Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation

This paper reports the production of a cellulase-free and alkali-stable xylanase in high titre from a newly isolated Bacillus pumilus SV-85S using cheap and easily available agro-residue wheat bran. Optimization of fermentation conditions enhanced the enzyme production to 2995.20 ± 200.00 IU/ml, which was 9.91-fold higher than the activity under unoptimized basal medium (302.2 IU/ml). Statistical optimization using response-surface methodology was employed to obtain a cumulative effect of peptone, yeast extract, and potassium nitrate (KNO₃) on enzyme production. A 2³ central composite design best optimized the nitrogen source at the 0 level for peptone and yeast extract and at the −α level for KNO₃, along with 5.38-fold increase in xylanase activity. Addition of 0.1% tween 80 to the medium increased production by 1.5-fold. Optimum pH for xylanase was 6.0. The enzyme was 100% stable over the pH range from 5 to 11 for 1 h at 37°C and it lost no activity, even after 3 h of incubation at pH 7, 8, and 9. Optimum temperature for the enzyme was 50°C, but the enzyme displayed 78% residual activity even at 65°C. The enzyme retained 50% activity after an incubation of 1 h at 60°C. Characteristics of B. pumilus SV-85S xylanase, including its cellulase-free nature, stability in alkali over a long duration, along with high-level production, are particularly suited to the paper and pulp industry.     

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.     

Enhanced production of an alkaline pectinase from Streptomyces sp. RCK-SC by whole-cell immobilization and solid-state cultivation

An alkalophilic Streptomyces sp. RCK-SC, which produced a thermostable alkaline pectinase, was isolated from soil samples. Pectinase production at 45 °C in shaking conditions (200 rev min⁻¹) was optimal (76,000 IU l⁻¹) when a combination of glucose (0.25% w/v) and citrus pectin (0.25% w/v) was added along with urea (0.25% w/v) in the basal medium devoid of yeast extract and peptone. All the tested amino acids and vitamins greatly induced pectinase production and increased the specific productivity of pectinase up to 550%. In an immobilized cell system containing polyurethane foam (PUF), the pectinase production was enhanced by 32% (101,000 IU l⁻¹) compared to shake flask cultures. In solid-state cultivation (SSC) conditions, using wheat bran as solid substrate, pectinase yield of 4857 IU g⁻¹ dry substrate was obtained at substrate-to-moisture ratio of 1:5 after 72 h of incubation. The partially purified pectinase was optimally active at 60 °C and retained 80% of its activity at 50 °C after 2 h of incubation. The half life of pectinase was 3 h at 70 °C. Pectinase was stable at alkaline pH ranging from 6.0 to 9.0 for more than 8 h at room temperature retaining more than 50% of its activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3

Streptomyces sp. QG-11-3, which produces a cellulase-free thermostable xylanase (96 IU ml⁻¹) and a pectinase (46 IU ml⁻¹), was isolated on Horikoshi medium supplemented with 1% w/v wheat bran. Carbon sources that favored xylanase production were rice bran (82 IU ml⁻¹) and birch-wood xylan (81 IU ml⁻¹); pectinase production was also stimulated by pectin and cotton seed cake (34 IU ml⁻¹ each). The partially purified xylanase and pectinase were optimally active at 60°C. Both enzymes were 100% stable at 50°C for more than 24 h. The half-lives of xylanase and pectinase at 70, 75 and 80°C were 90, 75 and 9 min, and 90, 53 and 7 min, respectively. The optimum pH values for xylanase and pectinase were 8.6 and 3.0, respectively, at 60°C. Xylanase and pectinase were stable over a broad pH range between 5.4 and 9.4 and 2.0 to 9.0, respectively, retaining more than 85% of their activity. Ca²⁺ stimulated the activity of both enzymes up to 7%, whereas Cd²⁺, Co²⁺, Cr³⁺, iodoacetic acid and iodoacetamide inhibited xylanase up to 35% and pectinase up to 63%; at 1 mM, Hg²⁺ inhibited both enzymes completely. Journal of Industrial Microbiology & Biotechnology (2000) 24, 396–402. Received 29 September 1999/ Accepted in revised form 02 February 2000     

Production of alkali-tolerant cellulase-free xylanase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. WLUN024 with wheat bran as the main substrate

An alkali-tolerant cellulase-free xylanase producer, WLI-11, was screened from soil samples collected from a pulp and paper mill in China. It was subsequently identified as a Pseudomonas sp. A mutant, WLUN024, was selected by consecutive mutagenesis by u.v. irradiation and NTG treatment using Pseudomonas sp. WLI-11 as parent strain. Pseudomonas sp. WLUN024 produced xylanase when grown on xylosidic materials, such as hemicellulose, xylan, xylose, and wheat bran. Effects of various nutritional factors on xylanase production by Pseudomonas sp. WLUN024 with wheat bran as the main substrate were investigated. A batch culture of Pseudomonas sp. WLUN024 was conducted under suitable fermentation conditions, where the maximum activity of xylanase reached 1245 U ml⁻¹ after incubating at 37 °C for 24 h. Xylanase produced by Pseudomonas sp. WLUN024 was purified and the molecular weight was estimated as 25.4 kDa. Primary studies on the characteristics of the purified xylanase revealed that this xylanase was alkali-tolerant (optimum pH 7.2–8.0) and cellulase-free. In addition, the xylanase was also capable of producing high quality xylo-oligosaccharides, which indicated its application potential in not only pulp bio-bleaching processes but also in the nutraceutical industry.     

Production of alkali tolerant cellulase free xylanase in high levels by Bacillus pumilus SV-205

The fermentation conditions were optimized for hyper production of xylanase from Bacillus pumilus SV-205. The bacterium secretes high levels (7382.7±1200 IU/mL) of cellulase-free xylanase using wheat bran led to 21.63 fold increase in activity. A combination of yeast extract and peptone stimulated highest xylanase production (2448.0 IU/mL) as compared to other combinations. The most important characteristic of the enzyme is its high pH stability (100%) over a broad pH range of 6-11 for 24h. Thermostability studies revealed that enzyme retained 65% activity after an incubation of 2h at 60°C. The level of production is remarkable as compared to earlier reports.     

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

Biobleaching of banana fibre pulp using <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C O1 xylanase produced from wheat bran under solid-state cultivation

A cellulase-free xylanase produced by Bacillus subtilis C 01 from wheat bran under solid-state cultivation was tested for its efficacy in biobleaching of raw banana fibre and banana pulp obtained through a mechanical pulping process. Banana pulp samples treated with crude xylanase (450 nkat g⁻¹ pulp) resulted in a 19.6% increase in the brightness as compared to untreated pulp. The presence of chromophores, hydrophobic compounds and an increased reducing sugar (10.79 mg g⁻¹ pulp) quantity in the bleached solution after enzymatic treatment indicated the removal of materials that were absorbed at 237 nm from the banana pulp.     

Studies on the production of nigerloxin using agro-industrial residues by solid-state fermentation

Nigerloxin, a new and potent lipoxygenase inhibitor, was discovered in our laboratory through solid-state fermentation of wheat bran by Aspergillus niger V. Teigh (MTCC-5166). The aim of this study is to investigate the possibility of using different agro-industrial residues as nutritional supplements along with wheat bran to enhance the production of nigerloxin. Nigerloxin produced by SSF was quantified spectrophotometrically at 292 nm. The results indicate that the inhibitor production was influenced by the type of solid substrate supplemented, moisture content, pH and size of the inoculum. Individually optimized supplements were tested in different combinations to determine their effects on nigerloxin production. A twofold increase in the production of nigerloxin (4.9 ± 0.3 mg gds⁻¹) was achieved by supplementing wheat bran with 10% w/w sweet lemon peel and 5% v/w methanol at optimized process parameters, that is, an initial moisture content of 65% v/w and incubation period of 6 days with an initial inoculum size of 2 ml (8 × 10⁵ spores gds⁻¹). Nigerloxin production was stable between pH of 4 and 5.     

Production of xylanolytic enzymes by <Emphasis Type="Italic">Aspergillus terricola</Emphasis> in stirred tank and airlift tower loop bioreactors

Fungi producing high xylanase levels have attracted considerable attention because of their potential industrial applications. Batch cultivations of Aspergillus terricola fungus were evaluated in stirred tank and airlift bioreactors, by using wheat bran particles suspended in the cultivation medium as substrate for xylanase and β-xylosidase production. In the stirred tank bioreactor, in physical conditions of 30°C, 300 rpm, and aeration of 1 vvm (1 l min⁻¹), with direct inoculation of fungal spores, 7,475 U l⁻¹ xylanase was obtained after 36 h of operation, remaining constant after 24 h. In the absence of air injection in the stirred tank reactor, limited xylanase production was observed (final concentration 740 U l⁻¹). When the fermentation process was realized in the airlift bioreactor, xylanase production was higher than that observed in the stirred tank bioreactor, being 9,265 U l⁻¹ at 0.07 vvm (0.4 l min⁻¹) and 12,845 U l⁻¹ at 0.17 vvm (1 l min⁻¹) aeration rate.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Highly thermo–halo–alkali-stable β-1,4-endoxylanase from a novel polyextremophilic strain of Bacillus halodurans

A novel bacterial isolate, capable of producing extracellular highly thermostable, halo-alkali-stable and cellulase-free xylanase, was isolated from soil and identified as Bacillus halodurans TSPV1 by polyphasic approach. The Plackett–Burman design identified wheat bran, lactose, tryptone and NaCl as the factors that significantly affect xylanase production, and thus, these were optimized by response surface methodology. The data analysis suggested that optimum levels of wheat bran (15–20 g L^?1), lactose (1.0–1.5 g L^?1), tryptone (2–2.5 g L^?1) and NaCl (7.0–8.0 g L^?1) support 6.75-fold higher xylanase production than that in the un-optimized medium. The xylanase is optimally active at 90 °C and pH 10, and stable for 4 h at 90 °C (T _1/2 60 h) over a broad range of NaCl concentrations (0–29 %). This is the first report on the isolation of polyextremophilic B. halodurans strain that produces thermo-halo-alkali-stable xylanase in submerged fermentation. This enzyme efficiently saccharifies agro residues like wheat bran and corncobs. Fifty-six percent of hemicellulose of wheat bran could be hydrolyzed by xylanase (100 U g^?1 substrate) along with cellulase (22 U FPase and 50 U CMCase g^?1). The xylanase, being thermo-alkali stable and cellulase free, can find applications in pre-bleaching of paper pulps and hydrolysis of xylan in agricultural residues.     

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.     

Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues

The production of an alkali-stable xylanase, with dual pH optima, from haloalkalophilic Staphylococcus sp. SG-13 has been enhanced using agro-residues in submerged fermentation and a biphasic growth system. The agro-residues such as wheat bran, sugarcane bagasse, corncobs and poplar wood when used as sole carbon source, improved the xylanase yield by five-fold as compared to xylose and xylan. Staphylococcus sp. SG-13 also produced equally good amounts of xylanase when grown simply in deionized water (pH 8.0) supplemented with agro-residues as sole carbon source. In the biphasic growth system (lower layer containing agricultural residue set in agar medium with liquid medium above it), the prime substrate, wheat bran (1% w/v), resulted in maximum xylanase production of 4525 U l^–1 (pH 7.5) and 4540 U l^–1 (pH 9.2) at an agar: broth ratio of 4.0 after 48 h of incubation at 37 °C under static conditions. In general, the cost-effective agro-residues were found to be more suitable inducers for xylanase production over expensive substrates like xylan.     

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.     

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.     

Statistical optimization for tannase production from <Emphasis Type="Italic">Aspergillus niger</Emphasis> under submerged fermentation

Statistically based experimental design was employed for the optimization of fermentation conditions for maximum production of enzyme tannase from Aspergillus niger. Central composite rotatable design (CCRD) falling under response surface methodology (RSM) was used. Based on the results of ‘one-at-a-time’ approach in submerged fermentation, the most influencing factors for tannase production from A. niger were concentrations of tannic acid and sodium nitrate, agitation rate and incubation period. Hence, to achieve the maximum yield of tannase, interaction of these factors was studied at optimum production pH of 5.0 by RSM. The optimum values of parameters obtained through RSM were 5% tannic acid, 0.8% sodium nitrate, 5.0 pH, 5 × 10⁷ spores/50mL inoculum density, 150 rpm agitation and incubation period of 48 h which resulted in production of 19.7 UmL⁻¹ of the enzyme. This activity was almost double as compared to the amount obtained by ‘one-at-a-time’ approach (9.8 UmL⁻¹).     

Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation

A study was taken up to evaluate the role of some fermentation parameters like inoculum concentration, temperature, incubation period and agitation time on ethanol production from kinnow waste and banana peels by simultaneous saccharification and fermentation using cellulase and co-culture of Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC 1077. Steam pretreated kinnow waste and banana peels were used as substrate for ethanol production in the ratio 4:6 (kinnow waste: banana peels). Temperature of 30°C, inoculum size of S. cerevisiae G 6% and (v/v) Pachysolen tannophilus MTCC 1077 4% (v/v), incubation period of 48 h and agitation for the first 24 h were found to be best for ethanol production using the combination of two wastes. The pretreated steam exploded biomass after enzymatic saccharification containing 63 gL⁻¹ reducing sugars was fermented with both hexose and pentose fermenting yeast strains under optimized conditions resulting in ethanol production, yield and fermentation efficiency of 26.84 gL⁻¹, 0.426 gg ⁻¹ and 83.52 % respectively. This study could establish the effective utilization of kinnow waste and banana peels for bioethanol production using optimized fermentation parameters.     

Strain improvement of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Rut C-30 for increased cellulase production

The strain of Trichoderma reesei Rut C-30 was subjected to mutation after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) for 6 h followed by UV irradiation for 15 min. Successive mutants showed enhanced cellulase production, clear hydrolysis zone and rapid growth on Avicel-containing plate. Particularly, the mutant NU-6 showed approximately two-fold increases in activity of both FPA and CMCase in shake flask culture when grown on basal medium containing peptone (1%) and wheat bran (1%). The enzyme production was further optimized using eight different media. When a mixture of lactose and yeast cream was used as cellulase inducer, the mutant NU-6 yielded the highest enzyme and cell production with a FPase activity of 6.2 U ml⁻¹, a CMCase activity of 54.2 U ml⁻¹, a β-glucosidase activity of 0.39 U ml⁻¹, and a fungal biomass of 12.6 mg ml⁻¹. It deserved noting that the mutant NU-6 also secreted large amounts of xylanases (291.3 U ml⁻¹). These results suggested that NU-6 should be an attractive producer for both cellulose and xylanase production.     

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315?±?16 IU/mL acidic xylanase, 290?±?20 IU/mL alkaline xylanase, and 88?±?9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10?mM MgSO₄, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60?hr at 200?rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3?hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6?hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.