Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7

A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH₄)H₂PO₄ 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively. Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application.     

Production of Keratinase by Free and Immobilized Cells of <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Strain PPKS-2: Partial Characterization and Its Application in Feather Degradation and Dehairing of the Goat Skin

An extremely alkaliphilic bacterial strain, Bacillus sp. PPKS-2, was isolated from rice mill effluents and screened for the production of extracellular keratinase. The maximum production of keratinase occurred after 48 h in shaking culture at pH 11.0 and 37 °C in a medium containing 0.5% soybean flour. The strain grew and produced alkaline keratinase using chicken feather and horn meal as the sole source of carbon and nitrogen. An addition of 0.1% soybean flour or feather hydrolysate and sodium sulfite to feather medium increased the production and complete solubilization of feather took place within 5 days under solid-state fermentation conditions. The partially purified enzyme displayed maximum activity at pH 11.0 and 60 °C in a broad range of NaCl, 0–16%, and was not inhibited by sodium dodecyl sulfate (10%), ethylenediaminetetraacetic acid (10 mM), H₂O₂ (15%), and other commercial detergents. Immobilization of the whole cells proved to be useful for continuous production of keratinase and feather degradation. The enzyme was effectively used to remove hair from goat hide. The strain PPKS-2 can be effectively used for solid waste management of poultry feather in submerged as well as solid-state fermentation.     

Optimization of Influential Parameters for Extracellular Keratinase Production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (MTCC9102) in Solid State Fermentation Using Horn Meal—A Biowaste Management

A Bacillus subtilis (MTCC9102) isolate was shown to produce significant amount of keratinase under optimized conditions in solid-state fermentation using Horn meal as a substrate. Optimized value for moisture, inoculum, and aeration were found to be 100% (v/w), 50% (v/w), and 150% (w/w), respectively, and the optimum nitrogen source was peptone and carbon source was dextrose. Maximum keratinolytic activity was observed at 48 h after incubation, and the optimum age (24 h) of inoculum was significant. The influence of cultivation temperature and initial pH of the medium on keratinase production revealed the optimum values for the temperature and pH as 37 °C and 7, respectively. Maximum keratinase activity of the crude extract was 15,972 U/mg/ml. These results indicate that this bacterial strain shows a high biotechnological potential for keratinase production in solid-state fermentation, and use of the horn meal as the substrate can be implemented for keratinous solid wastes management.     

Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation

Inulinase is an enzyme relevant to fructose production by enzymatic hydrolysis of inulin. This enzyme is also applied in the production of fructo-oligosaccharides that may be used as a new food functional ingredient. Commercial inulinase is currently obtained using inulin as substrate, which is a relatively expensive raw material. In Brazil, the production of this enzyme using residues of sugarcane and corn industry (sugarcane bagasse, molasses, and corn steep liquor) is economically attractive, owing to the high amount and low cost of such residues. In this context, the aim of this work was the assessment of inulinase production by solid state fermentation using by Kluyveromyces marxianus NRRL Y-7571. The solid medium consisted of sugar cane bagasse supplemented with molasses and corn steep liquor. The production of inulinase was carried out using experimental design technique. The effect of temperature, moisture, and supplements content were investigated. The enzymatic activity reached a maximum of 445 units of inulinase per gram of dry substrate.     

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.     

Production and Characterization of an Alkaline Thermostable Crude Lipase from an Isolated Strain of <Emphasis Type="Italic">Bacillus cereus</Emphasis> C<Subscript>7</Subscript>

A bacterial strain isolated from spoiled coconut and identified as Bacillus cereus was found capable of producing alkaline thermostable extracellular lipase. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 60 °C, 10 min, and 8.0 respectively. Common surfactants except Triton X 100 and cetyltrimethylammonium bromide have no or very little inhibitory effects on enzyme activity. The enzyme was found to be stable in presence of oxidizing agents and protease enzyme. The maximum lipase production was achieved at 30–33 °C, pH 8.0 on 24 h of fermentation using 50 ml medium in a 250-ml Erlenmeyer flask. The superior carbon and nitrogen sources for lipase production were starch (2%) and ammonium sulfate (nitrogen level 21.2 mg/100 ml), peptone (nitrogen level 297 mg/100 ml), and urea (nitrogen level 46.62 mg/100 ml) in combination, respectively. The maximum enzyme activity obtained was 33 ± 0.567 IU/ml.     

Induction of a High-Yield Lovastatin Mutant of Aspergillus terreus by 12C6+ Heavy-Ion Beam Irradiation and the Influence of Culture Conditions on Lovastatin Production Under Submerged Fermentation

Heavy-ion beams, possessing a wide mutation spectrum and increased mutation frequency, have been used effectively as a breeding method. In this study, the heavy-ion beams generated by the Heavy-Ion Research Facility in Lanzhou were used to mutagenize Aspergillus terreus CA99 for screening high-yield lovastatin strains. Furthermore, the main growth conditions as well as the influences of carbon and nitrogen sources on the growth and the lovastatin production of the mutant and the original strains were investigated comparatively. The spores of A. terreus CA99 were irradiated by 15, 20, 25, and 30 Gy of 80 MeV/u (12)C(6+) heavy-ion beams. Based on the lovastatin contents in the fermentation broth, a strain designated as A. terreus Z15-7 has been selected from the clone irradiated by the heavy-ion beam. When compared with the original strain, the content of lovastatin in the fermentation broth of A. terreus Z15-7 increased 4-fold. Moreover, A. terreus Z15-7 efficiently used the carbon and nitrogen sources for the growth and production of lovastatin when compared to the original strain. The maximum yield of lovastatin, 916.7 μg/ml, was obtained as A. terreus Z15-7 was submerged cultured in the chemically defined medium supplemented with 3% glycerol as a carbon source, 1% corn meal as an organic nitrogen source, and 0.2% sodium nitrate as an inorganic nitrogen source at 30 °C in the shake flask. The result shows that heavy-ion beam irradiation is an effective method for the mutation breeding of lovastatin production of A. terreus.     

Gene Cloning,Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9

An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R,R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25 °C and pH 6.0, which indicate its extreme suitability for industrial applications. Zn²⁺, Fe²⁺, and Mg²⁺ stimulated the activity of the purified enzyme, whereas Co²⁺, Cu²⁺, and Ni²⁺ inhibited enzyme activity. The K _m and V _max values for inulin hydrolysis were 1.72 mM and 21.69 μmol min^?1 mg^?1 protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 μmol min^?1 mg^?1 protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.     

Preparation and preliminary characterization of exopolysaccharides by yeast Rhodotorula acheniorum MC

The effects of various carbon and nitrogen sources on the synthesis of exopolysaccharides by Rhodotorula acheniorum MC were studied. The dynamic viscosity of cell-free culture broths during exopolysaccharide synthesis were measured. The highest values for the viscosity (10.14 MPa.s) and crude polysaccharide productivity (6.6 g/L) were obtained in a medium supplied with 5% sucrose. Ammonium sulfate was the most favorable nitrogen source for exopolysaccharide synthesis. The value of pH played a determinant role, and the obligatory condition for exopolysaccharide production was low (pH 1.7-2.0) during the fermentation. The chemical composition and sugar constituents of the crude exopolysaccharides were determined. Mannose was the main monosaccharide component, and its concentration was the highest (69.13%) in the crude exopolysaccharide synthesized in the medium that included 5% sucrose as a carbon source.     

Fermentation Characteristics of <Emphasis Type="Italic">Mortierella alpina</Emphasis> in Response to Different Nitrogen Sources

The fermentation characteristics of Mortierella alpina were investigated in response to various nitrogen sources. Influences on nitrogen source and glucose uptake rate, mycelial morphology of M. alpina, and pH of medium in relation to different nitrogen sources were discussed. Effects of different nitrogen sources on cell growth, fatty acid composition, arachidonic acid (ARA), and total lipid concentration were also evaluated. It revealed that the maximum nitrogen source uptake ratio was obtained when corn steep liquor was used as nitrogen source. When yeast extract was used as the sole nitrogen source, glucose was completely exhausted at the end of fermentation. The maximum dry cell weight obtained from medium with yeast extract as nitrogen source had the highest total lipid concentration. Sodium nitrate was the favorable nitrogen source for ARA accumulation, and the highest ARA percentage in total fatty acids was obtained, 35.9%. Urea was identified as the favorable nitrogen source for ARA production, the highest ARA concentration obtained from urea was 5.8 g/l. Compared with inorganic nitrogen sources, organic nitrogen compounds are favorable for both cell growth and total lipids accumulation.     

Adding Value to the Oil Cake as a Waste from Oil Processing Industry: Production of Lipase and Protease by <Emphasis Type="Italic">Candida utilis</Emphasis> in Solid State Fermentation

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level–three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of ≈25 U g^-1 and a protease activity value of 110 U g^-1 were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.     

Difructose anhydride-forming bacterial inulinase II and fructogenic fungal inulinase I

The reserve polymeric inulin from dahlia tuber (>12% or >60% yield, wet or dry basis, respectively) follows as an attractive source for both free fructose or difructofuranose anhydride (DFA III). Although DFA biological activity is not completely understood, there is interest in characterizing other DFA III-producers besidesArthrobacter ureafaciens. The inulinolytic bacterial isolate named “YLW,” owing to the yellow hue in agar slants, is such a producer. Its biochemical characterization showed the presence of galactosylated and mannosylated glycolipids associated with the bacterial cells. Immobilization of fungal inulinase I and bacterial inulinase II, the respective enzymatic catalysts for the production of fructose and DFA III by inulin hydrolysis, was attempted using controlled-pore silica (CPS). The effects of pH, temperature, and incubation time was analyzed and compared for both enzymes in the free and immobilized forms.

     

Production of chitinolytic enzymes with Trichoderma longibrachiatum IMI 92027 in solid substrate fermentation

Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0–5.0), and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, α-amylase, and β-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55°C, respectively. The enzyme totally lost its activity at 70°C in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70°C after a 60-min incubation in the presence of solid substrate fermentation solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities between pI 5.9 and 4.8, where N-acetyl-β-d-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI.     

A stable lipase fromCandida lipolytica

Although Upases have been intensively studied, some aspects of enzyme production like substrate uptake, catabolite repression, and enzyme stability under long storage periods are seldom discussed in the literature. This work deals with the production of lipase by a new selected strain ofCandida lipolytica. Concerning nutrition, it was observed that inorganic nitrogen sources were not as effective as peptone, and that oleic acid or triacylglycerides (TAG) were essential carbon sources. Repression by glucose and stimulation by oleic acid and long chain TAG (triolein and olive oil) were observed. Extracellular lipase activity was only observed at high levels at late stationary phase, whereas intracellular lipase levels were constant and almost undetectable during the cultivation period, suggesting that the produced enzyme was attached to the cell wall, mainly at the beginning of cultivation. The crude lipase produced by this yeast strain shows the following optima conditions: pH 8.0–10.0, temperature of 55°C. Moreover, this preparation maintains its full activity for at least 370 d at 5°C.     

Optimization of inulinase production by Kluyveromyces marxianus using factorial design

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (2^5–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K₂HPO₄), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K₂HPO₄ had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K₂HPO₄. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.     

A statistical approach using L<Subscript>25</Subscript> orthogonal array method to study fermentative production of clavulanic acid by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> MTCC 1142

Clavulanic acid is a naturally occurring antibiotic produced by Streptomyces clavuligerus. The present work reports on clavulanic acid production by Streptomyces clavuligerus MTCC 1142 using one-factor-at-a-time and L₂₅ orthogonal array. The one-factor-at-a-time method was adopted to investigate the effect of media components (i.e., carbon source, nitrogen source and inoculum concentration) and environmental factors such as pH for clavulanic acid production. Production of clavulanic acid by Streptomyces clavuligerus was investigated using seven different carbon sources (viz. glucose, sucrose, modified starch, rice-bran oil, soybean oil, palm oil, and glycerol) and six different nitrogen sources (viz. peptone, yeast extract, ammonium chloride, ammonium carbonate, sodium nitrate and potassium nitrate). A maximum yield of 140 μg/mL clavulanic acid was obtained in the medium containing soybean oil as a carbon source and yeast extract as nitrogen source. Subsequently, the concentration of soybean flour, soybean oil, dextrin, yeast extract and K₂HPO₄ were optimized using L₂₅ orthogonal array method. The final optimized medium produced 500 μg/mL clavulanic acid at the end of 96 h as compared to 140 μg/mL before optimization. Synthesis of precursor molecules as a metabolic driving force is of considerable importance in antibiotic synthesis. Attempts to increase the clavulanic acid synthesis by manipulating the anaplerotic flux on C₃ and C₅ precursors by supplementing the medium with arginine, ornithine, proline, valine, leucine, isoleucine, pyruvic acid and á-ketoglutarate were successful. Supplementing the optimized medium with 0.1 M arginine and 0.1 M leucine increased the yield of clavulanic acid further to 1100 μg/mL and 1384 μg/mL respectively.     

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.     

Partial Characterization of Inulinases Obtained by Submerged and Solid-State Fermentation Using Agroindustrial Residues as Substrates: A Comparative Study

Inulinase belongs to an important class of enzymes as it can be used to produce high-fructose syrups by enzymatic hydrolysis of inulin and fructooligosaccharides, which has been used as functional food. This work aimed to carry out a partial characterization of the crude enzymatic extract of two different inulinases, obtained by solid-state fermentation (SSF) and submerged fermentation (SmF), using agroindustrial residues as substrates. The crude enzymatic extract obtained by SmF showed an optimal pH and temperature for hydrolytic activity of 4.5 and 55?°C, respectively; and that obtained by SSF conducted to optimal pH and temperature of 5.0 and 55?°C, respectively. Both enzymes presented high thermostability, with a D value of 230.4 h and 123.1 h for SmF and SSF, respectively. The inulinase produced by SmF showed highest stability at pH?4.4, while inulinase obtained by SSF was more stable at pH?4.8. The results showed that inulinase obtained by SmF is less susceptible to pH effect and the inulinase obtained by SSF is more resistant to higher temperatures.     

Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation

This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Xylanase Production by Bacillus circulans D1 Using Maltose as Carbon Source

Bacillus circulans D1 is a good producer of extracellular thermostable xylanase. Xylanase production in different carbon sources was evaluated and the enzyme synthesis was induced by various carbon sources. It was found that d-maltose is the best inducer of the enzyme synthesis (7.05 U/mg dry biomass at 48 h), while d-glucose and d-arabinose lead to the production of basal levels of xylanase. The crude enzyme solution is free of cellulases, even when the microorganism was cultivated in a medium with d-cellobiose. When oat spelt xylan was supplemented with d-glucose, the repressive effect of this sugar on xylanase production was observed at 24 h, only when used at 5.0 g/L, leading to a reduction of 60% on the enzyme production. On the other hand, when the xylan medium was supplemented with d-xylose (3.0 or 5.0 g/L), this effect was more evident (80 and 90% of reduction on the enzyme production, respectively). Unlike that observed in the xylan medium, glucose repressed xylanase production in the maltose medium, leading to a reduction of 55% on the enzyme production at 24 h of cultivation. Xylose, at 1.0 g/L, induced xylanase production on the maltose medium. On this medium, the repressive effect of xylose, at 3.0 or 5.0 g/L, was less expressive when compared to its effect on the xylan medium.