Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 × His tag was found to be 22.6 U mg^?1 of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 °C, respectively and the inulinase was stable in the pH range of 3–8 and in the temperature range of 0–50 °C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g^?1 of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10 h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L^?1 citric acid and 5.3 g L^?1 iso-citric acid from inulin while 68.9 g L^?1 of citric acid and 4.1 g L^?1 iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.     

Partial purification of extracellular exo-inulinase from Ulocladium atrum

Eight fungal species were cultivated on the Czapek liquid medium and a good starting extracellular and intracellular exo-inulinase were selected. Extracellular inulinase from Ulocladium atrum was prepared in the presence of 1% inulin source and 0.2% sodium nitrate as the best carbon and nitrogen sources. Incubation for the U. atrum was increased till it reached its maximum (36 U/ml) at the sixth day of incubation at 30 °C which was the best temperature for the production of exo-inulinase. Effect of all metal ions inhibited inulase production by U. atrum. Exo-inulinase was purified by using ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose. Three active inulinase forms INI, INII and INIII were resolved, each for DEAE cellulose. The specific activity of INI was 1915 U/mg protein which represented 2.65-fold purification over the crude extract with 42.8% recovery pooling of INI placed on CM cellulose chromatography and INI was resolved into INIa, INIb and INIc. The specific activity of INIa was 2479.2 U/mg protein which represented 3.43-fold purification over the crude extract with 24.2% recovery.     

Heterologous expression of a newly screened β-agarase from Alteromonas sp. GNUM1 in Escherichia coli and its application for agarose degradation

The gene of agaG1 from Alteromonas sp. GNUM1 encoding a β-agarase (AgaG1) was heterologously expressed in E. coli BL21 (DE3). The recombinant strain was cultured at 37 °C and then AgaG1 was expressed at 25 °C and 0.5 mM IPTG. The optimum conditions for AgaG1 to hydrolyze agarose were pH 7.0 and 40 °C. The main products of agarose hydrolysis by AgaG1 were confirmed to be neoagarobiose and neoagarotetraose. A new agarose hydrolysis process using AgaG1 was developed, in which the reaction temperature was adjusted stepwise to avoid gelation problem with no chemical pretreatment step. The enzyme AgaG1 was found to be very effective and highly selective. When 10.0 g/L agarose was hydrolyzed, 98% of the agarose added was converted to 3.8 and 6.4 g/L of neoagarobiose and neoagarotetraose, respectively.     

Purification of a laccase from fungus combs in the nest of Odontotermes formosanus

A new enzyme was isolated from the fungus combs in the nest of Odontotermes formosanus and identified as a laccase. The single laccase was purified with a purification factor of 16.83 by ammonium sulphate precipitation and anion exchange chromatography, to a specific activity of 211.11 U mg⁻¹. Its molecular mass was 65 kDa. The optimum pH value and temperature were 4.0 °C and 10 °C with ABTS as the substrate, respectively. The enzyme activity stabilized at temperatures between 10 °C and 30 °C and decreased rapidly when the temperature was above 30 °C. The V_max and K_m values were 3.62 μmol min⁻¹ mg⁻¹ and 119.52 μM, respectively. Ethanol concentration affected laccase activity, inhibiting 60% of enzyme activity at a concentration of 70%. Metal ions of Mg²⁺, Ba²⁺ and Fe²⁺ showed inhibition on enzyme activity of 17.2%, 5.3% and 9.4%, respectively, with the increase of metal ions concentration from 1 mM to 5 mM. Especially Fe²⁺ strongly inhibited enzyme activity up to 89% inhibition at a concentration of 1 mM.     

Cloning,expression and characterization of highly thermo- and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes, belong to glycoside hydrolase family 13. A gene corresponding to MA in Geobacillus caldoxylosilyticus TK4 (GcaTK4MA) was cloned into pET28a(+) vector and expressed in Escherichia coli with 6xHis-tag at the N-terminus. Herein, we report on the biochemical properties of a new thermo- and pH-stable MA. GcaTK4MA has similar properties those of other MAases in terms of the primary structure, preference for CD over starch and having an extra domain at its N- and C-terminals. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. The purified enzyme exhibited optimal activity for β-CD hydrolysis at 50 °C and pH 7.0. When the enzyme was separately incubated at 4 °C and 50 °C in the buffer solutions (pH 3.0–9.0) up to 7 days, it was seen that the enzyme had the higher stability at 50 °C than 4 °C. The enzyme retained about 80% of its original activity when it was incubated at 50 °C for 7 days. The enzyme activity was significantly inhibited by SDS and EDTA at the final concentration of 1%. These results suggest that this is the first reported MA having an extremely pH- and thermal stabilities.     

Biochemical characterization of a lichenase from Penicillium occitanis Pol6 and its potential application in the brewing industry

The purification and characterization of an extracellular lichenase from the fungus Penicillium occitanis Pol6 were studied. The strain produced the maximum level of extracellular lichenase (45 ± 5 U ml⁻¹) when grown in a medium containing oat flour (2%, w/v) at 30 °C for 7 days. The purified enzyme EG_L showed as a single protein band on SDS–PAGE with a molecular mass of 20 kDa. Its N-terminal sequence of 10 amino acid residues was determined as LDNGAPLLNV. The purified enzyme showed an optimum activity at pH 3.0 and 50–60 °C. The half-lives of EG_L at 60 °C and 70 °C were 80 min and 21 min, respectively. Substrate specificity studies revealed that the enzyme is a true β-1,3-1,4-d-glucanase. The enzyme hydrolyzed lichenan to yield trisaccharide, and tetrasaccharide as the main products. Under simulated mashing conditions, addition of EG_L (20 U/ml) or a commercial β-glucanase (20 U/ml) reduced the filtration time (25% and 21.3%, respectively) and viscosity (10% and 8.18%, respectively). These characteristics indicate that EG_L is a good candidate in the malting and brewing industry.     

Characterization of a thermophilic l-arabinose isomerase from Thermoanaerobacterium saccharolyticum NTOU1

l-Arabinose isomerase (EC 5.3.1.4, l-AI) mainly catalyzes the reversible aldose–ketose isomerization between l-arabinose and l-ribulose. l-AIs can also catalyze other reactions, such as the conversion of d-galactose to d-tagatose. In this study, the araA gene encoding l-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant l-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7–7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55–65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn²⁺ or 0.05 mM Co²⁺, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 l-AI has the lowest demand for metal ions among all characterized thermophilic l-AIs. This thermophilic l-AI shows a potential to be used in industry to produce d-tagatose from d-galactose.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

Biology and thermal requirements of Chrysoperla genanigra (Neuroptera: Chrysopidae) reared on Sitotroga cerealella (Lepidoptera: Gelechiidae) eggs

Chrysoperla genanigra Freitas is a common green lacewing associated with melon pests in the Northeastern Brazil. All life stages of this recently described species were studied under a range of constant temperature conditions (17, 21, 25, 29, 33, 35 and 37 °C), a photoperiod of 12 h:12 h (L:D) and 70 ± 10% relative humidity. Adults of C. genanigra were fed on a diet consisting of a 1:1 (v/v) mixture of brewer’s yeast and honey, while larvae were provided with eggs of Sitotroga cerealella (Olivier) ad libitum. The duration of preimaginal development of the species was inversely proportional to temperature and ranged from approximately 63 days at 17 °C to 15 days at 35 °C. The percentage of adult emergence varied from 6.7% at 17 °C to 76.7% at 25 °C, although no larvae were able to complete development at 37 °C. The lower thermal threshold for total preimaginal development was approximately 10.8 °C and the thermal requirement was 336.7 degree-days. Egg production, along with the longevity of both males and females, were significantly affected by temperature. It is concluded that the best temperature for rearing C. genanigra is 25 °C, with the lowest preimaginal mortality and the highest egg production (992.7 eggs/female).     

Cold storage of the egg parasitoids Trissolcus basalis (Wollaston) and Telenomus podisi Ashmead (Hymenoptera: Scelionidae)

Adults of Trissolcus basalis and Telenomus podisi were stored either at 15 or 18 °C after their immature development had been completed at 18 or 25 °C. Longevity of the parasitoids in the storage temperatures was evaluated, as well as fecundity and longevity following their return to 25 °C after different periods in reproductive diapause. Temperature during immature development influenced female longevity and highest mean longevity was obtained for females that developed to the adult stage at 25 °C and then were stored at 15 °C (ca. 13 months for T. basalis and 10 months for Te. podisi). For adults of T. basalis that developed at 25 °C, storage periods of 120 or 180 days at 15 or 18 °C did not affect fecundity. The fecundity of T. basalis females that developed at 18 °C and were stored for 120 days at 15 or 18 °C was not affected; however, after remaining for 180 days, fecundity was reduced in ca. 30 and 50%, respectively. Storage of Te. podisi adults at 15 or 18 °C significantly reduced fecundity. It is concluded that adults of T. basalis can be stored in the adult stage at 15 or 18 °C between two soybean crop seasons for mass production purposes, aiming the biological control of stink bugs.     

Comparison of the life tables and predation rates of Harmonia dimidiata (F.) (Coleoptera: Coccinellidae) fed on Aphis gossypii Glover (Hemiptera: Aphididae) at different temperatures

The life histories and predation rates of the ladybird beetle Harmonia dimidiata (F.) were compared among beetles kept at 15, 20, and 25 °C. The beetles were fed on Aphis gossypii Glover and were maintained at 70 ± 10% RH and a 14:10 (L:D) h photoperiod. According to the age-stage, two-sex life table, the net reproductive rates (R₀) were 147.4, 98.7, and 62.5 offspring for beetles kept at 15, 20, and 25 °C, respectively. Additionally, we employed both the jackknife and bootstrap techniques for estimating the means, variances, and standard errors of the population parameters. The sample means of R₀ and the other population parameters obtained using the bootstrap technique fit a normal distribution, but the jackknife technique generated biologically meaningless zero values for R₀. The net predation rates were 10963, 13050, and 7492 aphids for beetles kept at 15, 20, and 25 °C, respectively. For a comprehensive comparison of predation potential, we incorporated both the finite rate and the predation rate into the finite predation rate. When both the growth rate and the predation rate were considered, our results showed that H. dimidiata is a more efficient biological control agent for A. gossypii at 20 and 25 °C than at 15 °C.     

Characteristics of cold-adaptive endochitinase from Antarctic bacterium Sanguibacter antarcticus KOPRI 21702

The psychrotrophic Sanguibacter antarcticus KOPRI 21702^T, isolated from Antarctic seawater, produced a cold-adapted chitinolytic enzyme that is a new 55 kDa family 18 chitinase (Chi21702). Chi21702 exhibited high activities toward pNP-(GlcNAc)₂ and pNP-(GlcNAc)₃ with no activity for pNP-GlcNAc, indicating that it prefers chitin chains longer than dimers, just as endochitinases do. A mixture of GlcNAc and GlcNAc₂ was produced as a main product by Chi21702 activity from chitin oligosaccharides and swollen chitin, while less GlcNAc₃ was produced. These results show that Chi21702 has an endochitinase activity, randomly hydrolyzing chitin at internal sites. Chi21702 displayed chitinase activity at 0–40 °C (optimal temperature of 37 °C), maintained its activity at pH 4–11 (optimal pH of 7.6). Interestingly, Chi21702 exhibited relative activities of 40% and 60% at 0 and 10 °C, respectively, in comparison to 100% at 37 °C, which is higher than those of the previously characterized, cold-adapted, chitinases from bacterial strains.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.     

Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu²⁺, Mn²⁺, Mg²⁺, and Na⁺ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K_d values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.     

Characterization,purification, and temperature/pressure stability of polyphenol oxidase extracted from plums (Prunus domestica)

In this study, polyphenol oxidase (PPO) was extracted from Prunus domestica and partially purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and ion exchange chromatography. The final purification step revealed a 32.81-fold purification, and the molecular mass was estimated to be 65 kDa by SDS-PAGE. The purified PPO showed enzymatic activity mainly toward five substrates, namely catechol, catechin, 4-methyl catechol, chlorogenic acid, and L-3,4-dihydroxyphenylalanine, whereas it showed no activity toward caffeic acid, ferulic acid, p-coumaric acid, p-cresol, and l-tyrosine. The optimum pH and temperature values were 6.0 and 25 °C, respectively. The enzyme showed high stability in the pH range of 5.0–7.0 and in the temperature range of 25–65 °C. The most effective inhibitors of this enzyme were found to be ascorbic acid and l-cysteine. The thermal inactivation followed a first-order kinetic model, with activation energy of E_a 150.46 ± 1.29 kJ/mol. PPO extracted from plum showed stability at high pressure, with enzyme activation at 500 MPa.     

Purification and characterization of an extracellular cholesterol oxidase from a Bordetella species

A soil-isolated bacterium (strain B4) was identified as a species of Bordetella and deposited with the China General Microbiological Culture Collection (code, CGMCC 2229). The bacterium grew in a mineral medium, on cholesterol as a sole source of carbon and energy. Only one metabolite of cholesterol was accumulated in detectable amounts during the strain growth. It was identified as 4-cholesten-3-one. Cholesterol oxidase (COD) (EC 1.1.3.6), which catalyzes cholesterol into this metabolite, was evidenced from the strain. The conditions of the bacterium growth were optimized for extracellular enzyme production, which then reached around 1700 UL⁻¹ within 24 h culturing. The enzyme was purified from the spent medium of the strain to homogeneity on SDS-PAGE, and characterized. Its molecular mass, as estimated by this technique, was 55 kDa. COD showed an optimum activity at pH 7.0. It was completely stable at pH 5.0 and 4 °C for 48 h, and retained 80% at least of its initial activity at pH 4.0 or at a pH of 6.0–10.0. The optimum temperature for its reaction was 37 °C. The thermal stability of COD was appreciable, as 90% or 80% of its initial activity was recovered after 1 h or 2 h incubation at 50 °C. Ag⁺ or Hg⁺ at 1 mM, was inhibitor of COD activity, while Cu²⁺, at the same concentration, was activator. The COD K_m, determined at 37 °C and pH 7.0, was 0.556 mM. The enzyme was stable at pH 7.0 and 37 °C during 24 h mechanical shaking in the presence of 33% (v/v) of either of the solvents, dimethylsulfoxide, ethyl acetate, butanol, chloroform, benzene, xylene or cyclohexane.     

Immobilization of trypsin on polysaccharide film from Anacardium occidentale L. and its application as cutaneous dressing

Polysaccharide obtained from Anacardium occidentale L. gum was used for trypsin entrapment using cellulose (gaze) as a support and this preparation was applied as cutaneous wound healing. Trypsin release in vitro and the influence of pH and temperature on activity, stability and storage time of entrapped enzyme were evaluated. The preparation showed that it was still capable to release enzyme even after 48 h. Entrapped enzyme presented an optimal pH and temperature of 8.6 and 55 °C, respectively. Also, it was stable at high temperature (45 °C for 60 min) and wide range of pH, retaining 80% of its initial activity when stored for 28 days at 25 °C. Histopathological analysis of mice skin wound healing under the entrapped trypsin preparation treatment showed an acceleration of fibroblast proliferation, neovascularization of granulation tissue and stimulating effect on the epithelium formation compared to the skin wound under the treatment using preparations without trypsin. These results demonstrate that the trypsin–polysaccharide–cellulose preparation could be used in cutaneous dressing applications for wound healing.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.