Junmei Ding

Properties of a Newly Identified Esterase from Bacillus sp. K91 and Its Novel Function in Diisobutyl Phthalate Degradation

The widely used plasticizer phthalate esters (PAEs) have become a public concern because of their effects on environmental contamination and toxicity on mammals. However, the biodegradation of PAEs, especially diisobutyl phthalate (DiBP), remains poorly understood. In particular, genes involved in the hydrolysis of these compounds were not conclusively identified. In this study, the CarEW gene, which encodes an enzyme that is capable of hydrolyzing ρ-nitrophenyl esters of fatty acids, was cloned from a thermophilic bacterium Bacillus sp. K91 and heterologously expressed in Escherichia coli BL21 using the pEASY-E2 expression system. The enzyme showed a monomeric structure with a molecular mass of approximately 53.76 kDa and pI of 4.88. The enzyme exhibited maximal activity at pH 7.5 and 45°C, with ρ-NP butyrate as the best substrate. The enzyme was fairly stable within the pH range from 7.0 to 8.5. High-pressure liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were employed to detect the catabolic pathway of DiBP. Two intermediate products were identified, and a potential biodegradation pathway was proposed. Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.

Characterization of two glycoside hydrolase family 36 α-galactosidases: novel transglycosylation activity, lead-zinc tolerance, alkaline and multiple pH optima, and low-temperature activity.

Two α-galactosidases, AgaAJB07 from Mesorhizobium and AgaAHJG4 from Streptomyces, were expressed in Escherichia coli. Recombinant AgaAJB07 showed a 2.9-fold and 22.6-fold increase in kcat with a concomitant increase of 2.3-fold and 16.3-fold in Km in the presence of 0.5mM ZnSO4 and 30.0mM Pb(CH3COO)2, respectively. Recombinant AgaAHJG4 showed apparent optimal activity at pH 8.0 in McIlvaine or Tris-HCl buffer and 9.5 in glycine-NaOH or HCl-borax-NaOH buffer, retention of 23.6% and 43.2% activity when assayed at 10 and 20°C, respectively, and a half-life of approximately 2min at 50°C. The activation energies for p-nitrophenyl-α-d-galactopyranoside hydrolysis by AgaAJB07 and AgaAHJG4 were 71.9±0.8 and 48.2±2.0kJmol(-1), respectively. Both AgaAJB07 and AgaAHJG4 exhibited transglycosylation activity, but they required different acceptors and produced different compounds. Furthermore, potential factors for alkaline and multiple pH optima and low-temperature adaptations of AgaAHJG4 were presumed.

Metagenomic analysis of the Rhinopithecus bieti fecal microbiome reveals a broad diversity of bacterial and glycoside hydrolase profiles related to lignocellulose degradation

BackgroundThe animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host and the diet adopted by the host. Although the importance of gut microbiota of humans has been well demonstrated, there is a paucity of research regarding non-human primates (NHPs), especially herbivorous NHPs.ResultsIn this study, an analysis of 97,942 pyrosequencing reads generated from Rhinopithecus bieti fecal DNA extracts was performed to help better understanding of the microbial diversity and functional capacity of the R. bieti gut microbiome. The taxonomic analysis of the metagenomic reads indicated that R. bieti fecal microbiomes were dominated by Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria phyla. The comparative analysis of taxonomic classification revealed that the metagenome of R. bieti was characterized by an overrepresentation of bacteria of phylum Fibrobacteres and Spirochaetes as compared with other animals. Primary functional categories were associated mainly with protein, carbohydrates, amino acids, DNA and RNA metabolism, cofactors, cell wall and capsule and membrane transport. Comparing glycoside hydrolase profiles of R. bieti with those of other animal revealed that the R. bieti microbiome was most closely related to cow rumen.ConclusionsMetagenomic and functional analysis demonstrated that R. bieti possesses a broad diversity of bacteria and numerous glycoside hydrolases responsible for lignocellulosic biomass degradation which might reflect the adaptations associated with a diet rich in fibrous matter. These results would contribute to the limited body of NHPs metagenome studies and provide a unique genetic resource of plant cell wall degrading microbial enzymes. However, future studies on the metagenome sequencing of R. bieti regarding the effects of age, genetics, diet and environment on the composition and activity of the metagenomes are required.

Cold-active and NaCl-tolerant exo-inulinase from a cold-adapted Arthrobacter sp. MN8 and its potential for use in the production of fructose at low temperatures

An exo-inulinase gene was cloned from Arthrobacter sp. MN8, a cold-adapted bacterium isolated from lead-zinc-rich soil. The gene was expressed in Escherichia coli BL21(DE3). The resultant 505-residue polypeptide (InuAMN8) showed the highest identity (81.1%) with the putative levanase from Arthrobacter phenanthrenivorans Sphe3 (ADX73279) and shared 57.8% identity with the exo-inulinase from Bacillus sp. snu-7 (AAK00768). The purified recombinant InuAMN8 (rInuAMN8) showed an apparently optimal activity at 35°C, and 75.3%, 39.4%, and 15.8% of its maximum activity at 20°C, 10°C, and 0°C, respectively. After pre-incubation for 60 min at 50°C and 55°C, the rInuAMN8 exhibited 69.8% and 17.7% of its initial activity, respectively. The apparent Km values of rInuAMN8 towards inulin were 2.8, 1.5, 1.2, 5.3, and 8.2 mM at 0°C, 10°C, 20°C, 30°C, and 35°C, respectively. Inulin and Jerusalem artichoke tubers were effectively hydrolyzed to release fructose by rInuAMN8 at 0°C, 10°C, and 35°C. Compared with its hyperthermophilic and thermophilic counterparts, the exo-inulinase had less aromatic amino acid F and more hydrophobic amino acid A. In addition, the purified rInuAMN8 retained 127.9%-88.4% inulinase activity at 3.5%-15.0% (w/v) NaCl concentrations. Zn(2+) and Pb(2+) at 10 mM exhibited little or no effect on the enzyme activity. This paper is the first to report a cold-active and/or NaCl-tolerant exo-inulinase from the genus Arthrobacter. The exo-inulinase rInuAMN8 shows a potential for use in the production of fructose at low temperatures.

Molecular and Biochemical Characterization of a Novel Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti.

Xylanases sourced from different bacteria have significantly different enzymatic properties. Therefore, studying xylanases from different bacteria is important to their applications in different fields. A potential xylanase degradation gene in Massilia was recently discovered through genomic sequencing. However, its xylanase activity remains unexplored. This paper is the first to report a xylanase (XynRBM26) belonging to the glycosyl hydrolase family (GH10) from the genus Massilia. The gene encodes a 383-residue polypeptide (XynRBM26) with the highest identity of 62% with the endoxylanase from uncultured bacterium BLR13. The XynRBM26 expressed in Escherichia coli BL21 is a monomer with a molecular mass of 45.0 kDa. According to enzymatic characteristic analysis, pH 5.5 is the most appropriate for XynRBM26, which could maintain more than 90% activity between pH 5.0 and 8.0. Moreover, XynRBM26 is stable at 37°C and could maintain at least 96% activity after being placed at 37°C for 1 h. This paper is the first to report that GH10 xylanase in an animal gastrointestinal tract (GIT) has salt tolerance, which could maintain 86% activity in 5 M NaCl. Under the optimum conditions, Km, Vmax, and kcat of XynRBM26 to beechwood xylan are 9.49 mg/ml, 65.79 μmol/min/mg, and 47.34 /sec, respectively. Considering that XynRBM26 comes from an animal GIT, this xylanase has potential application in feedstuff. Moreover, XynRBM26 is applicable to high-salt food and seafood processing, as well as other high-salt environmental biotechnological fields, because of its high catalytic activity in high-concentration NaCl.

Characterization of a Glycoside Hydrolase Family 27 α-Galactosidase from Pontibacter Reveals Its Novel Salt-Protease Tolerance and Transglycosylation Activity.

α-Galactosidases are of great interest in various applications. A glycoside hydrolase family 27 α-galactosidase was cloned from Pontibacter sp. harbored in a saline soil and expressed in Escherichia coli. The purified recombinant enzyme (rAgaAHJ8) was little or not affected by 3.5-30.0% (w/v) NaCl, 10.0-100.0 mM Pb(CH3COO)2, 10.0-60.0 mM ZnSO4, or 8.3-100.0 mg mL(-1) trypsin and by most metal ions and chemical reagents at 1.0 and 10.0 mM concentrations. The degree of synergy on enzymatic degradation of locust bean gum and guar gum by an endomannanase and rAgaAHJ8 was 1.22-1.54. In the presence of trypsin, the amount of reducing sugars released from soybean milk treated by rAgaAHJ8 was approximately 3.8-fold compared with that treated by a commercial α-galactosidase. rAgaAHJ8 showed transglycosylation activity when using sucrose, raffinose, and 3-methyl-1-butanol as the acceptors. Furthermore, potential factors for salt adaptation of the enzyme were presumed.

Identification and Characterization of A New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis

UNLABELLED Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic β-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic β-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA. IMPORTANCE Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic β-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family.

The 340-cavity in neuraminidase provides new opportunities for influenza drug development: A molecular dynamics simulation study

Influenza neuraminidase (NA) is a pivotal target for viral infection control. However, the accumulating of mutations compromise the efficacy of NA inhibitors. Thus, it is critical to design new drugs targeted to different motifs of NA. Recently, a new motif called 340-cavity was discovered in NA subtypes close to the calcium binding site. The presence of calcium is known to influence NA activity and thermostability. Therefore, the 340-cavity is a putative ligand-binding site for affecting the normal function of NA. In this study, we performed molecular dynamics simulations of different NA subtypes to explore the mechanism of 340-loop formation. Ligand-binding site prediction and fragment library screening were also carried out to provide evidence for the 340-cavity as a druggable pocket. We found that residues G342 and P/R344 in the 340-loop determine the size of the 340-cavity, and the calcium ion plays an important role in maintaining the conformation of the 340-loop through contacts with G345 and Q347. In addition, the 340-cavity is predicted to be a ligand-binding site by metaPocket, and a sequence analysis method is proposed to predict the existence of the 340-cavity. Our study shows that the 340-cavity is not an occasional or atypical domain in NA subtypes, and it has potential to function as a new hotspot for influenza drug binding.

Identification and Characterization of a New Alkaline SGNH Hydrolase from a Thermophilic Bacterium Bacillus sp. K91

est19 is a gene from Bacillus sp. K91 that encodes a new esterase. A comparison of the amino acid sequence showed that Est19 has typical Ser-Gly-Asn-His (SGNH) family motifs and could be grouped into the SGNH hydrolase family. The Est19 protein was functionally cloned, and expressed and purified from Escherichia coli BL21(DE3). The enzyme activity was optimal at 60°C and pH 9.0, and displayed esterase activity towards esters with short-chain acyl esters (C₂-C₆). A structural model of Est19 was constructed using phospholipase A1 from Streptomyces albidoflavus NA297 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of the typical catalytic triad Ser49-Asp227-His230, which were further investigated by site-directed mutagenesis. To the best of our knowledge, Est19 is a new member of the SGNH hydrolase family identified from thermophiles, which may be applicable in the industrial production of semisynthetic β-lactam antibiotics after modification.

Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase

A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0–9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30–32.5 °C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20 °C, respectively, and the Ea, ΔG* (30 °C), Km (30 °C) and kcat (30 °C) values for hydrolysis of sucrose by rInvHJ14 was 47.6 kJ mol−1, 57.6 kJ mol−1, 62.9 mM and 746.2 s−1, respectively. The enzyme also showed strong sucrose tolerance. rInvHJ14 preserved approximately 50% of its highest activity in the presence of 2045.0 mM sucrose. Furthermore, potential factors for low-temperature-active and alkaline adaptations of rInvHJ14 were presumed. Compared with more thermostable homologs, rInvHJ14 has a higher frequency of glycine residues and a longer loop but a lower frequency of proline residues (especially in a loop) in the catalytic domain. The catalytic pockets of acid invertases were almost negatively charged while that of alkaline rInvHJ14 was mostly positively charged.