Recombinant Expression and Characterization of A Novel Cold-Adapted β-Galactosidase from Paenibacillus polymyxa

Objective: To analyze a novel cold-adapted β-galactosidase, which provided the basis for producing low lactose dairy products or synthesizing galactooligosaccharides (GOS) at low temperatures. Methods: The β-galactosidase (PpBgal42A) gene was cloned from the genome of Paenibacillus polymyxa. The recombinant plasmid (pET-28a-PpBgal42A) was constructed and successfully expressed in E.coli BL21(DE3). After purification by affinity chromatography, the enzymatic properties of recombinant β-galactosidase were studied. To evaluate the transglycosylation ability of PpBgal42A, the concentration of each component was determined by high performance liquid chromatography with the conversion rate of GOS as evaluation index. Results: PpBgal42A shared the highest identity of 59.9% with the GH family 42 β-galactosidase from Alicyclobacillus acidocaldarius. After purification, the specific activity of PpBgal42A was 163.7 U/mg and the molecular weight was about 79 kDa. Its optimal pH and temperature were pH7.5 and 35 ℃, respectively. It was stable in the range of pH7.0~9.5 and 4~35 ℃. It was rapidly inactivated at 50 ℃. The half-life of PpBgal42A at 35 ℃ was 1777 min. PpBgal42A synthesized 31.6% of GOS using 350 g/L lactose as the substrate at 30 ℃ for 6 h. Conclusion: PpBgal42A is a novel cold-adapted enzyme. It can hydrolyze lactose and synthesize galactooligosaccharides, which has potential application value

Biochemical characterization of a novel β-galactosidase from Lacticaseibacillus zeae and its application in synthesis of lacto-N-tetraose

ABSTRACT: Lacto-N-tetraose (LNT) is one of the most important components of human milk oligosaccharides, which has various beneficial health effects. β-Galactosidase is an important enzyme used in dairy processing. The transglycosylation activity of β-galactosidases offers an attractive approach for LNT synthesis. In this study, we reported for the first time the biochemical characterization of a novel β-galactosidase (LzBgal35A) from Lacticaseibacillus zeae. LzBgal35A belongs to glycoside hydrolases (GH) family 35 and shared the highest identity of 59.9% with other reported GH 35 members. The enzyme was expressed as soluble protein in Escherichia coli. The purified LzBgal35A displayed optimal activity at pH 4.5 and 55°C. It was stable within the pH range of 3.5 to 7.0 and up to 60°C. Moreover, LzBgal35A could catalyze the synthesis of LNT via transferring the galactose residue from o-nitrophenyl-β-galactopyranoside to lacto-N-triose II. Under optimal conditions, the conversion rate of LNT reached 45.4% (6.4 g/L) within 2 h, which was by far the highest yield of LNT synthesized through a β-galactosidase-mediated transglycosylation reaction. This study demonstrated that LzBgal35A has great potential application in LNT synthesis

Co-treatment of nicotinamide mononucleotide and neoagarooligosaccharide mitigates aging-induced cognitive impairment by promoting mitochondrial dynamics

The synergistic effects of nicotinamide mononucleotide (NMN) and neoagarooligosaccharides (NAOS) on mitochondrial dysfunction, downstream apoptosis, and inflammation were evaluated in both senescence-accelerated mouse prone 8 (SAMP8) mice and H2O2-induced human lung fibroblast (MRC-5) cells. Co-treatment of NMN and NAOS effectively ameliorated the learning and memory impairment in the passive avoidance and the Morris water maze tests. MNN and NAOS supplementation increased the expression levels of mitochondrial fusion proteins in the brain of SAMP8 mice, including OPA1 (by 37.4%) and Mitofusin 2 (by 99.7%). Moreover, co-treatment of NMN and NAOS significantly inhibited cell apoptosis as shown by the reduced protein expression levels of Bax and Caspase-3, down-regulated P53/P21/P16 pathway in both SAMP8 mice and H2O2-induced MRC-5 cells. At the same time, dietary intervention of NMN combined with NAOS attenuated the inflammatory response via down-regulating TLR4/MyD88/NF-κB pathway. Conclusively, the synergistic efficacy of the combination of NMN and NAOS can improve cognitive impairment by modulating mitochondrial dysfunction

Releasing Bioactive Compounds from Brown Seaweed with Novel Cold-Adapted Alginate Lyase and Alcalase

Seaweeds are considered to be third-generation renewable biomasses, the comprehensive utilization of which has drawn increasing attention in recent years. A novel cold-active alginate lyase (VfAly7) was identified from Vibrio fortis and biochemically characterized for brown seaweed utilization. The alginate lyase gene was high-level expressed in Pichia pastoris, with an enzyme yield of 560 U/mL and a protein content of 9.8 mg/mL by high-cell density fermentation. The recombinant enzyme was most active at 30 °C and pH 7.5, respectively. VfAly7 was a bifunctional alginate lyase with both poly-guluronate and poly-mannuronate hydrolysis activities. On the basis of VfAly7, a bioconversion strategy for the utilization of brown seaweed (Undaria pinnatifida) was developed. The obtained AOSs showed stronger prebiotic activity towards tested probiotics when compared to that of commercial fructooligosaccharides (FOSs), while the obtained protein hydrolysates displayed strong xanthine oxidase inhibitory activity with IC50 of 3.3 mg/mL. This study provided a novel alginate lyase tool as well as a biotransformation route for the utilization of seaweeds

Biochemical characterization of a first fungal esterase from Rhizomucor miehei showing high efficiency of ester synthesis.

Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis.A novel esterase-encoding gene from Rhizomucor miehei (RmEstA) was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide belonging to the hormone-sensitive lipase (HSL) family IV and showing highest similarity (44%) to the Paenibacillus mucilaginosus esterase/lipase. Recombinant RmEstA was purified to homogeneity: it was 34 kDa by SDS-PAGE and showed optimal pH and temperature of 6.5 and 45°C, respectively. The enzyme was stable to 50°C, under a broad pH range (5.0-10.6). RmEstA exhibited broad substrate specificity toward p-nitrophenol esters and short-acyl-chain triglycerols, with highest activities (1,480 U mg(-1) and 228 U mg(-1)) for p-nitrophenyl hexanoate and tributyrin, respectively. RmEstA efficiently synthesized butyl butyrate (92% conversion yield) when immobilized on AOT-based organogel.RmEstA has great potential for industrial applications. RmEstA is the first reported esterase from Rhizomucor miehei

Directed evolution of a β-N-acetylhexosaminidase from Haloferula sp. for lacto-N-triose II and lacto-N-neotetraose synthesis from chitin

In our previous study, a β-N-acetylhexosaminidase (HaHex74) from Haloferula sp. showing high human milk oligosaccharides (HMOs) synthesis ability was identified and characterized. In this study, HaHex74 was further engineered by directed evolution and site-saturation mutagenesis to improve its transglycosylation activity for HMOs synthesis. A mutant (mHaHex74) with improved transglycosylation activity (HaHex74-Asn401Ile/His394Leu) was obtained and characterized. mHaHex74 exhibited maximal activity at pH 5.5 and 35 °C, respectively, which were distinct from that of HaHex74 (pH 6.5 and 45 °C). Moreover, mHaHex74 showed the highest LNT2 conversion ratio of 28.2% from N,N’-diacetyl chitobiose (GlcNAc2), which is 2.2 folds higher than that of HaHex74. A three-enzyme cascade reaction for the synthesis of LNT2 and LNnT from chitin was performed in a 5–L reactor, and the contents of LNT2 and LNnT reached up to 15.0 g Lsingle bond1 and 4.9 g Lsingle bond1, respectively. Therefore, mHaHex74 maybe a good candidate for enzymatic synthesis of HMOs