New Ionic Liquids with Buffering and Chelating Abilities for Enzyme Engineering

Ionic liquids (ILs) with buffering and chelating abilities were designed and synthesized on the basis of ethylenediaminetetraacetic acid (EDTA) for the development of buffered enzymatic IL systems and for enzymatic reaction in heavy metal containing aqueous system. Transesterification activity of Candida antarctica lipase B dissolved in the hydroxyl-functionalized IL was buffer dependent. High activity and outstanding stability was obtained with the buffered enzymatic IL systems for the transesterification. In heavy metal containing aqueous system, EDTA IL buffers as Hg2+ chelators protected horseradish peroxidase (HRP) against Hg2+-induced denaturation and precipitation. Higher pH favored the protection, while at lower pH the protection diminished. We can conclude that the new ILs possess both buffering and chelating abilities.

Rapid biosorption and reduction removal of Cr(Ⅵ) from aqueous solution by dried seaweeds

Four types of common seaweeds(Laminaria japonica,Undaria pinnatifida,Porphyra haitanensis,and Gracilaria lemaneiformis) were examined to remove Cr(Ⅵ) ions from aqueous solution.The experimental parameters that affected the biosorption process including pH,biomass dosage,contact time and temperature were investigated via batch experiments.The surface characteristics of seaweeds before and after Cr(Ⅵ) adsorption were studied with scanning electron microscopy and Fourier transform infrared spectroscopy.The results show that an initial solution with the pH of 1.0 is most favorable for Cr(Ⅵ) adsorption.Rapid adsorption is observed in the initial stage and adsorption equilibrium state is reached within 1 h.The adsorption efficiency by Porphyra haitanensis is the maximum among four types of seaweed powders,followed by Laminaria japonica and Undaria pinnatifida with biosorption efficiency up to 90%.The removal rate of Gracilaria lemaneiformis is less than 60%.The kinetic data obtained using the seaweeds are found to follow pseudo-second order kinetic model.Experimental sorption data adequately correlate with the Langmuir model.FTIR indicates that amino and carboxyl groups play an important role in the process of Cr(Ⅵ) adsorption and a large percentage of Cr(Ⅵ) ions are reduced by reductive groups on the surface of seaweeds.

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.

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, thermo-phileGeobacillussp.EPT3 was isolated from a deep-sea hydrothermal field in the East Pacific. A thermo-stable superoxide dismutase （SOD） from this strain was purified to homogeneity by steps of fractional am-monium 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 fromGeobacillussp. 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＆#176;C and 60＆#176;C. At tested conditions,SOD was relatively stable in the presence of some inhibitors and denaturants, such asβ-mercaptoethanol （β-ME）, dithiothreitol （DTT）, phenylmethylsulfonyl fluoride （PMSF）, urea, and guanidine hydrochloride.Geobacillussp. 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.

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.

Transformation of coccolithophorid Emiliania huxleyi harboring a marine virus(Coccolithoviruses)serine palmitoyltransferase(SPT)gene by electroporation

Emiliania huxleyi is the most prominent modern coccolithophore,a group of marine unicellular eukaryotes that play a critical role in ocean biogeochemistry.Coccolithoviruses are large double stranded DNA viruses,which is responsible for the demise of large oceanic blooms formed by E.huxleyi.E.huxleyi virus(EhVs)acquired a series of enzyme-coding genes predicted to be involved in the sphingolipid biosynthesis by horizontal gene transfer between virus-host.Currently,there is limited experimental validation identifying the functions of these genes in EhV.Genetic transformation of eukaryotic cells is a powerful tool to get an insight into gene functions of the studied organisms.Serine palmitoyltransferase(SPT)catalyzes the first committed step in de novo sphingolipid biosynthetic pathway.Here,a novel vector system for the transformation of E.huxleyi was designed.It contained fragments of promoter and terminator sequences of E.huxleyi endogenic fucoxanthin chlorophyll a/c-binding protein gene“fcp”and harbored EhV-99B1 spt gene.The resultant recombinant transformation vectors pEhux-I-spt and pEhux-II were co-transferred into E.huxleyi BOF92 by electroporation.Transformants were obtained upon glufosinate-ammonium selection,and confirmed by Southern hybridization,genome PCR,qRT-PCR and Western blot screening of spt gene,which indicated that spt gene was integrated into the nuclear genome and was expressed at the mRNA and protein levels.The expression of the viral spt gene led to differences in lipid compositions analyzed using thin-layer chromatography(TLC).The results present the genetic transformation system for E.huxleyi,providing additional genetic resource with potential for exploring basic biological questions such as the virus-host interactions.

Green synthesis of Au@WSe_(2) hybrid nanostructures with the enhanced peroxidase-like activity for sensitive colorimetric detection of glucose

We report a colorimetric method for glucose detection based on Au nanoparticle-decorated WSe_(2)(Au@WSe_(2))hybrid nanostructures.These hybrid structures are easily synthesized by simply stirring HAuCl_(4) precursor with WSe_(2) nanosheets in aqueous solution.Owing to strong synergistic catalytic effects of Au nanoparticles and WSe_(2) nanosheets,the Au@WSe_(2) hybrid nanostructures exhibit enhanced peroxidase-like activity(about 2-fold higher compared to WSe_(2) nanosheets alone)for 3,3',5,5'-tetramethylbenzidine oxidation by H_(2)O_(2).Based on the highly catalytical property,the colorimetric method for glucose detection is established by coupling glucose oxidase(GOx).The detection limit of glucose is 3.66 pM.Moreover,the proposed colorimetric method is applicable to glucose detection in serum samples and is promising for applications in biomedical fields.

Gold nanoparticle-decorated MoSe_(2) nanosheets as highly effective peroxidase-like nanozymes for total antioxidant capacity assay

A visual colorimetric detection strategy is reported for total antioxidant capacity(TAC)assay by using 3,3',5,5'-tetramethylbenzidine(TMB)oxidation as chromogenic substrate based on gold nanoparticle-decorated MoSe_(2) nanosheets(Au@MoSe_(2)).Au@MoSe_(2) nanostructures exhibit high peroxidase-like activity and can catalyze H_(2)O_(2)to oxidize TMB.Based on inhibition effect of ascorbic acid(AA)on TMB oxidation,a facile and sensitive colorimetric method was developed for AA detection.Under optimal conditions,the proposed method showed a sensitivity for AA in a concentration range from 2 to 120μM and limit of detection was 0.41μM.Furthermore,the method was employed for TAC assay in actual samples,including commercial beverages and vitamin C tablets.This work represents a model in nanostructure design and will lead to further development of TAC assay in evaluation of antioxidant food quality.