Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp.AG1

An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.

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. ZH1 was cloned and overexpressed in Escherichia coll. 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℃. Almost no decrease in esterase activity was observed at 60℃ for 3 h, and over 40% of activity was still maintained after incubation at 90℃ 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.

Substitution of His260 residue alters the thermostability of Pseudoalteromonas carrageenovora arylsulfatase

This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, two mutants of H260L and D84A/H260L showed enhanced thermal stability than the wild-type predecessor (WT). Site-directed mutagenesis demonstrated that only amino acid residue at Position 260 plays an important role in the thermostability of P. carrageenovora arylsulfatase. Thermal inactivation analysis showed that the half-life (t1/2) values at 55°C for H260L, H260I, H260Q, H260F and H260R were 40.6, 48.4, 30.9, 29.1 and 34.5 min, respectively, while that of WT was 9.1 min. Structure modeling demonstrated that the additional hydrogen bonds and/or optimization of surface charge-charge interactions could be responsible for the increased thermostability imparted by H260L, H260I, H260Q, H260F and H260R.