In this study, three kinds of milk were treated with the β-galactosidase Bgal1-3 (4 U/mL), resulting in 7.2–9.5 g/L galactooligosaccharides (GOS) at a lactose conversion of 90–95%. Then, Bgal1-3 was secreted from Pichia pastoris X33 under the direction of an α-factor signal peptide. After cultivation for 144 h in a flask culture with shaking, the extracellular activity of Bgal1-3 was 4.4 U/mL. Five more signal peptides (HFBI, apre, INU1A, MF4I, and W1) were employed to direct the secretion, giving rise to a more efficient signal peptide, W1 (11.2 U/mL). To further improve the secretion yield, recombinant strains harboring two copies of the bgal1-3 gene were constructed, improving the extracellular activity to 22.6 U/mL (about 440 mg/L). This study successfully constructed an engineered strain for the production of the β-galactosidase Bgal1-3, which is a promising catalyst in the preparation of prebiotic-enriched milk.Keywords: galactooligosaccharide; gut microbiota; Pichia pastoris; prebiotic-enriched milk; signal peptide W1; β-galactosidase;

In this study, the twin-arginine (Tat) signal peptide PhoD was used to direct the secretion of the β-galactosidase Bgal1-3 into the growth medium of an engineered strain of Bacillus subtilis 168. After 24 h of cultivation, the extracellular activity reached 1.15 U/mL, representing 78% of the total activity. Bgal1-3 was exported via both Tat-dependent and Tat-independent pathways. To improve the secretion amounts, two more copies of the target gene were inserted into the designated loci on the chromosome, further improving the extracellular enzymatic activity to 2.15 U/mL. The engineered strain with three copies of bgal1-3 was genetically stable after 150 generations. To the best of our knowledge, this is the first report on the functional secretion of a heterologous protein via both Tat-dependent and Tat-independent pathways mediated by a Tat signal peptide in B. subtilis. Furthermore, this study provides us with a markerless engineered strain for the production of β-galactosidase.

A novel β-galactosidase (Bgal1-3) was isolated from a marine metagenomic library and then its cross-linked enzyme aggregates (CLEAs) were prepared. The enzymatic properties of Bgal1-3-CLEAs were studied and compared with that of the free enzyme. The thermostability and storage stability of Bgal1-3 were significantly improved after it was immobilized as CLEAs. The galactose-tolerance of the enzyme was also enhanced after the immobilization, which could relieve the inhibitory effect and then tends to be beneficial for the galacto-oligosaccharides (GOS) synthesis. Moreover, higher GOS yield was achieved (59.4 ± 1.5%) by Bgal1-3-CLEAs compared to the free counterpart (57.1 ± 1.7%) in an organic–aqueous biphasic system. The GOS content and composition of the syrups synthesized by the free enzyme and Bgal1-3-CLEAs were similar and they both contained at least seven different oligosaccharides with the degree of polymerization (DP) ranging between 3 and 9. Furthermore, Bgal1-3-CLEAs maintained 82.1 ± 2.1% activity after ten cycles of reuse; the GOS yield of the tenth batch was 52.3 ± 0.3%, which was still higher than that of the most former reports. To the best of our knowledge, this is the first report on the GOS synthesis using CLEAs of β-galactosidase in an organic–aqueous biphasic system. The study not only further expands the application scope of CLEA, but also provides a potential catalyst for the synthesis of GOS with low cost.

To improve the thermostability of EstF27, two rounds of random mutagenesis were performed. A thermostable mutant, M6, with six amino acid substitutions was obtained. The half-life of M6 at 55 °C is 1680 h, while that of EstF27 is 0.5 h. The Kcat/Km value of M6 is 1.9-fold higher than that of EstF27. The concentrations of ferulic acid released from destarched wheat bran by EstF27 and M6 at their respective optimal temperatures were 223.2 ± 6.8 and 464.8 ± 11.9 μM, respectively. To further understand the structural basis of the enhanced thermostability, the crystal structure of M6 is determined at 2.0 Å. Structural analysis shows that a new disulfide bond and hydrophobic interactions formed by the mutations may play an important role in stabilizing the protein. This study not only provides us with a robust catalyst, but also enriches our knowledge about the structure–function relationship of feruloyl esterase.

Prebiotic galacto-oligosaccharides (GOS) were effectively synthesized from lactose in organic–aqueous biphasic media by a novel metagenome-derived β-galactosidase BgaP412. A maximum GOS yield of 46.6% (w/w) was achieved with 75.4% lactose conversion rate in the cyclohexane/buffer system [95:5 (v/v) cyclohexane/buffer] under the optimum reaction conditions (initial lactose concentration = 30% (w/v), T = 50 °C, pH 7.0, and t = 8 h). The corresponding productivity of GOS was approximately 17.5 g L–1 h–1. The GOS mixture consisted of tri-, tetra-, and pentasaccharides. Trisaccharides were the chief component of reaction products. These experimental results showed that a low water content, a high initial lactose concentration, and an elevated reaction temperature could significantly promote the transgalactosylation activity of β-galactosidase BgaP412; at the same time, the enhanced GOS yield in an organic–aqueous biphasic system is because of the fact that thermodynamic equilibrium can be shifted to the synthetic direction by reversing the normal hydrolysis.

A novel gene (designated as tan410) encoding tannase was isolated from a cotton field metagenomic library by functional screening. Sequence analysis revealed that tan410 encoded a protein of 521 amino acids. SDS−PAGE and gel filtration chromatography analysis of purified tannase suggested that Tan410 was a monomeric enzyme with a molecular mass of 55 kDa. The optimum temperature and pH of Tan410 were 30 °C and 6.4. The activity was enhanced by addition of Ca2+, Mg2+ and Cd2+. In addition, Tan410 was stable in the presence of 4 M NaCl. Chlorogenic acid, rosmarinic acid, ethyl ferulate, tannic acid, epicatechin gallate and epigallocathchin gallate were efficiently hydrolyzed by recombinant tannase. All of these excellent properties make Tan410 an interesting enzyme for biotechnological application.

A novel β-galactosidase gene, zd410, was isolated by screening a soil metagenomic library. Sequence analysis revealed that zd410 encodes a protein of 672 amino acids with a predicted molecular weight of 78.6 kDa. The recombinant ZD410 was expressed and purified in Pichia pastoris, with a yield of ca. 300 mg from 1 L culture. The purified enzyme displayed optimal activity at 38°C and pH 7.0. Given that the enzyme had 54% of the maximal activity at 20°C and 11% of the maximal activity at close to 0°C, ZD410 was regarded as a cold-adapted β-galactosidase. ZD410 displays high enzymatic activity for its synthetic substrate-ONPG (o-nitrophenyl-β-d-galactopyranoside, 243 U/mg) and its natural substrate-lactose (25.4 U/mg), while its activity was slightly stimulated by addition of Na+, K+, or Ca2+ at low concentrations. ZD410 is a good candidate of β-galactosidases for food industry after further study.

Lac591, a gene encoding a novel multicopper oxidase with laccase activity, was identified through activity-based functional screening of a metagenomic library from mangrove soil. Sequence analysis revealed that lac591 encodes a protein of 500 amino acids with a predicted molecular mass of 57.4 kDa. Lac591 was overexpressed heterologously as soluble active enzyme in Escherichia coli and purified, giving rise to 380 mg of purified enzyme from 1 l induced culture, which is the highest expression report for bacterial laccase genes so far. Furthermore, the recombinant enzyme demonstrated activity toward classical laccase substrates syringaldazine (SGZ), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP). The purified Lac591 exhibited maximal activity at 55°C and pH 7.5 with guaiacol as substrate and was found to be stable in the pH range of 7.0–10.0. The substrate specificity on different substrates was studied with the purified enzyme, and the optimal substrates were in the order of 2, 6-DMP > catechol > α-naphthol > guaiacol > SGZ > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). The alkaline activity and highly soluble expression of Lac591 make it a good candidate of laccases in industrial applications for which classical laccases are unsuitable, such as biobleaching of paper pulp and dyestuffs processing.

Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes) and developing various biotechnological applications.The pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45–49%). The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3), purified and characterized. The molecular mass of the native enzyme was approximately 31 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the Pye3 indicated molecular mass of 31 kDa and 31.5 kDa, respectively, suggesting that the Pye3 is a monomer. The purified Pye3 not only degraded all pyrethroid pesticides tested, but also hydrolyzed ρ-nitrophenyl esters of medium-short chain fatty acids, indicating that the Pye3 is an esterase with broader specificity. The Km values for trans-Permethrin and cis-permethrin are 0.10 μM and 0.18 μM, respectively, and these catalytic properties were superior to carboxylesterases from resistant insects and mammals. The catalytic activity of the Pye3 was strongly inhibited by Hg2+, Ag+, ρ-chloromercuribenzoate, whereas less pronounced effect was observed in the presence of divalent cations, the chelating agent EDTA and phenanthroline.A novel pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, the broader substrate specificities and higher activity of the pyrethroid-hydrolyzing esterase (Pye3) make it an ideal candidate for in situ for detoxification of pyrethroids where they cause environmental contamination problems. Consequently, metagenomic DNA clone library offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria.

•A new gene encoding feruloyl esterase (Tan410) was isolated from a soil metagenomic library.•Tan410 was found not only show feruloyl esterase activity, but also show tannase activity, indicating novel properties for feruloyl esterase.•The novel feruloyl esterase revealed moderate thermostability.A gene (tan410) encoding a feruloyl esterase was isolated by screening a cotton soil metagenomic library. Sequence analysis revealed that tan410 encodes a protein of 520 amino acids with a predicted molecular weight of 55 kDa. The gene was further expressed in Escherichia coli BL21 (DE3) using a pET expression system. The recombinant enzyme was purified and characterized. Its optimum temperature and pH were 35 °C and 7.0, respectively. Tan410 activity was enhanced by the addition of Mn2+, Mg2+, NH4+ and Ni2+. Besides ethyl ferulate, methyl caffeate, and methyl p-coumarate, Tan410 can also hydrolyze methyl gallate, tannic acid, epicatechin gallate, and epigallocatechin gallate which makes Tan410 an interesting enzyme for biotechnological applications.Download full-size image

•A novel esterase, EstWSD, was isolated from a soil metagenomic library.•EstWSD and its closest homologues belong to a new bacterial lipolytic enzyme family.•EstWSD is stable in 0.5–4.5 M NaCl, suggesting that it is a halotolerant esterase.•EstWSD is stable in dimethylsulfoxide, ρ-xylene, hexane, heptane, and octane.Soil metagenome conceals a great variety of unexploited genes for industrially important enzymes. To identify novel genes conferring lipolytic activity, one metagenomic library comprising of 200,000 transformants were constructed. Among the 48,000 clones screened, 19 clones which exhibited lipolytic activity were obtained. After sequence analysis, 19 different lipolytic genes were identified. One of these genes, designated as estWSD, consisted of 1152 nucleotides, encoding a 383-amino-acid protein. Multiple sequence alignment and phylogenetic analysis indicated that EstWSD and its closest homologues may constitute a new family of bacterial lipolytic enzymes. The best substrate for the purified EstWSD among the ρ-nitrophenol esters examined was ρ-nitrophenol butyrate. Recombinant EstWSD displayed a pH optimum of 7.0 and a temperature optimum of 50 °С. This enzyme retained 52% of maximal activity after incubation at 50 °C for 3 h. Furthermore, EstWSD also exhibited salt tolerance with over 51% of its initial activity in the presence of up to 4.5 M NaCl for 1 h. In particular, this enzyme showed remarkable stability in 15% and 30% dimethylsulfoxide, ρ-xylene, hexane, heptane, and octane even after incubation for 72 h. To our knowledge, it is the first report to find a novel esterase belonging to a new lipolytic family and possessing such variety of excellent features. All these characteristics suggest that EstWSD may be a potential candidate for application in industrial processes.Download full-size image

A novel gene (designated as est816) encoding an esterase was isolated from a Turban Basin metagenomic library with a functional screening method. Sequence analysis revealed that est816 encoded a protein of 271 amino acids with a predicted molecular mass (Mr) of 29.9 kDa and was expressed in Escherichia coli BL21 (DE3) in soluble form. The optimum pH and temperature of the recombinant Est816 were 7.5 and 60 °C, respectively. The enzyme was stable in the pH range of 5.0–9.0 and at temperatures below 50 °C. The residual activity of Est816 was 47.7% when stored at 25 °C for 5 months. The enzyme could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl acetate with the highest activity (364 U/mg). It could also degrade medium to long-chain AHLs at the concentration of 1 mM in half an hour with more than 90% degradation efficiency. This is the first report to construct one metagenomic library from Turban Basin to obtain one esterase, which belongs to family V esterases/lipases and has AHL-lactonase activity. The recombinant enzyme displayed broad substrate spectrum, high activity and thermostability. These excellent properties make it an attractive enzyme for quorum quenching.Graphical abstractDownload full-size imageHighlights► A novel thermotolerant estersase gene est816 was cloned by metagenomics. ► Est816 was overexpressed in E. coli BL21 (DE3) in soluble form. ► Est816 was stable in the pH range of 5.0–9.0 and at temperatures below 50 °C. ► Est816 had outstanding storage stability with the residual activity 47.7% after stored at 25 °C for 5 months. ► The enzyme could degrade medium to long-chain AHLs at the concentration of 1 mM in half an hour with more than 90% degradation efficiency.

A novel organic solvent-stable and thermotolerant lipase gene (designated ostl28) was cloned from a metagenomic library and overexpressed in Escherichia coli BL21 (DE3) in soluble form. OSTL28 contained 262 amino acids with relative molecular mass 30.1 kDa and isoelectric point 9.7. The optimum pH and temperature of the OSTL28 were 7.5 and 60 °C, respectively. OSTL28 was stable in the pH range of 4.5–9.5 and at temperatures below 65 °C. The enzyme could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl laurate with the highest activity of 236 U/mg (54,000 U/L). The recombinant OSTL28 was highly resisted to organic solvents, especially glycerol and methanol. The metal ions, with the exception of Hg2+ and Ag+, did not have any influence on enzyme activity, whereas non-ionic surfactants and Al3+ slightly activated the enzyme. These features indicate that it is a potential biocatalyst for biodiesel production.Graphical abstractDownload full-size imageHighlights► A novel thermotolerant lipase gene OSTL28 was cloned by metagenomics. ► OSTL28 was overexpressed in Escherichia coli BL21 (DE3) in soluble form. ► OSTL28 was stable in the pH range of 4.5–9.5 and at temperatures below 65 °C. ► OSTL28 was highly resisted to organic solvents, especially glycerol and methanol. ► Non-ionic surfactants and Al3+ slightly activated the enzyme.

•Effects of pH, incubation temperature, nitrogen and carbon sources, inorganic salts on tannase production were detailed described.•The properties of tannases were summarized, including molecular weight, effect of temperature and pH, effect of metal ions, effect of organic solvents and surfactants.•Tannase protein sequences and molecular architecture were described in detail.•Novel developments in the field of tannase including metagenomic approach and directed evolution were discussed to get novel tannases or improve properties of the existing tannase.Tannases, tannin acylhydrolases, are an important group of biotechnologically relevant enzymes which were utilized in a number of industrial applications, including the manufacture of instant tea, beer, fruit juices, some wines and gallic acid production. Tannases are by and large produced by microorganisms including Aspergillus, Paecilomyces, Lactobacillus and Bacillus. Tannases are generally produced on tannic carbon such as tannic acid, wheat bran, tea and coffee husk extract. Microbial tannases are mostly induced extracellular enzyme and produced by submerged fermentation and solid-state fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography in addition to reverse micelle. Most tannases can act in a wide range of temperature and pH, although tannases with acidic pH optima are more common. A sequence-based classification spreads tannases in many families thus reflecting the variety of molecules. Furthermore, tannase from Lactobacillus plantarum had been characterized by three-dimensional architecture. In recent years, a novel approach, metagenomic, was developed to exploring novel tannase from natural communities.Download full-size image

•A novel glycosyltransferase (Glyt110) was obtained from a metagenomic library.•It is an unusual enzyme with β-galactosidase activity but grouped into family GT4.•The specific activities toward ONPG and lactose were 314 ± 18.3 and 32 ± 2.7 U/mg, respectively.•A GOS yield of 47.2% (w/w) was achieved by Glyt110 using lactose as substrate.•Glyt110 may be a potential candidate for industrial production of GOS.Glycosyltransferases and glycoside hydrolases are two diversified groups of carbohydrate-active enzymes (CAZymes) in existence, they serve to build and break down the glycosidic bonds, respectively, and both categories have formed many sequence-based families. In this study, a novel gene (glyt110) conferring β-galactosidase activity was obtained from a metagenomic library of Turpan Basin soil. Sequence analysis revealed that glyt110 encoded a protein of 369 amino acids that, rather than belonging to a family typically known for β-galactosidase activity, belonged to glycosyltransferase family 4. Because of this unusual sequence information, the novel gene glyt110 was subsequently expressed in Escherichia coli BL21(DE3), and the recombinant enzyme (Glyt110) was purified and characterized. Biochemical characterization revealed that the β-galactosidase activity of Glyt110 toward o-nitrophenyl-β-d-galactopyranoside (ONPG) and lactose were identified to be 314 ± 18.3 and 32 ± 2.7 U/mg, correspondingly. In addition, Glyt110 can synthesize galacto-oligosaccharides (GOS) using lactose as substrate. A GOS yield of 47.2% (w/w) was achieved from 30% lactose solution at 50 °С, pH 8.0 after 10 h reaction. However, Glyt110 was unable to glycosylate either N-acetylated saccharides or lactose and galactose using UDP-gal as sugar donor, and its glycosyltransferase activity needs further investigation. These results indicated that Glyt110 is an unusual enzyme with β-galactosidase activity but phylogenetically related to glycosyltransferase. Our findings may provide opportunities to improve the insight into the relationship between glycosyltransferases and glycoside hydrolases and the sequence-based classification.