Yanbing Zhu

Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1

The gene (741 bp) encoding carboxylesterase from the thermophilic bacterium Geobacillus sp. ZHl was cloned and overexpressed in Escherichia coli. The purified recombinant protein presented a molecular mass of about 40 kDa by SDS-PAGE analysis. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. Among the p-nitrophenyl esters tested, the carboxylesterase presented preference for p-nitrophenyl caprylate, but hydrolyzed p-nitrophenyl butyrate more efficiently. When p-nitrophenyl butyrate was used as a substrate, the recombinant carboxylesterase exhibited highest activity at pH 8.0 and 60°C. Almost no decrease in esterase activity was observed at 60°C for 3 h, and over 40% of activity was still maintained after incubation at 90°C for 3 h. These results indicate that Geobacillus sp. ZH1 recombinant esterase was thermostable. The enzymatic activity was inhibited by the addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism. Except SDS and xylene, this esterase showed stability toward other tested detergents and organic solvents. Cloning, expression, and biochemical characterization of Geobacillus sp. ZH1 carboxylesterase lay a good foundation for its structural characterization and industrial application.

Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates

A novel alginate-degrading marine bacterium Microbulbifer sp. ALW1 was isolated from rotten brown alga. An extracellular alginate lyase was purified to electrophoretic homogeneity and had a molecular mass of about 26.0 kDa determined by SDS-PAGE and size exclusion chromatography. This enzyme showed activities towards both polyguluronate and polymannuronate indicating its bifunctionality while with preference for the former substrate. Using sodium alginate as a substrate, strain ALW1 alginate lyase was optimally active at 45 °C and pH 7.0. It was stable at 25 °C, 30 °C, 35 °C and 40 °C, but not stable at 50 °C. This alginate lyase showed good stability over a broad pH range (5.0-9.0). The enzyme activity was increased to 5.1 times by adding NaCl to a final concentration of 0.5M. Strain ALW1 alginate lyase produced disaccharide (majority) and trisaccharide from alginate indicating that this enzyme could be a good tool for preparation of alginate oligosaccharides with low degree of polymerization (DP). The alginate oligosaccharides displayed the scavenging abilities towards radicals (DPPH, ABTS(+) and hydroxyl) and the reducing power. Therefore, the hydrolysates exhibited the antioxidant activity and had potential as a natural antioxidant.

Characterization of an alkaline β-agarase from Stenotrophomonas sp. NTa and the enzymatic hydrolysates

An extracellular agarase from marine bacterium Stenotrophomonas sp. NTa was purified to homogeneity. By size exclusion chromatography and SDS-PAGE analysis, the enzyme was determined to be a homodimer with monomeric molecular mass of 89.0 kDa. The optimal temperature and pH of strain NTa agarase were 40 °C and 10.0, respectively. It exhibited striking stability across a wide pH range of 5.0-11.0. Agarase from Stenotrophomonas sp. NTa had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. The Km and Vmax for agar were 11.3mg/ml and 25.4 U/mg, respectively. Thin layer chromatography analysis, mass spectrometry, and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that strain NTa agarase was a β-agarase that degraded agarose into neoagarobiose, neoagarotetraose and neoagarohexaose as the predominant products, as well as a small amount of 3,6-anhydro-α-L-galactose. This is the first to present evidence of agarolytic activity in strain from genus Stenotrophomonas.

Expression and biochemical characterization of recombinant α-l-rhamnosidase r-Rha1 from Aspergillus niger JMU-TS528

A putative cDNA of α-l-rhamnosidase was PCR-cloned from Aspergillus niger JMU-TS528 and further extracellular over-expressed in Pichia pastoris GS115. The activity of the recombinant α-l-rhamnosidase r-Rha1 was 711.9U/mL, eightfold higher than the native α-l-rhamnosidase from A. niger JMU-TS528. r-Rha1 is a N-glycosylated protein of 90kDa and possesses broad substrate specificities by hydrolyzing α-1,2, α-1,3 α-1,4, and α-1,6 linkages to β-d-glucosides. This is the first report presenting that α-l-rhamnosidase showed activity on four kinds of glucosidic linkages. Compared with other previously characterized α-l-rhamnosidases, r-Rha1 showed a good thermostability and wide range of pH-stability with the optimum pH of 5.0 and temperature of 60°C. r-Rha1 activity was not greatly affected by representative metal ions and other detected effectors and showed excellent tolerance abilities against glucose and ethanol. These beneficial characteristics of r-Rha1 suggest that r-Rha1 should be considered a potential new biocatalyst for food and drug industrial applications.

Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6

A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249‐amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p‐nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p‐nitrophenyl butyrate. When p‐nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H‐257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X‐100, sodium deoxycholate, urea, and guanidine hydrochloride.

Characterization of an arylsulfatase from a mutant library of Pseudoalteromonas carrageenovora arylsulfatase

A library of Pseudoalteromonas carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, one mutant strain was obtained whose arylsulfatase had improved thermal stability. Protein sequence analysis revealed one amino acid substitution of H260L. The mutant arylsulfatase (named H260L) retained higher residual activity than wild-type enzyme (named WT) after incubation at 45, 50, 55 and 60°C for 60min. Thermal inactivation analysis showed that the half-life (t1/2) value at 55°C for H260L was 40.6min, while that of WT was 9.1min. When p-nitrophenyl sulfate was used as a substrate, the optimal reaction temperature and pH for the mutant enzyme were 55°C and pH 8.0, respectively. H260L was stable over the pH range of 6.0-9.0. Inhibition assay with EDTA indicated that metal ions play an important role during the catalytic process of the mutant enzyme. The desulfation ratio against agar of Gracilaria lemaneiformis was 82%.

Improvement thermostability of Pseudoalteromonas carrageenovora arylsulfatase by rational design

This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A total of 10 single-site mutants were chosen using the PoPMuSiC program, and two mutants of K253N and P314T showed enhanced thermal stability. By saturation mutagenesis and thermostability analysis, K253H and P314T were the best mutants at the two sites. Combinational mutations of K253H, P314T and H260L were subsequently introduced, and the best mutant of K253H/H260L was selected. Thermal inactivation analysis showed the half-life (t1/2) value at 55°C for K253H/H260L was 7.7-fold that of the wild-type enzyme (WT), meanwhile this mutant maintained the specific enzyme activity. Structure modeling demonstrated that the additional hydrogen bonds, optimization of surface charge-charge interactions, and increasing of hydrophobic interaction could account for the improved thermostability imparted by K253H/H260L.

Purification and biochemical characterization of manganesecontaining superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

Thermostable SOD is a promising enzyme in biotechnological applications. In the present study, thermophile Geobacillus sp. EPT3 was isolated from a deep-sea hydrothermal field in the East Pacific. A thermostable superoxide dismutase (SOD) from this strain was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography, and Phenyl-Sepharose chromatography. SOD was purified 13.4 fold to homogeneity with a specific activity of 3 354 U/mg and 11.1% recovery. SOD from Geobacillus sp. EPT3 was of the Mn-SOD type, judged by the insensitivity of the enzyme to both KCN and H2O2. SOD was determined to be a homodimer with monomeric molecular mass of 26.0 kDa. It had high thermostability at 50°C and 60°C. At tested conditions, SOD was relatively stable in the presence of some inhibitors and denaturants, such as β-mercaptoethanol (β-ME), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), urea, and guanidine hydrochloride. Geobacillus sp. EPT3 SOD showed striking stability across a wide pH range from 5.0 to 11.0. It could withstand denaturants of extremely acidic and alkaline conditions, which makes it useful in the industrial applications.

β-Agarase immobilized on tannic acid-modified Fe3O4 nanoparticles for efficient preparation of bioactive neoagaro-oligosaccharide

β-Agarase was immobilized by using tannic acid modified-Fe3O4 magnetic nanoparticles (TA-MNPs) as a supportmaterial. The MNPs were synthesized by improved chemical coprecipitation method and modified with TA for agarase immobilization. TA-MNPs and immobilized β-agarase were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and thermal gravimetric analysis (TGA), all of which indicated the successful surface modification of MNPs with TA and the immobilization of β-agarase. The optimal immobilization conditions for 25 mg TA-MNPs included 100 r/min oscillation speed, immobilization time of 2 h, immobilization temperature of 15 °C, and initial β-agarase concentration of 3 mL (480 U). Immobilized β-agarase showed better pH and thermal stability and excellent reusability than the free enzyme. Results revealed the promising application of β-agarase-TA-MNPs for the preparation of neoagaro-oligosaccharides with different averagepolymerizationdegrees and varying activities in the antioxidant.

Improving the thermostability by introduction of arginines on the surface of α-L-rhamnosidase (r-Rha1) from Aspergillus niger

To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutants enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry.