Characterization of a Novel α-l-Arabinofuranosidase from Ruminococcus albus 7 and Rational Design for Its Thermostability.

An α-l-arabinofuranosidase (Abf) encoding gene was obtained via genomic mining from a Ruminococcus albus strain. The specific activity of this GH 51 Abf was 73.3 U/mg at pH 6.0 and 50 °C. The modification of Abf, aimed at improving thermostability, was performed through different strategies. Structure-based rational design using the PoPMuSiC and the Enzyme Thermal Stability System (ETSS) predicted thermal stability of Abf and enhanced the half-life of thermal inactivation (t1/2) at 50 °C for K208W more than 11.1 times versus the wild-type (WT). Sequence-based rational design was also conducted by substituting histidine with lysine at various sites. Among eight mutants, the t1/2 at 50 °C of H337K was prolonged by 5.0-fold, and the specific activity of this mutant was increased to 121.8 U/mg. In addition, the mutant H337K was utilized with some enzymes to extract pectin from apple pomace. The enzymatically produced pectin got less moisture and ash, milder pH, and higher viscosity than its acid-extracted counterpart, indicating that Abf has an application prospect in pectin production.

An Acid-Adapted Endo-α-1,5-L-arabinanase for Pectin Releasing.

An arabinanase gene was cloned by overlap-PCR from Penicillium sp. Y702 and expressed in Pichia pastoris. The recombinant enzyme was named AbnC702 with 20 U/mg of endo-arabinanase activity toward linear α-1,5-L-arabinan. The optimal pH and temperature of AbnC702 were 5.0 and 50 °C, respectively. The recombinant AbnC702 was highly stable at pH 5.0-7.0 and 50 °C. It could retain about 72.3 % of maximum specific activity at pH 5.0 after incubation for 2.5 h, which indicated AbnC702 was an acid-adapted enzyme. The K m and V max values were 24.8 ± 4.7 mg/ml and 88.5 ± 5.6 U/mg, respectively. A three-dimensional structure of AbnC702 was made by homology modeling, and the counting of acidic/basic amino residues within the region of 10 Å around the active site, as well the hydrogen bonds within the area of 5 Å around the active site, might theoretically interpret the acid adaptability of AbnC702. Analysis of hydrolysis products by thin layer chromatography (TLC) combined with high-performance liquid chromatography (HPLC) verified that the recombinant AbnC702 was an endo-1,5-α-L-arabinanase, which yielded arabinobiose and arabinotriose as major products. AbnC702 was applied in pectin extraction from apple pomace with synergistic action of α-L-arabinofuranosidase.

Effects of droplet size on the oxidative stability of oil-in-water emulsions.

The effects of droplet size and emulsifiers on oxidative stability of polyunsaturated TAG in oil-in-water (o/w) emulsions with droplet sizes of 0.806 +/- 0.0690, 3.28 +/- 0.0660, or 10.7 +/- 0.106 microm (mean +/- SD) were investigated. Hydroperoxide contents in the emulsion with a mean droplet size of 0.831 microm were significantly lower than those in the emulsion with a mean droplet size of 12.8 microm for up to 120 h of oxidation time. Residual oxygen contents in the headspace air of the vials containing an o/w emulsion with a mean droplet size of 0.831 microm were lower compared with those of the emulsion with a mean droplet size of 12.8 microm. Hexanal developed from soybean oil TAG o/w emulsions with smaller droplet size showed significantly lower residual oxygen contents than those of the larger droplet size emulsions. Consequently, oxidative stability of TAG in o/w emulsions could be controlled by the size of oil droplet even though the origins of TAG were different. Spin-spin relaxation time of protons of acyl residues on TAG in o/w emulsions measured by H NMR suggested that motional frequency of some acyl residues was shorter in o/w emulsions with a smaller droplet size. The effect of the wedge associated with hydrophobic acyl residues of emulsifiers was proposed as a possible mechanism to explain differences in oxidative stability between o/w emulsions with different droplet sizes.