Statistical optimization,partial purification,and characterization of coffee pulp β-glucosidase and its application in ethanol production

Extracellular β-glucosidase was produced using coffee pulp as a sole carbon source by Penicillium verrucosum by solid state fermentation and 897.36±59 U/g enzyme activity was obtained. Increase in 2.21-fold of enzyme activity on optimizing the bioprocess parameters by response surface methodology based on central composite rotatable design is illustrated. Maximum production level of 1,991.17 U/g was obtained with optimum values of pH 4.2, moisture 66.8%, and fermentation duration of 56 h. The enzyme was partially purified and the enzyme activity was optimum at 50°C temperature and at pH 6. The metal ions such as Mg²⁺, Zn²⁺, Ca²⁺, K⁺, detergents, and chelator such as EDTA were effective and further increased the β-glucosidase activity. On application of β-glucosidase for simultaneous saccharifiation and fermentation, 3.3% ethanol was obtained. Thus, this study provides insight on exploitation of P. verrucosum for synthesis of of β-glucosidase using coffee pulp which is available abundantly in coffee processing industries.

     

Pectinesterase Activity and Proximate Analyses of Sea Buckthorn Juices

Three varieties of sea buckthorn fruit were harvested and pressed to obtain juices. These were analyzed for pectin methylesterase activity, moisture, nitrogen, oil, pH, total acid, and °Brix. Yields of press juice for Hippophae rhamnoides ssp. rhamnoides L. varieties Luchistaya, Prozrachnaya, and ssp. mongolica Rousi cv. Indian Summer were 68%, 69%, and 66% w/w, respectively. Differences in juice composition were found to be in moisture content, °Brix, pH, and in total acid with the Luchistaya variety having the highest acid levels and lowest pH. Pectin methylesterase (PME) was present in all sea buckthorn juices with initial activity levels of 6.8 to 14.0 μ equivalents per min per 100 g of juice at pH 8 and 23 °C. Activity was pH‐dependent with little PME activity at pH 3 to 5 and the highest activity at pH 8. The cream, pellet, and serum layers of centrifuged juices all contained PME. Heat treatment reduced PME activity in the juices by 1 decimal reduction or less.     

Bioconversion of shrimp shell waste for the production of antioxidant and chitosan used as fruit juice clarifier

The pH, proteolytic activity, extent of demineralisation and deprotenisation of shrimp waste were studied during 7 days of fermentation using Pseudomonas aeruginosa A2. After 3 days, pH dropped from 7.0 to 4.4 and then remained constant. Simultaneously, a demineralisation of 92% was achieved. However, protease activity reached its highest level (1230 U mL^?1) after 1 day of incubation, and a protein removal of 90% was achieved. Chitin obtained was converted to chitosan. This chitosan, with 73% deacetylation, was tested for clarification of different fruit juices. It was observed that low concentrations of chitosan (below to 1%) greatly increase the clarity of juices without affecting the nutritional value. The antioxidant activity of the hydrolysates produced during fermentation was tested. Hydrolysate obtained after 3 days showed the strongest scavenging activity (90%), which was comparable to the positive control BHA; however, that obtained after 1 day exhibited the highest ferric‐reducing antioxidant power (OD 700 nm = 1.7).     

Production and Application of Xylanase from Penicillium sp. Utilizing Coffee By-products

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K _m 5.6 mg/mL, and V _max 925 μmol mg⁻¹ min⁻¹ with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.     

Properties of an Alkaline‐Tolerant,Thermostable Xylanase from Streptomyces chartreusis L1105, Suitable for Xylooligosaccharide Production

Abstract: An extracellular xylanase was purified to homogeneity from a culture of Streptomyces chartreusis L1105 by a 2‐step method of ammonium sulfate precipitation and carboxymethyl sepharose fast‐flow chromatography (CMSFF). The xylanase was purified by 6.86‐fold, with a recovery yield of 31.96%. The purified xylanase appeared as a single protein band on SDS‐PAGE with a molecular mass of about 34.2 kDa. The optimum temperature and pH of the purified xylanase activity were 70 °C and 7.2 respectively. The xylanase was more stable under alkaline conditions and retained more than 80% activity after 30 min incubation at pH 6 to 10. It also showed specific activity towards different xylans. Hydrolysis of oat‐spelt and corn‐cob xylans by the xylanase yielded xylobiose and xylotriose as principle products without the formation of xylose. These properties indicate that the purified xylanase may potentially be useful in biotechnological applications, such as xylooligosaccharide preparation. This is the first report about the purification and characterization of a xylanase from S. chartreusis.     

Selectivity for water-unextractable arabinoxylan and inhibition sensitivity govern the strong bread improving potential of an acidophilic GH11 Aureobasidium pullulans xylanase

An acidophilic xylanase of Aureobasidium pullulans (XAPI) was recombinantly produced, characterised and its functionality in bread making compared with that of Bacillus subtilis Xyn A xylanase (XBS), an enzyme frequently used in bread making. Prominent characteristics of XAPI were its high specific activity towards arabinoxylan (AX), its relative preference for hydrolysis of water-unextractable AX (WU-AX), its optimal activity under acidic conditions and its sensitivity towards TAXI (Triticum aestivum xylanase inhibitor) and XIP (xylanase inhibiting protein). Optimally developed dough containing XAPI had a considerably drier feel after mixing and was less sticky after fermentation than dough treated with XBS. Both xylanases improved bread loaf volume by 24% under the conditions of the process. In spite of the higher dosage of XAPI necessary to obtain this result, the similar loaf volume increase coincided with less solubilisation of WU-AX and even lesser degradation of solubilised AX (S-AX) and water-extractable AX (WE-AX) by XAPI than by XBS during the bread making process. This is probably due to different dynamics and extent of xylanase inhibition in the dough matrix for both xylanases. AX solubilisation by the acidophilic XAPI was not boosted by the drop in pH in the dough during fermentation. Results show that significant bread loaf volume increase goes hand in hand with excellent dough properties when a limited solubilisation of WU-AX during mixing is coupled to retention of a high molecular mass of S-AX and WE-AX during the rest of the process.     

Purification and characterisation of β‐mannanase from Lactobacillus plantarum (M24) and its applications in some fruit juices

β‐Mannanase was purified 2619.05‐fold from the Lactobacillus plantarum (M24) bacterium by ammonium sulphate precipitation and ion exchange chromatography (DEAE‐Sephadex). The purified enzyme gave two protein bands at a level of approximately 36.4 and 55.3 kDa in the SDS‐PAGE. The purified mannanase enzyme has shown its maximum activity at 50 °C and pH 8, and it has been also determined that the enzyme was stable at 5–11 pH range and over 50 °C. The V_max and K_m values have been identified as 82 mg mannan mL^?1 and 0.178 mm , respectively. The effects of some metal ions such as Fe²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ on the mannanase enzyme have been also investigated, and it has been determined that all metal ions had significant effects on the activation of the mannanase enzyme. In addition, the effectiveness of the purified mannanase enzyme on the clarification of some fruit juices such as orange, apricot, grape and apple has been investigated. During the clarification processes, the enzyme was more effective than crude extracts on the clarification of the peach juice with a ratio of 223.1% at most.     

Effect of Storage on the Physico‐Chemical and Antioxidant Properties of Strawberry and Kiwi Leathers

Strawberry and kiwi leathers were used to develop a new healthy and preservative‐free fruit snack for new markets. Fruit puree was dehydrated at 60 °C for 20 h and subjected to accelerated storage. Soluble solids, titratable acidity, pH, water activity (a_w), total phenolic (TP), antioxidant activity (AOA) and capacity (ORAC), and color change (browning index) were measured in leathers, cooked, and fresh purees. An untrained panel was used to evaluate consumer acceptability. Soluble solids of fresh purees were 11.24 to 13.04 °Brix, whereas pH was 3.46 to 3.39. Leathers presented an a_w of 0.59 to 0.67, and a moisture content of 21 kg water/100 kg. BI decreased in both leathers over accelerated storage period. TP and AOA were higher (P ≤ 0.05) in strawberry formulations. ORAC decreased 57% in strawberry and 65% in kiwi leathers when compared to fruit puree. TP and AOA increased in strawberries during storage. Strawberry and Kiwi leathers may be a feasible new, natural, high antioxidant, and healthy snack for the Chilean and other world markets, such as Europe, particularly the strawberry leather, which was preferred by untrained panelists.     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

ISOLATION AND PROPERTIES OF PROTEASE FROM SACCHAROMYCES CARLSBERGENSIS

ABSTRACT Proteases from autolyzed Saccharomyces carlsbergensis were partially purified by ammonium sulfate precipitation and hydroxyapatite column chromatography. pH optima at pH 3.0 and 6.8 were observed. A 7.4-fold purification was achieved for the protease with neutral pH optimum. The general properties of this enzyme were studied. It displayed optimum activity and stability at 37°C and pH 6. It was inhibited by Fe⁺²and Fe⁺³, N-ethylmaleimide (NEM), phenylmethanesulfonyl fluoride (PMSF), and sodium chloride (NaCl). It was activated by β-mercaptoethanol and urea.     

INDUCTION, PROPERTIES AND APPLICATION OF XYLANASE ACTIVITY FROM SCLEROTINIA SCLEROTIORUM S2 FUNGUS

Extracellular xylanase activity was produced in a submerged culture by Sclerotinia sclerotiorum S2 fungus, on wheat bran as an inducing substrate. The enzyme was partially purified and biochemically characterized. The optimum pH and temperature for the activity were 5.5 and 65C, respectively. The xylanase was stable over a pH range of 4.0–9.0 and retained more than 75% of its activity after incubation for 24 h without substrate. The enzyme was stable at 50C and 60C for 80 min of incubation at pH 5.0, while the half‐life was 20 min at 70C. The novel xylanase activity was useful as an aid in orange juice clarification. The clarification was observed with concomitant production of reducing sugars (0.350 mg/mL from juice) after 24 h of incubation of juice with the xylanase. Xylanase aided juice clarification, which resulted in a 27% decrease in insoluble materials.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

Enzymatic Clarification of Fruit Juices by Fungal Pectin Lyase

The activity of pectic enzymes was measured in culture filtrates of four selected fungal strains. Pectin lyase (PL) activity was produced by all fungi, but pectinesterase activity was not detected in culture filtrates of the strain Penicillium expansum F16. The use of crude enzymes resulted in good clarification of apple juice as evaluated by an increase in transmittance at 660 nm. PL activities were partially purified by dye-affinity chromatography on Blue Sepharose. The enzyme was bound to the column and eluted in a single peak, free of PE activity, and not coinciding with the major amount of protein. Apple, grape, and passion fruit juices were effectively clarified by these partially purified enzymes. Methanol content was determined by gas-liquid chromatography, being undetectable in the clarified juices. These pectic enzymes demonstrate potential for use in fruit juice processing.     

Production of feed enzymes from citrus processing waste by solid‐state fermentation with Eupenicillium javanicum

Bingtang sweet orange processing waste was utilised to produce four feed enzymes (Endoglucanase, β‐glucosidase, pectinase and xylanase) by the solid‐state fermentation (SSF) with Eupenicillium javanicum. The factors related with SSF including moisture content, temperature, initial pH, time, carbon source (0.5 g), nitrogen sources (0.05 g), inorganic mineral salts (0.1 g) were investigated separately. The corresponding optimal condition was: moisture content 80% (w/w), temperature 30 °C, natural pH, time 96 h, wheat bran 0.5 g, (NH₄)₂SO₄ 0.05 g or NaNO₃ 0.05 g, CaCl₂ 0.1 g. The L₉(3⁴) orthogonal experiment results showed that the optimal condition for producing above multiple enzymes was: moisture content 80% (w/w), temperature 30 °C, wheat bran 1 g, (NH₄)₂SO₄ 0.05 g, NaNO₃ 0.05 g, CaCl₂ 0.1 g, fermentation time 96 h and natural pH. Under this condition, the average activity of Endoglucanase (CMCase), β‐glucosidase, pectinase and xylanase by E. javanicum could reach 46.80, 49.64, 51.87 and 106.42 U g^?1, respectively, which was significantly higher than those in single factor experiments. Our present results demonstrated that E. javanicum could also be an effective and useful fungus for multienzyme preparation especially for β‐glucosidase and xylanase from citrus processing wastes.     

Characterization of purified xylanase from finger millet (<Emphasis Type="Italic">Eleusine coracana-</Emphasis>Indaf 15) malt

Xylanase (E.C. 3.2.1.8) was purified to apparent homogeneity from 96 h finger millet (Eleusine coracana, Indaf-15) malt by a three step purification procedure via ammonium sulphate fractionation, DEAE-cellulose ion exchange and Sephadex G-75 gel permeation chromatographies with a recovery of 4.0% and fold purification of 60. Xylanase, having a molecular weight of 29 ± 2 kDa was found to be monomeric on SDS-PAGE. pH optimum of the enzyme was found to be in the range of 5.0–5.5. The activation energy was 25 kJmol⁻¹. Xylanase showed maximum stability at 35 °C in a pH range of 5.0–6.0. K _m and V _max of purified xylanase were found to be 0.2% and 4.5 μmol min⁻¹, respectively. Metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺, Fe²⁺, Ag²⁺ and Ni²⁺ enhanced xylanase activity at 5 mM concentration. p-chloromercuribenzoate, citric, oxalic and boric acids inhibited the enzyme in concentration dependent manner. The mode of action of xylanase was found to be “endo” as determined by the analysis of products liberated from larchwood xylan by ESI-MS and H¹NMR. In vitro studies using Bifidobacterium and Lactobacillus sp. confirmed the prebiotic activity of the xylo-oligosaccharides.     

High-Efficient Expression and Pilot Scale Fermentation of Streptomyces Xylanase from a Constitutive Pichia pastoris Vector

To develop an efficient Streptomyces xylanase-expressing system necessary for large-scale industrial production, a DNA fragment encoding the endo-β-l,4-xylanase (XYL1) from Streptomyces sp. S38 was cloned into an expression vector pGAPZαA to generate a recombinant plasmid pGAPZαA-XYL1. The plasmid pGAPZαA-XYL1 was transformed into Pichia pastoris X-33. The secreted XYL1 protein was confirmed to be completely consistent to sequence as reported previously (GenBank accession number X98518.1). The secreted XYL1 protein was purified using the Q-Sepharose FF chromatography and Sephadex G-25 resin, with an estimated purity of greater than 90%. We also found that in a 50 L fermentor, the average level and enzymatic activity of secreted xylanases were up to 1.15 ± 0.09 mg/mL and 1528 ± 59 IU/mL, respectively. The pH and temperature for optimal XYL1 expression were 6.0 and 55°C, respectively. The Km and Vmax values of XYL1 were 2.0 mg/mL and 5000 μmol min^?1 mg^?1, respectively. In addition, the XYL1 was found to have good thermal stability and wide pH adaptability, suggesting that it may be a useful candidate for various commercial applications. The high-efficient expression and pilot scale fermentation of XYL1 in this study, together with enzymatic studies, provide a strong basis for future large-scale industrial production.     

Stability and Activity of β-Galactosidase in Sonicated Cultures of Lactobacillus delbrueckii ssp. bulgaricus 11842 as Affected by Temperature and Ionic Environments

Stability of β‐galactosidase and related lactose hydrolyzing activity in sonicated cultures of Lactobacillus bulgaricus 11842 was investigated in the presence of Na⁺ or K⁺ ions. After sonication in Na⁺ or K⁺ buffers at various pH levels, cultures were held at various temperatures, before adding test lactose solutions. Hydrolysis was monitored by cryoscopy. Cultures sonicated in K⁺ buffer had higher activity and stability than those in Na⁺ buffer; both were highest at pH 6 and 7. Stability was unaffected at pH 6 and 7 below 56 °C. Holding at 61 °C for 60 min caused 70% activity loss with K⁺ ions at pH 6 and 7, while with Na⁺, activity loss was almost complete.     

Biochemical properties of type I sourdough affected by wheat bran dietary fibre during fermentation

The supplementation of dietary fibre in sourdough is beneficial to increase the nutritional quality of fermented foods. The influence of different wheat bran dietary fibre (WBDF) levels (0%, 3%, 6%, 9%, and 12%) on the biochemical characteristics of sourdough during fermentation was investigated. This study showed that WBDF was slightly resistant to the reduced pH caused by the initial fermentation of sourdough dough. After 6 h of fermentation, whether or not it contained WBDF, the dough’s amylase activity was relatively stable (P > 0.05). However, in the samples containing WBDF, it took longer for the amylase activity to increase from 200 to 300 U g⁻¹. The content of free amino acids in the dough showed a tendency to first decrease and then increase with the metabolic activity of microorganisms, and WBDF could accelerate this rate. This study also provided evidence that insoluble dietary fibre may stimulate the growth and reproduction of lactic acid bacteria.     

Purification of New β-Galactosidase from Enterococcus faecium MTCC 5153 with Transgalactosylation Activity

A new β-galactosidase (β-gal) was purified from a lactic acid bacterial strain of Enterococcus faecium MTCC5153 by chromatographic techniques. The purified enzyme had a specific activity of 24.06 U/mg of protein with k _m and V_max values of 2 mM and 18.2 mM/min/mg of protein, respectively. The yield of purified β-gal was 10.65% and estimated molecular weight found to be ～90 kDa, consisting of two homodimeric subunits of 43kDa. The enzyme was stable in pH range of 8.0–9.0 with an optimum pH of 8 and the optimum temperature of 40°C. The enzyme was activated in the presence of metal ions such as Mg⁺², Mn⁺², Ca⁺², K⁺ and Na⁺ and was inhibited by Zn⁺², Co⁺² and Cu⁺². Chemical modifiers (N-bromosuccinamide and Diethylpyro carbonate) inactivated the enzyme indicating the role of tryptophan and histidine moieties for activity. The purified β-gal was able to synthesize oligosaccharides from lactose. This study suggests that the β-gal of Enterococcus faecium MTCC5153 could be applied in dairy industry for hydrolysis of lactose and to improve its digestibility. β-gal of probiotic cultures are of particular interest due to their transgalactosylation properties.     

木聚糖酶对冷冻面团和馒头品质的影响

本实验研究了木聚糖酶对冻藏1d、7d、14d的冷冻面团和馒头品质特性的影响;并采用差示量热扫描仪(DSC)测定了冷冻面团的可冻结水(冰)含量,讨论了木聚糖酶改善冷冻面团品质的热力学机制。研究结果表明,随着冻藏时间的延长,面团的发酵力、酵母存活率、馒头比容、抗老化能力均呈现一定的下降趋势;在相同的冻藏时间下,木聚糖酶对面团的发酵特性和馒头品质改善作用明显;木聚糖酶含量为80mg.kg_-1时,可冻结水(冰)含量最低;添加木聚糖酶后,冰晶颗粒更加细小均匀。这说明适量的木聚糖酶能够有效地降解水不溶性阿拉伯木聚糖,使更多的水分保留在面筋网络结构中,从而抑制低温面筋网络结构和酵母细胞的破坏作用,改善馒头品质。