Biocatalytic potential of protease-resistant phytase of Aspergillus oryzae SBS50 in ameliorating food nutrition

Production of protease-resistant phytase by Aspergillus oryzae SBS50 was optimized in solid state fermentation using wheat bran as substrate. An integrated statistical optimization approach involving the Placket–Burman design followed by response surface methodology was employed. Among all the variables tested, incubation period, triton X-100, moisture ratio, and magnesium sulphate were identified as significant and further optimized using response surface methodology that resulted in 3.35-fold improvement in phytase production from 55.43 to 185.75 U/g dry mouldy bran (DMB). Optimal conditions for maximum phytase production (185.75 U/g DMB) included wheat bran 10 g per 250 ml flask moistened with 35 ml distilled water supplemented with 3.0% triton X-100, 0.04% magnesium sulphate, 1.0% sucrose and 0.5% yeast extract incubated at 30?°C for an incubation time of 48 h. Phytase titers were sustainable (179.55 to 185.75 U/g DMB), when the mould was grown in shake flasks of varied volumes and enamel-coated metallic trays under optimized conditions. Fermentation time was reduced to half from 96 h to 48 h after optimization resulting in a 6.7-fold enhancement in the phytase productivity from 577.39 to 3868.75 U/Kg/h and thus, reducing the cost of enzyme production. Phytase released inorganic phosphate, reducing sugars and soluble proteins from different food samples in a time dependent manner as a result of phytate hydrolysis.     

Isolation and characterization of Aspergillus sp. for the production of extracellular polysaccharides by response surface methodology

In this study, Aspergillus sp. was isolated for the production of extracellular polysaccharide. The process parameters were initially optimized by traditional methods. The cheap substrate, wheat bran was used for the production of extracellular polysaccharide in solid state fermentation. Supplementation of (1%, w/w) maltose, gelatin enhanced EPS production (5.36?mg/g). The salts such as, Cu²⁺ (4.9?mg/g), Ca²⁺ (3.5?mg/g), Zn²⁺ (2.9?mg/g), Mn²⁺ (3.4?mg/g) and Mg²⁺ (1.8?mg/g) stimulated EPS production. In two level full factorial experimental designs, the EPS yield varied from 3.18 to 11.65?mg/g wheat bran substrate with various combinations of the components supplemented with wheat bran substrate. Among these selected factors in central composite design, maltose significantly influenced on extracellular polysaccharide production.     

发酵麸皮多糖酶辅助提取工艺优化及其生物活性研究

通过响应面法优化提取发酵麸皮多糖的工艺,并评价其体外益生和抗氧化活性。以发酵麸皮多糖的得率为响应值,采用纤维素酶酶解与水浴浸提相结合的方法提取发酵麸皮多糖,以纤维素酶添加量、料液比、水浴浸提温度、水浴浸提时间为试验因素建立数学模型,筛选最佳提取工艺条件。通过测定还原力、DPPH和·OH自由基的清除能力对比发酵和未发酵麸皮多糖的体外抗氧化活性,并通过测定嗜酸乳杆菌、植物乳杆菌、两歧双歧杆菌的生长对比发酵和未发酵麸皮多糖的体外益生活性。结果表明,发酵麸皮多糖最佳提取工艺为:料液比1∶16(w/v),酶添加量1 000 U/g,水浴浸提温度90℃,水浴浸提时间60 min,在此条件下发酵麸皮多糖的得率实测值为73. 35%。发酵麸皮多糖具有较强的DPPH和·OH自由基的清除能力,可促进嗜酸乳杆菌、植物乳杆菌和两歧双歧杆菌的生长。     

Optimization and characterization of extracellular cellulase produced by Bacillus pumilus MGB05 isolated from midgut of muga silkworm (Antheraea assamensis Helfer)

Mature larvae of Antheraea assamensis were collected from different locations of Assam to isolate the cellulolytic gut microflora. Altogether sixty cellulase degrading bacteria were isolated on agar plates containing microcrystalline cellulose as the sole carbon source. Among them, ten isolates showed hydrolyzing zone on agar plates containing carboxy methyl cellulose (CMC) after staining with Congo-red. Isolate MGB05 exhibited the highest CMCase activity (0.262?U/mL) at 72?h of incubation under submerged condition. FPase and β-glucosidase activity were 0.012?U/mL and 3.71?U/mL respectively. It showed maximum FPase (0.022?U/mL) activity on the 3rd day of incubation in the media containing wheat bran as a carbon source. β-glucosidase production was also found to be highest with wheat bran (20.03?U/mL) at 48?h of incubation. The optimum pH and temperature of FPase activity of MGB05 were found at 6.0 and 50?°C respectively while for β-glucosidase activity, it was maximum at pH?6.0 under 50?°C. In addition, metal ion Mg⁺⁺ and Ca⁺⁺ enhanced FPase activity up to 110.92% (0.026?U/mL) and 105.31% (0.025?U/mL) respectively. In-vitro antimicrobial bioassay of the most potent cellulolytic bacteria (MGB05) also showed high antimicrobial activity against Escherichia coli (2.9?cm) and Pseudomonas aeruginosa (3.0?cm). The isolate MGB05 has been identified based on 16S rDNA homology as Bacillus pumilus MGB05 with accession KP298708.2. Results encompass the prospective beneficial role of gut-microflora on digestion and disease resistance, which might be a potential probiotic component to enhance silk productivity.     

Agricultural wastes as substrates for β-glucosidase production by Talaromyces thermophilus: Role of these enzymes in enhancing waste paper saccharification

In the present study, we investigated a potent extracellular β-glucosidases secreted by the thermophilic fungal strain AX4 of Talaromyces thermophilus, isolated from Tunisian soil samples. This strain was selected referring to the highest thermostability of its β-glucosidases compared to the other fungal isolates. The β-glucosidase production was investigated by submerged fermentation. The optimal temperature and initial pH for maximum β-glucosidase production were 50°C and 7.0, respectively. Several carbon sources were assayed for their effects on β-glucosidase production, significant yields were obtained in media containing lactose 1% (3.0?±?0.36?U/ml) and wheat bran 2% (4.0?±?0.4?U/ml). The combination of wheat bran at 2% and lactose at 0.8% as carbon source enhanced β-glucosidase production, which reached 8.5?±?0.28?U/ml. Furthermore, the β-glucosidase-rich enzymatic juice of T. thermophilus exhibited significant synergism with Trichoderma reesei (Rut C30) cellulases for pretreated waste paper (PWP) hydrolysis. Interestingly, the use of this optimal enzymatic cocktail increased 4.23 fold the glucose yield after saccharification of waste paper. A maximum sugar yield (94%) was reached when using low substrate (2%) and enzyme loading (EC1).     

Optimization of medium composition for alkali-stable xylanase production by Aspergillus fischeri Fxn 1 in solid-state fermentation using central composite rotary design

Response surface methodology and central composite rotary design (CCRD) was employed to optimize a fermentation medium for the production of alkali-stable cellulase-free xylanase by Aspergillus fischeri in solid-state fermentation at pH 9.0 with wheat bran as substrate. The four variables involved in this study were sodium nitrite, potassium dihydrogen phosphate, magnesium sulphate and yeast extract. The statistical analysis of the results showed that, in the range studied, only sodium nitrite had a significant effect on xylanase production. The optimized medium containing (in g/l) NaNO(2)-7.0, K2HPO(4)-1.0, MgSO(4)-0.5 and yeast extract-5.0 resulted in 1.9-fold increased level of alkali-stable xylanase (1024 U/g wheat bran) production compared to initial level (540 U/g) after 72 h of fermentation, whereas its value predicted by the quadratic model was 931 U/g. The level of protease activity was considerably decreased in optimized medium, thus helping to preserve the xylanase activity and demonstrating another advantage of applying statistical experimental design.     

Concomitant production of cellulase and xylanase by thermophilic mould <Emphasis Type="Italic">Sporotrichum thermophile</Emphasis> in solid state fermentation and their applicability in bread making

Sporotrichum thermophile BJAMDU5 secreted high titres of xylanolytic and cellulolytic enzymes in solid state fermentation using mixture of wheat straw and cotton oil cake (ratio 1:1) at 45?°C, pH 5.0 after 72 h inoculated with 2.9?×?10⁷ CFU/mL conidiospores. Supplementation of solid medium with lactose and ammonium sulphate further enhanced the production of hydrolytic enzymes. Among different surfactants studied, Tween 80 enhanced the production of all enzymes [3455 U/g DMR (dry mouldy residue), 879.26 U/g DMR, 976.28 U/g DMR and 35.10 U/g DMR for xylanase, CMCase (Carboxymethylcellulase), FPase (Filter paper activity) and β-glucosidase, respectively] as compared to other surfactants. Recycling of solid substrate reduced the production of all these enzymes after second cycle. End products analysis by TLC showed the ability of hydrolytic enzymes of S. thermophile to liberate monomeric (xylose and glucose) as well as oligomeric (xylobiose, cellobiose and higher ones) sugars. Supplementation of enzyme resulted in improved nutritional properties of the bread. Formation of oligomeric sugars by xylanase enzyme of S. thermophile BJAMDU5 make it a good candidate in food industry.     

Response surface methodology for the optimization of <Emphasis Type="Italic">α</Emphasis>-amylase production by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. ML12 using agricultural byproducts

Amylases constitute one of the most important groups of enzymes for commercial use. In the present study, production of α-amylase was optimized using a newly isolated actinobacterial strain from the coral reef environment of the Gulf of Mannar Biosphere Reserve, India. It was identified as Streptomyces sp. ML12 based on chemotaxonomy, cultural and morphological characteristics, carbon source utilization and 16S rRNA gene sequencing. Fermentation variables were selected in accordance with the Plackett-Burman design and were optimized by response surface methodology. Five significant variables (rice bran and wheat bran — both agricultural byproducts, sodium chloride, magnesium sulphate and incubation period) were selected for the optimization via central composite design. The optimal features were rice bran (5.5 g/100 mL), wheat bran (5.3 g/100 mL), sodium chloride (2.8 g/100 mL), magnesium sulphate (1.4 g/100 mL) and 8 days of incubation period. Optimization of the medium with the above tested features increased the amylase yield by 4.4-fold.     

Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.     

Production of tyrosinase by Auricularia auricula using low cost fermentation medium

Low cost fermentation media using agricultural by-products (wheat bran extract, rice bran extract and soybean meal extract) as a major nutrient source, were evaluated for the production of tyrosinase from the fungus Auricularia auricula in submerged culture. In single-factor experiments, three components (wheat bran extract, casein and CuSO₄) were chosen to further optimize medium composition using response surface methodology (RSM). The central composite experimental results showed the following optimum medium composition: wheat bran extract 36.0 %, casein 1.1 g/l and CuSO₄ 0.13 g/l. Under these conditions, the highest tyrosinase activity was 17.22 U/ml, which was 2.1 fold higher than that obtained using the non-optimized medium. The present study is the first to report the statistical optimization of medium composition for production of tyrosinase by A. auricula using cheaper wheat bran extract as a major nutrient source. These results might provide a reference for the development of a cost-effective medium for commercial production of tyrosinase.     

Statistical optimization of production conditions of alkaline pectin lyase from Bacillus cereus using response surface methodology

Alkaline pectin lyase finds applications in the degumming and retting of plant fibres, textile industry and pectic wastewater treatment where it degrades highly methylesterified pectin without prior action of any other pectinase. Response surface methodology (RSM) has been frequently utilized for the optimization of production process of industrially important enzymes from microbes. In the present work, fermentation conditions for the production of pectin lyase from Bacillus cereus were optimized using the factorial and central composite design of RSM. The cubic order polynomial regression model was found to be adequate and significant with a determination coefficient R² of 0.9505 (p?相似文献   

响应面法优化黑曲霉HDF05产β-葡萄糖苷酶过程参数

为获得黑曲霉Aspergillus niger HDF05菌株较高的β-葡萄糖苷酶酶活,对其发酵条件进行了优化。采用Plackett-Burman实验设计考察关键发酵操作参数对产酶的影响。继而采用最陡爬坡路径逼近最大响应区域,并结合中心组合实验和响应面对4个显著性因素进行分析。Plackett-Burman实验结果表明,发酵温度、装液量、麦麸和 (NH4)2SO4浓度对β-葡萄糖苷酶合成影响显著。通过响应面分析得到一元二阶方程,对方程求解得到优化的发酵过程参数：发酵温度为28 ℃,装液量为71.4 mL/250 mL,麸皮浓度为36 g/L,(NH4)2SO4浓度为5.5 g/L。采用该优化的过程参数,菌株的最大产β-葡萄糖苷酶活力可达60.06 U/mL,较优化前提高了23.9％。将黑曲霉HDF05产生的β-葡萄糖苷酶用于酸解玉米芯纤维残渣的酶解实验中,可明显降低纤维二糖的积累,48 h内可使玉米芯纤维素残渣酶解得率达到80.4％。     

Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2?U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2?U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65?°C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60?°C for almost 90?min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.     

Response surface methodology based optimization of keratinase from Bacillus velezensis strain ZBE1 and nanoparticle synthesis,biological and molecular characterization

The increasing demands of keratinases for biodegradation of recalcitrant keratinaceous waste like chicken feathers has lead to research on newer potential bacterial keratinases to produce high-value products with biological activities. The present study reports a novel keratinolytic bacterium Bacillus velezensis strain ZBE1 isolated from deep forest soil of Western Ghats of Karnataka, which possessed efficient feather keratin degradation capability and induced keratinase production. Production kinetics depicts maximum keratinase production (11.65 U/mL) on 4th day with protein concentration of 0.61 mg/mL. Effect of various physico-chemical factors such as, inoculum size, metal ions, carbon and nitrogen sources, pH and temperature influencing keratinase production were optimized and 3.74 folds enhancement was evidenced through response surface methodology. Silver (AgNP) and zinc oxide (ZnONP) nanoparticles with keratin hydrolysate produced from chicken feathers by the action of keratinase were synthesized and verified with UV–Visible spectroscopy that revealed biological activities like, antibacterial action against Bacillus cereus and Escherichia coli. AgNP and ZnONP also showed potential antioxidant activities through radical scavenging activities by ABTS and DPPH. AgNP and ZnONP revealed cytotoxic effect against MCF-7 breast cancer cell lines with IC₅₀ of 5.47 µg/ml and 62.26 µg/ml respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the thermostability, structure and surface attributes. The study suggests the prospective applications of keratinase to trigger the production of bioactive value-added products and significant application in nanotechnology in biomedicine.     

Enhancement of prodigiosin synthetase (PigC) production from recombinant Escherichia coli through optimization of induction strategy and media

PigC is a synthetase that catalyzes the condensation of 4-methoxy-2,2′-bipyrrole-5-carboxyaldehyde and 2-methyl-3-amylpyrrole to produce prodigiosin, which has a wide variety of impressive biological properties. In this study, we optimized PigC production from engineered Escherichia coli BL21(DE3). Investigation of different induction strategies revealed that autoinduction was the most appropriate method for PigC expression. As a result, PigC activity was elevated to 75.7?U/mL, nearly 2.1-fold higher than that with induction by isopropy-β-d-thiogalactoside. To achieve maximum enzyme production, the automedium components were optimized. “Single-factor experiments” showed that PigC production was greatly affected by the concentrations of glucose, yeast extract, and lactose. The Box–Behnken design for response surface methodology was then used to determine the optimal concentrations of these three components. According to a statistical approach, the optimum values of the three most influential parameters were 0.73?g/L glucose, 13.17?g/L yeast extract, and 5.86?g/L lactose. In the optimized automedium, the best PigC activity was obtained at 179.3?U/mL, which was 2.4-fold higher than using the initial medium. This study maximized PigC production as a foundation for further study and future industrial application.     

Immobilization of keratinolytic metalloprotease from Chryseobacterium sp. strain kr6 on glutaraldehyde-activated chitosan

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support (qm) and dissociation constant (Kd) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at 65 degrees C. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.     

High-level xylanase production by alkaliphilic Bacillus pumilus ASH under solid-state fermentation

Bacillus pumilus ASH produced a high level of an extracellular and thermostable xylanase enzyme when grown using solid-state fermentation (SSF). Among a few easily available lignocellulosics tested, wheat bran was found to be the best substrate (5,300 U/g of dry bacterial bran). Maximum xylanase production was achieved in 72 h (5,824 U/g). Higher xylanase activity was obtained when wheat bran was moistened with deionized water (6,378 U/g) at a substrate-to-moisture ratio of 1:2.5 (w/v). The optimum temperature for xylanase production was found to be 37°C. The inoculum level of 15% was found to be the most suitable for maximum xylanase production (7,087 U/g). Addition of peptone stimulated enzyme production followed by yeast extract and mustard oil cake, whereas glucose, xylose and malt extract greatly repressed the enzyme activity. Repression by glucose was concentration-dependent, repressing more than 60% of the maximum xylanase production at a concentration of 10% (w/v). Cultivation in large enamel trays yielded a xylanase titre that was slightly lower to that in flasks. The enzyme activity was slightly lower in SSF than in SmF but the ability of the organism to produce such a high level of xylanase at room temperature and with deionized water without addition of any mineral salts in SSF, could lead to substantial reduction in the overall cost of enzyme production. This is the first report on production of such a high level of xylanase under SSF conditions by bacteria.     

Two-step sequential optimization for production of ionic liquid stable cellulase from Bacillus subtilis I-2

The cost-effective bulk production of cellulases with desirable characteristics e.g., ionic liquid (IL)-stability, thermal, and pH stability is highly desirable. This study reports the optimization of cultural and environmental variables for enhanced production of an IL-stable, broad pH range, and thermo-stable cellulase from Bacillus subtilis I-2 employing low-cost agro-industrial wastes. Furthermore, combined interactive effects of different variables on enzyme yield were investigated using response surface methodology. The optimal levels of carbon and nitrogen sources were determined (% w/v, wheat bran 2.0, potato peel 1.5, cotton seed cake 0.8, and soybean meal 0.8) for enhanced cellulase yield. Further, optimization of environmental variables (temperature 48.41?°C, pH 7.0, and agitation rate 180 rpm) lead to overall 4.1-fold (76– 315.90 U/ml) enhancement of cellulase yield.     

Screening and selection of media components for lactic acid production using Plackett–Burman design

Plackett–Burman design was used to efficiently select important media components influencing lactic acid production in a two step screening procedure. A total of 36 screening experiments were conducted for studying the effect of various media components such as carbon and nitrogen (simple and complex) sources, minerals/buffering agents and a specific inducer for the production of lactic acid by Lactobacillus plantarum NCIM 2084. The eleven ingredients chosen after the first screening experiments were further screened by a Plackett-Burman design consisting of 12 experiments. Liquefied starch, wheat bran extract, ammonium nitrate, manganese sulphate and sodium acetate were chosen as promising ingredients for further optimisation studies. The highest yield of 41.9?g/l of lactic acid was obtained at the end of 24 hours of fermentation which corresponded to 90% conversion, on the basis of sugar supplied.     

Influence of Medium Composition on Xylitol Bioproduction from Wheat Straw Hemicellulosic Hydrolysate

Summary Xylose-to-xylitol batch bioconversions from wheat straw hemicellulosic hydrolysate were carried out in Erlenmeyer flasks in order to assess the influence of medium composition (hydrolysate concentration, supplementation with ammonium sulphate, calcium chloride and rice bran extract, and initial pH) on xylitol production, productivity and yield. By using the screening design and the response surface methodologies, the statistically significant variables influencing the bioconversion were selected and linear models were fitted to the experimental data. According to the results, the best conditions to perform the bioconversion consisted in using a threefold concentrated hydrolysate supplemented with ammonium sulphate (1.0 g/l) and rice bran extract (5.0 g/l), whose pH was adjusted to 6.0 prior to inoculation. Under these conditions, a xylitol production of 24.17 g/l was observed after 72 h of fermentation, resulting in a productivity of 0.34 g/l h and in a bioconversion yield of 0.49 g/g.