltose, soybean protein hydrolysate (SPH), isoleucine, proline, KH₂PO₄ and MgSO₄. Fractional factorial designs (FFD) and the path of steepest ascent were effective in searching for the major factors and optimum medium composition. By a 2^6–1 FFD, supplementary isoleucine was shown to have a negative effect on enzyme production. The effects of the other five factors were further investigated by a central composite design and the optimum composition was found to be 1.10% maltose, 0.13% SPH, 0.15% proline, 0.38% KH₂PO₄, and 0.05% MgSO₄. When the strain was cultivated in the optimum medium, enzyme production increased 60% compared with ordinary medium. Proline was verified as being a significant factor in promoting enzyme production.     

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K _m and K _cat of the enzyme were 0.31 mM and 3.01 s⁻¹, respectively. Fe³⁺ could enhance the enzyme activity, whereas Ag⁺, Hg²⁺, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement of uric acid.     

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.     

Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2

Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO₄, 1.4% K₂HPO₄, 0.02% CaCl₂·2H₂O, 0.002% FeSO₄·7H₂O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F₂ fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70°C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90°C, the t_1/2 values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90°C, the enzyme retained its activity up to 8 h in the presence of 1mM MnCl₂. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The K _{m app} values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the V_max values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F₂ chitosanase at 24 h incubation (70°C) were pentasaccharide (GlcN)₅ and hexasaccharide (GlcN)₆. The prelimiaary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium. Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.     

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.     

Role of casein on induction and enhancement of production of a bacterial milk clotting protease from an indigenously isolated Bacillus subtilis

Aims: To isolate and enhance the yield of a bacterial milk clotting protease (MCP) through process optimization and scale up. Materials and Results: Bacillus subtilis was isolated as MCP producer with good milk clotting activity (MCA) per proteolytic activity (PA) index. The enzyme production was inducible with casein and enhanced with fructose and ammonium nitrate resulting in 571·43 U ml^?1 of enzyme. Conclusions: Medium containing 4% fructose, 0·75% casein, 0·3% NH₄NO₃ and 10 mmol l^–1 CaCl₂, pH 6·0, inoculated with 4% (v/v) inoculum, incubated at 37°C, 200 rev min^?1 for 72 h gave maximum production. A 6·67‐fold increase in MCP yield with very high MCA per PA index was observed after final optimization indicating similarity to rennets. Significance and Impact of the Study: Mostly fungal MCPs have been reported. The MCA and MCA per PA index of this bacterium is comparable to that of many fungal reports and better than quite a few bacterial MCPs. Thus, this enzyme by B. subtilis has good probability of successful use in cheese production.     

Nutritional status of Aureobasidium sp. ATCC 20524 for the production of β-fructofuranosidase

Sucrose at 10 to 20% (w/v) was the best carbon source for the production of -fructofuranosidase by Aureobasidium sp. ATCC 20524. At higher concentrations, it arrested growth. Glucose and fructose were also good carbon sources for the enzyme production. Yeast extract at 1.5 to 2% (w/v) was the best nitrogen source for the enzyme production and for cell growth. Addition of NaNO₃ (1 to 2%, w/v) and MgSO₄·7H₂O (0.5 to 1.5%, w/v) to the cultivation medium increased the intracellular enzymatic activity. The total enzymatic activity and cell growth reached 1.2×10⁴ U/flask and 2.5 g dry cell/flask, respectively after 48 h.Sachio Hayashi, Yoshihiko Shimokawa, Masaharu Nonoguchi, Yoshiyuki Takasaki and Kiyohisa Imada are with the Department of Industrial Chemistry, Faculty of Engineering, Miyazaki University, 1-1 Nishi, Gakuen Kibanadai, Miyazaki, 889-21 Japan. Hideo Ueno is with the Nippon Oligo Corporation, 588 Izumisawa, Jyohana-chyo, Tonami-gun, Toyama, 939-18, Japan.     

Comparison of β-glucosidase activities in different Debaryomyces strains

Summary β-Glucosidase production by Debaryomyces vanrigii and Debaryomyces hansenii was studied using two media. Cellobiose was found to stimulate the biosynthesis of the enzyme, while NH₄NO₃ (1.0 g/l) and NH₄Cl (1.26 g/l) were the best nitrogen sources for D. hansenii and D. vanrigii respectively. Optimal conditions for enzyme activity were established in relation to pH, temperature and enzyme stability. Thermal and pH stability studies show that β-glucosidase from D. vanrigii was more stable at pH 4.5–5.0 at 50°C, while that enzyme from D. hansenii was stable at pH 6.5 at 35°C. This feature may be advantageous in the commercial application by hydrolysing cellobiose, the potent inhibitor of cellulose solubilizing enzymes.