Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.     

Aminopeptidase from jumbo squid (Dosidicus gigas) hepatopancreas: purification,characterisation, and casein hydrolysis

An aminopeptidase (AP) was partially purified from jumbo squid (Dosidicus gigas) hepatopancreas with 154.24‐fold and yield of 6.15%. The purification procedure consisted of ammonium sulphate fractionation and DEAE‐Sephacel chromatography. The enzyme was approximately 48–53 kDa as estimated by SDS‐PAGE. With l ‐leu‐p‐NA, it had optimum activity at pH 8.0 and 30 °C. The K_m and V_max/K_m values of the enzymes for l ‐leu‐p‐NA were 0.326 mm and 2787 at 37 °C, respectively. Activation energy (E_a) of the enzyme was 53.50 kJ M^?1.The AP showed activity against seven synthetic substrates: l ‐proline>l ‐methionine>Ac. l ‐γ‐glutamic>l ‐glycine>l ‐leucine>l ‐alanine>l ‐lysine‐p‐NA. The enzyme was strongly inhibited by Bestatin, partially inhibited by a metal‐chelating agent and by PCMB, a cystein protease inhibitor. Zn²⁺ and (or) Ca²⁺ seemed to be its metal cofactor(s). Incubation of casein with the partially purified AP resulted in a degree of hydrolysis of 6%.     

Partial characterization of β‐ d‐xylosidase from wheat malts

Arabinoxylans (AXs) from wheat malts potentially affect beer quality and production. β‐ d ‐Xylosidase is a key enzyme that degrades the main chains of AXs to produce xylose. This study performed a partial characterization of β‐ d ‐xylosidase from wheat malts. The optimal temperature was 70 °C and the enzyme exhibited excellent thermostability, that is, residual activities were 92.6% at 60 °C for 1 h. The enzyme was stable over a pH range of 3.0–6.0 and showed optimum activity at pH 3.5 and 4.5. Kinetic parameters K_m and V_max of wheat malt β‐ d ‐xylosidase against p‐nitrophenyl‐xyloside were 1.74 mmol L⁻¹ and 0.76 m m min⁻¹, respectively. The enzyme activity was severely inhibited by Cu²⁺, moderately inhibited by Mn²⁺, Mg²⁺, Al³⁺, Ca²⁺, Ba²⁺ and Na⁺ and mildly inhibited by Fe³⁺ and Fe²⁺. The partial enzymatic characterization achieved in this study can be used as a theoretical basis for purifying β‐ d ‐xylosidase from wheat malts. Copyright © 2015 The Institute of Brewing & Distilling     

EFFECT OF TWO POLAR ORGANIC-AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE-FUNCTION RELATIONSHIPS OF PROTEASES II. CHYMOSIN AND MUCOR MIEHEI PROTEINASE

The kinetics of chymosin and Mucor miehei proteinase (Mmp) catalyzed proteolysis of sodium (Na) caseinate, as well as their intrinsic structural properties, were examined in two polar organic-aqueous solvent systems. Chymosin and Mmp in standard buffer (10 mM phosphate, pH 6.0), 5% (v/v) ethanol (EtOH) in standard buffer and 5% (v/v) acetonitrile (ACN) in standard buffer were used in the investigation. Kinetic parameters for the hydrolysis of Na-caseinate by chymosin showed that, relative to standard buffer, the K_m in both 5% EtOH in standard buffer and 5% ACN in standard buffer increased significantly (p ≤ 0.05) to nearly the same level from 4.8 mg/ml to 7.5 mg/ml and 7.7 mg/ml, respectively. No significant changes (p > 0.05) were observed in V_max in 5% EtOH (12.8 μmoles/min.mg) or 5% ACN (11.8 μmoles/min.mg) compared with standard buffer (12.8 μmoles/min.mg) resulting in V_max/K_m values reduced to a similar extent in both organic solvents. For Mmp, a glycoprotein, K_m increased (p ≤ 0.05) in 5% ACN in standard buffer (5.3 mg/ml, yet it decreased (p ≤ 0.05) in 5% EtOH in standard buffer (3.1 mg/ml) compared with standard buffer (4.2 mg/ml). The solvent-induced decrease in V_max for Mmp was somewhat larger in 5% EtOH in standard buffer than 5% ACN in standard buffer, from 17.3 μmoles/min.mg (standard buffer) to 14.0 μmoles/min.mg (p ≤ 0.05) in 5% EtOH in standard buffer but was not significantly different (p > 0.05) in 5% ACN in standard buffer (15.7 μmoles/min.mg). For both chymosin and Mmp, changes in kinetic parameters appeared to correspond with solvent-induced structural changes as evidenced by near-UV circular dichroism (CD) spectroscopy; higher K_m corresponded to a higher ellipticity in the near-UV CD spectra (240–320 nm), which may indicate a decreased protein flexibility. The fact that chymosin and Mmp behaved differently in these organic solvents indicates that factors other than polarity of the media may also have been involved in this phenomenon since both 5% EtOH in standard buffer and 5% ACN in standard buffer solutions were of the same polarity based on E_T(30) scale. Different specificities for Na-caseinate hydrolysis between chymosin and Mmp, as well as altered specificities of both enzymes for the substrate caused by organic solvents, were demonstrated in SDS-PAGE peptide maps as differences in banding patterns. Differential scanning calorimetric studies on chymosin and Mmp in the two solvent systems showed destabilization (lowered temperature of denaturation) of the enzymes in both 5% EtOH in standard buffer and 5% ACN in standard buffer relative to standard buffer.     

α‐l‐Rhamnosidase from Aspergillus flavus MTCC‐9606 isolated from lemon fruit peel

An α‐l ‐rhamnosidase producing fungal strain has been isolated from decaying lemon fruit. The fungal strain has been identified as Aspergillus flavus. The α‐l ‐rhamnosidase has been purified from the culture filtrate of the fungal strain using ultra filtration and cation exchange chromatography on carboxy methyl (CM) cellulose. The molecular mass of the purified enzyme determined by SDS–PAGE analysis was 41 kDa. The K_m values of the enzyme using p‐nitrophenyl‐α‐l ‐rhamnopyranoside and naringin as the substrates were 1.89 and 1.6 mm respectively. The pH and temperature optima of the enzyme were 11.0 and 50 °C respectively. The effects of various chemical species present in grape fruit juice and wine on the activity of the enzyme have been determined.     

Multiple forms of β‐galactosidase from mango (Mangifera indica L Alphonso) fruit pulp

Mango (Mangifera indica L cv Alphonso) was found to contain three isoforms (I, II and III) of β‐galactosidase which, upon purification on Sephadex G‐200, had relative abundances of 44, 38 and 18%, respectively. The total specific activity increased from 20 to 727 µmol l^?1 upon purification, representing a ～36‐fold increase with a recovery of 0.28 U U^?1. The optimal pH for activity and stability were in the ranges 3.6–4.3 and 4–6.2, respectively. The optimal temperature for β‐galactosidase activity was between 42 and 47 °C with T_m in the range 45–51 °C. The K_m for pNP‐β‐galactopyranoside was 0.98, 1.11 and 0.95 mM , and V_max was 0.56, 0.53 and 0.35 µmol pNP min^?1, respectively for isoforms I, II and III. Hg²⁺ caused strong inhibition, whereas galacturonic acid, galactose, xylose, fucose and mannose slightly inhibited the activity of β‐galactosidase isoforms. The apparent molecular weights by GPC were 78, 58 and 91 kDa for isoforms I, II and III, respectively. The ability of these isoforms to degrade the endogenous substrate (arabinogalactan) possibly suggests a role in pectin dissolution during tissue softening/fruit ripening. Copyright © 2004 Society of Chemical Industry     

ENZYMATIC PROPERTIES OF A PROTEASE FROM THE HEPATOPANCREAS OF SHRIMP, PENAEVS OMENTAIM

The protease purified from hepatopancreas of shrimp, Penaeus orientals, had high proteolytic activity in the pH range of 7.0 to 9.5. Temperature optimum for hydrolysis of casein was 70C. The protease was stable at neutral and alkaline pH and unstable at acidic pH. The apparent Michaelis‐Menten constant (K_m) and the turnover number (V_max) of the protease on hydrolysis of N‐CBZ‐L‐tyrosine p‐nitrophenyl ester (CBZ‐Tyr‐NE) and N‐CBZ‐L‐phenylalanine p‐nitrophenyl ester (CBZ‐Phe‐NE) ‐were similar, however, those for N‐CBZ‐L‐cysteine p‐nitropher.yl ester (CBZ‐Cys‐NE) were different. K_m and V_max for hydrolysis of casein by the protease were determined to be 0.31% and 5.21s^‐1, respectively. The N‐terminal sequence of the protease showed higher homology with the collagenase of crab and trypsins from Crustacea. Myosin heavy chain (MHC) was the primary substrate during proteolysis with the protease. Actin/tropomyosin were degraded progressively during 2 h incubation but to a lesser extent than MHC.     

Characterization of an extracellular β‐d‐fructofuranosidase produced by Aspergillus niveus during solid‐state fermentation (SSF) of cassava husk

β‐d ‐Fructofuranosidases are biotechnologically important enzymes produced by various organisms. Here, Aspergillus niveus produced an extracellular β‐d ‐fructofuranosidase during SSF of cassava husk. This enzyme was purified 8.5‐fold (recovery of 5.2%). A 37‐kDa protein band was observed after 8% SDS‐PAGE. Native molecular mass is 91.2 kDa. Optimal temperature and pH of activity were 55°C and 4.5, respectively. The enzyme was stable at 50°C for 1 hr, and 80% of its activity was retained after 1 hr at pH 8.0. The enzymatic activity was improved by Mn²⁺, was resistant to most solvents, and was inhibited by Triton X‐100 and Tween 20. K_m and V_max with sucrose were 22.98 mM and 120.48 U/mg of protein, respectively. With Mn²⁺, these values were 16.31 mM and 0.30 U/mg of protein. The enzyme did not hydrolyze inulin and for this reason can be considered a true invertase. Thus, A. niveus β‐d ‐fructofuranosidase holds promise for invert sugar production.

Practical applications

β‐d ‐Fructofuranosidase is an enzyme that can be applied to different industrial sectors, especially food and beverage industries. It is responsible for the hydrolysis of sucrose and yields an equimolar mixture of D‐glucose and D‐fructose, named as inverted sugar syrup, with broad applications in the confectionery industry. The Aspergillus niveus enzyme hydrolyzed only sucrose here and can be considered a true invertase, showing its potential for application to invert sugar production. Besides, the use of cassava husk for enzyme production means an interesting utilization route of this agroindustrial residue. Thus, characterization of this enzyme is an important step for identification of its potential for practical applications.     

EFFECT OF TWO POLAR ORGANIC-AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE-FUNCTION RELATIONSHIP OF PROTEASES I. PEPSIN

The effect of various organic-aqueous solvent systems on the kinetic parameters, intrinsic spectral properties, thermal stability, and proteolytic patterns of porcine pepsin were studied. Two substrates, Z-His-Phe(-NO₂)-Phe-OMe and sodium (Na-) caseinate, were chosen. Three different buffer compositions were used in the investigation: (1) the standard buffers (20 mM formate buffers, pH 2.1 and 10 mM phosphate buffer, pH 5.7); (2) 5% (v/v) ethanol (EtOH) in the standard buffers; (3) 5% (v/v) acetonitrile (ACN) in the standard buffer. Relative to pepsin in formate buffer (pH 2.1), the K_m for Z-His-Phe(-NO₂)-Phe-OMe in 5% EtOH increased from 0.57 mM to 1.03 mM (p ≤ 0.05), while no significant difference was observed in 5% ACN (p > 0.05). The solvent-induced decrease in V_max was much larger in 5% ACN than in 5% EtOH, from 48.0 nmoles/min.mg to 12.3 nmoles/min.mg (p > 0.05) and 35.0 nmoles/min.mg (p > 0.05), respectively, as compared to standard buffer. Relative to pepsin in 10 mM phosphate buffer (pH 5.7), the K_m for Na-caseinate in both 5% EtOH and 5% ACN increased from 4.1 mg/ml to 7.8 mg/ml and 6.2 mg/ml (p ≤ 0.05), respectively while only 5% ACN caused a significant decrease in V_max compared with standard buffer, from 11.8μmoles/min.mgto 7.6 μmoles/min.mg (p ≤ 0.05). Changes in kinetic parameters generally corresponded with solvent-induced structural changes as evidenced by circular dichroism (CD) spectroscopy, a low K_m corresponding to low ellipticity in the near-UV CD spectra (240–320 nm) possibly indicative of a greater protein flexibility. The above results were attributed to differences in properties other than polarity of the solvent systems since the polarity of both 5% EtOH and 5% ACN solutions, as measured by E_T(30) scale, was the same. Differential scanning calorimetric studies of pepsin in the different solvent systems showed destabilization of the enzyme in the organic solutions relative to standard buffer, i.e., lowered temperature of denaturation. Altered specificity of pepsin for Na-caseinate hydrolysis in the presence of the various organic solvents was demonstrated in SDS-PAGE peptide maps as differences in the banding patterns.     

The effect of thermal treatment on β‐glucosidase inactivation in vanilla bean (Vanilla planifolia Andrews)

The conditions for enzyme activity (pH and temperature) and kinetic parameters for the thermal inactivation of β‐glucosidase enzyme in vanilla beans have been investigated. The maximum enzyme activity was detected at pH 6.5 and 38 °C. The values obtained for V_max and K_m were 62.05 units and 2.07 mm, respectively. When hot water treatment (the most practical method of vanilla bean killing) was applied, β‐glucosidase treated at pH 6.0 and 60 °C for 3 min lost 51% of activity, while at 70 °C for 90 s the enzyme lost 60% of activity and at 80 °C for 30 s the enzyme lost 48% of its activity. When vanilla beans were cured in an oven at 60 °C for 36 to 48 h all β‐glucosidase activity was lost.     

Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013

The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell‐bound). Both enzymes were tested for their hydrolytic ability to p‐nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium‐ (C12) and long‐ (C18) chain fatty acids were used as substrates. Cell‐bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p‐nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p‐nitrophenyl decanoate > p‐nitrophenyl caprylate > p‐nitrophenyl laurate > p‐nitrophenyl palmitate > p‐nitrophenyl stearate. The cell‐bound lipase indicates preference for p‐nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell‐bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K_m) and maximum reaction rate (V_max) for p‐nitrophenyl palmitate hydrolysis of cell‐bound lipase were significantly higher (K_m 2.462 mM and V_max 0.210 U/g/min) than those of extracellular lipase (K_m 0.406 mM and V_max 0.006 U/g/min). Copyright © 2010 John Wiley & Sons, Ltd.     

Purification and characterisation of carrot (Daucus carota L) pectinesterase

A pectinesterase isoform with an alkaline isoelectric point of over 8.66 was detected in crude extracts of carrot. The enzyme was purified by ion exchange and molecular exclusion chromatography. The molecular weight of the isoform was 25 kDa, determined in native conditions by filtration through Sephadex G‐75 SF. The enzyme showed a high affinity for its substrate, with K_m and V_max values of 0.031 mg ml^?1 and 6.77 units respectively for apple pectin. The pectinesterase activity exhibited an optimum around pH 7.4 and was activated by metallic ions, with optimum activities at NaCl concentrations between 130 and 330 mM and at CaCl₂ concentrations between 15 and 50 mM . The enzyme was activated most by Ca²⁺ and exhibited a greater tolerance of high concentrations of Na⁺. Comparison of its heat stability with other pectinesterases of vegetable origin indicated that the purified isoform was very thermolabile, being rendered inactive by heating for 5 min at 70 °C. The enzyme was inhibited by high concentrations of polygalacturonic acid and competitively inhibited by D ‐galacturonic acid, with a K_i value of 1 mM . Copyright © 2003 Society of Chemical Industry     

Cultivar and Maturity Effects on Muskmelon ( Cucumis melo) Colour,Texture and Cell Wall Polysaccharide Composition

Cell wall polysaccharides (CWP) of two types of melons were isolated and purified. Fractionations were performed using cyclohexanetrans-1,2-diamine tetraacetate (CDTA), Na₂CO₃, guanidinium thiocyanate (GTC) and KOH. Alditol acetate derivatives of neutral sugars from each CWP fraction were prepared and analysed by gas chromatography. Trifluoro-acetic acid insoluble fractions were analysed colorimetrically and uronic acid was determined. The CDTA and Na₂CO₃ fractions were found to be composed of typical pectic materialscontaining primarily galacturonic acid with the neutral sugars arabinose, galactose, rhamnose and a smaller amount of xylose. As maturity increased, CDTA fraction yields increased, though total neutral sugar CWP compositions decreased. GTC and KOH fractions were typical of hemicellulose, and contained principally xylose, glucose, galactose, mannose and fucose, with very small amounts of uronic acid, arabinose and rhamnose. The residues contained principally glucose and galactose, with smaller amounts of mannose, xylose, arabinose and fucose. With the exception of xylose and glucose, all neutral sugars decreased significantly during ripening in both the Cantaloupe and Honey Dew melons. Total uronic acid did not change as maturity increased, except for Cantaloupe, where total uronic acid decreased from the ripe to overripe stages. Relationships between firmness, drip loss and other composition measurements, as well as the total CWP sugar composition, were also determined. Only the CDTA fraction yields were negatively correlated with the changes in firmness of both melons and positively correlated with changes in drip loss as maturity increased.     

Isolation and properties of 5′-nucleotidase isolated from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

The enzyme 5′-nucleotidase of jumbo squid (Dosidicus gigas) mantle was purified and its SDS–PAGE showed a single band of 33 kDa, whereas a protein with a molecular mass of 107 kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with V_max of 1.16 μM/min/mg of protein, K_m of 1.49 mM, K_cat of 3.48 μM of P_ι s⁻¹ and K_cat/K_m relation of 356.52 ((mol/L)⁻¹ s⁻¹). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a K_m of 6.34 mM, V_max of 0.19 μM/min/mg of protein a K_cat of 0.3388 mol of P_ι s⁻¹ and K_cat/K_m relation of 53.44 ((mol/L)⁻¹ s⁻¹). The low K_m in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.     

Rapid Purfication of β -Galactosidase (Aspergillus Niger) from a Commercial Preparation

β-Galactosidase (A. niger) was purified from a commercial source in order to study the protein nature of the enzyme and some of its kinetic properties. The enzyme was rapidly purified by acetone precipitation, gel filtratior, and affinity chromatography. The specific activity of the purified enzyme was twice as high as that found in previous studies. The K_m and V_max for o-nitrophenyl β-D-galactopyranoside were 2.02 mM and 345 μmoles/min/mg protein respectively at pH 4.5 and 37°C. The procedure described yields a highly active enzyme which may be suitable for immobilization and hydrolysis of lactose. The molecular weight of the enzyme was 117,000 and the isolectric point was 4.9. The enzyme appears to be a glycoprotein and may contain multiple molecular forms.     

Characterization of L-Arabinose Isomerase in Bacillus subtilis,a GRAS Host,for the Production of Edible Tagatose

L-Arabinose isomerase (AI; E.C. 5.3.1.4), a commercial enzyme for the production of edible tagatose in vitro and ribulose production in vivo, has been studied using enzymes expressed in an Escherichia coli system, which might cause noxious by-products in food. To ensure food safety in the tagatose manufacturing process, we studied an AI expression system in Bacillus subtilis. The AI gene from Geobacillus stearothermophilus (GSAI) was expressed in Bacillus subtilis, a GRAS host used in the production of fermented soybean in Korea, after subcloning into a Bacillus subtilis - E. coli shuttle vector, and was characterized after purification. The activities of the crude enzyme extract and a purified sample were 0.15 U/mg protein and 2.7 U/mg protein, respectively. The optimal pH and the optimal temperature for arabinose and galactose as substrates were pH 8.0 and 60°C, respectively, the same as those for GSAI in an E. coli expression system. Substrate affinities (K_m) for arabinose and galactose were 77 mM and 279 mM, respectively, whereas in the E. coli expression system, they were 100 mM and 578 mM, respectively. Catalytic efficiencies (k_cat/K_m) for arabinose and galactose were 58.3 and 11.4 mM^?1 min^?1, respectively. The potential use of GSAI expressed in a GRAS host for the production of edible tagatose is discussed in light of these results.     

The yeasts of the genus Spathaspora: potential candidates for second‐generation biofuel production

Yeasts of the Spathaspora clade have the ability to convert d ‐xylose to ethanol and/or xylitol. This is an important trait, as these yeasts may be used to produce bioethanol from lignocellulosic biomass or as a source of new d ‐xylose metabolism genes for recombinant industrial strains of Saccharomyces cerevisiae. The core group of the genus Spathaspora has 22 species, both formally described and not yet described. Other species, such as Sp. allomyrinae, Candida alai, C. insectamans, C. lyxosophila, C. sake, Sp. boniae and C. subhashii are weakly associated with this clade, based on LSU rRNA gene D1/D2 sequence analyses. Spathaspora passalidarum, Sp. arborariae, Sp. gorwiae and Sp. hagerdaliae produce mostly ethanol from d ‐xylose, whereas the remaining species within the Spathaspora clade already tested for this property may be considered xylitol producers. Among the d ‐xylose‐fermenting Spathaspora species, Sp. passalidarum is the best ethanol producer, displaying high ethanol yields and productivities when cultured in media supplemented with this pentose under oxygen‐limited or anaerobic conditions. The species also exhibits rapid d ‐xylose consumption and the ability to ferment glucose, xylose and cellobiose simultaneously. These characteristics suggest that Sp. passalidarum is a potential candidate for domestication and use in the fermentation of lignocellulosic materials. Copyright © 2017 John Wiley & Sons, Ltd.     

CHARACTERIZATION OF TRYPSIN PURIFIED FROM THE PYLORIC CAECA OF THE SOUTHWEST ATLANTIC WHITE CROAKER Micropogonias furnieri (Sciaenidae)

A southwest Atlantic croaker protease was purified from the pyloric caeca by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The enzyme was classified as a trypsin on the basis of molecular weight, its ability to hydrolyze synthetic substrates N-α-benzoyl-arginine-p-nitroanilide (BAPA) and tosylarginine methyl ester (TAME), and its inhibition by known trypsin inhibitors. The isolated enzyme has a single band on SDS-PAGE with an estimated molecular mass of 24 kDa. Croaker trypsin activity was stable between pH 5 and pH 11 for 30 min at 0C, 10C and 25C and its maximal activity against BAPA was at pH 9.5. Thermostability was observed to about 55C for 30 min at pH 7.8 and its temperature optimum was 60C. Substrate turnover number was 850 BAPA units per μmol trypsin, and the K_mwas 0.081 mM at 25C. For the hydrolysis of TAME, V_maxwas 9273 units per μmol trypsin and the K_m was 0.155 mM at 25C. The catalytic efficiency V_max/K_mwas higher than that of trypsin from fish living in colder waters.