Search

Fucoidan oligosaccharides possesses diverse physicochemical and biological activities. Specific glycoside hydrolases are valuable tools for degrading polysaccharides to produce oligosaccharides. In this study, BcFucA, a novel fucosidase belonging to GH95 family from Bacillus cereus 2-8, was cloned into pET21a vector, expressed in E. coli BL21 (DE3) and characterized. The protein consists of 1136 amino acid residues encoded by 3411 bases and has a molecular weight of 125.35 kDa. The optimal temperature and pH of this enzyme are 50 °C and pH 4.0. In addition, this study showed that the unknown function domain (encoding Lys261-Thr681) defined as a linker is quite important for its activity. The obtained novel enzyme BcFucA will contribute to the effective degradation of fucoidan and future industrial applications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Metabolomics is a potent tool to assist in identifying the function of unknown genes through analysis of metabolite changes in the context of varied genetic backgrounds. However, the availability of a universal unbiased profiling analysis is still a big challenge. In this study, we report an optimized metabolic profiling method based on gas chromatography-mass spectrometry for Escherichia coli. It was found that physiological saline at -80°C could ensure satisfied metabolic quenching with less metabolite leakage. A solution of methanol/water (21:79, v/v) was proved to be efficient for intracellular metabolite extraction. This method was applied to investigate the metabolome difference among wild-type E. coli, its yfcC deletion, and overexpression mutants. Statistical and bioinformatic analysis of the metabolic profiling data indicated that the expression of yfcC potentially affected the metabolism of glyoxylate shunt. This finding was further validated by real-time quantitative polymerase chain reactions showing that expression of aceA and aceB, the key genes in glyoxylate shunt, was upregulated by yfcC. This study exemplifies the robustness of the proposed metabolic profiling analysis strategy and its potential roles in investigating unknown gene functions in view of metabolome difference., (Copyright © 2014 Elsevier Inc. All rights reserved.)

With the help of sepiolite, a unique method for transforming DNA into bacteria, based on the Yoshida effect, has been developed recently. However, we confronted many problems when this newest method was tried. Only a few transformants could be obtained even when 100 ng of plasmid pET15b was used, and a successful result seemed difficult to repeat. To address this problem, we optimized the operating method and could achieve about 15,000 transformants using the same amount of plasmid, which could match the efficiency gained using the calcium chloride transformation method. Meanwhile, the results could also be reproduced well. In the same way, carbon nanotubes were used to attain more than 15,000 transformants in the same situation. Therefore, the transformation method could be extended to other nanomaterials. Meanwhile, compared with the mechanism previously reported, we verified quite a different principle for the mechanism responsible for such a transformation. In sum, this unique transformation can be developed to become the third widely-used transformation method in laboratories in addition to the chemical method and electroporation.

Recombinant human interleukin-1 receptor antagonist (rHuIL-1ra) was produced in E. coli as an inclusion body. rHuIL-1ra was purified to Over 98% purity by anion exchange chromatography after on-column refolding. The optimized processes produced more than 2 g pure refolded rHuIL-1ra per 1 l culture, corresponding to a 44% recovery, without an intermediate dialysis step. Refolded rHuIL-1ra had full biological activity with the MTT assay. An intramolecular disulfide linkage in the oxidized recombinant protein was suggested by data from HPLC and non-reducing SDS-PAGE.

Mature human interleukin-11 (HuIL-11) is a cytokine consisting of 178 amino acid residues that results from scission of the N-terminal signal peptide, consisting of 21 amino acid residaues, from the corresponding nascent polypeptide. A DNA fragment encoding a truncated HuIL-11 (trHuIL-11), with an additional 5 amino acid residues removed from the N-terminus, was cloned into vector pGEX-2T between the BamHI site and the EcoRI site. Upon transformation with Escherichia coli BL21, the construct over-produced a glutathione S-transferase (GST)-fused protein in a soluble form after IPTG induction. The fusion protein was initially fractionated with butyl-Sepharose 4 fast flow column and by affinity chromatography using a GSH-Sepharose 4B column. On-site enzymatic release with thrombin gave the target protein at 96% purity as judged by SDS-PAGE and HPLC. Expression of the interleukin as a GST-fused protein thus greatly improved downstream processing. Subsequent biological activity assay suggested that trHuIL-11 had similar activity profile to the naturally produced sample and may be a promising candidate for further development as biopharmaceutical.

Konjac glucomannan oligosaccharide has attracted much attention due to its broad biological activities. Specific glucomannan degrading enzymes are effective tools for the production of oligosaccharides from konjac glucomannan. However, there are still few reports of commercial enzymes that can specifically degrade konjac glucomannan. The gene ppgluB encoding a glucomannanase consisting of 553 amino acids (61.5 kDa) from Paenibacillus polymyxa 3–3 was cloned and heterologous expressed in Escherichia coli BL21 (DE3). The recombinant PpGluB showed high specificity for the degradation of konjac glucomannan. Moreover, the hydrolytic products of PpGluB degrade konjac glucomannan were a series of oligosaccharides with degrees of polymerisation of 2–12. Furthermore, the biochemical properties indicated that PpGluB is the optimal active at 45 to 55 °C and pH 5.0–6.0, and shows highly pH stability over a very broad pH range. The present characteristics indicated that PpGluB is a potential tool to be used to produce oligosaccharides from konjac glucomannan. [ABSTRACT FROM AUTHOR]

Abstract: The nucleotide sequence encoding bovine enterokinase light chain (EK) from Chinese northern yellow bovine was isolated. Two single-nucleotide mutations, namely, C245G and A528T were identified. The gene encoding the Pro82Arg/Glu176Asp variant of known bovine EK was fused with glutathione S-transferase and overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with IPTG and glucose. Effective fusion protein purification, refolding, auto-catalytic cleavage and mature EK recovery were described. The specific activity of the purified EK was determined as 110±10U/mg, which was comparable to a specific activity of ⩾20U/mg of the E. coli expressed EK sample provided by Sigma (Cat. No. E4906). This procedure produced approximately 53mg of EK per 500mL of cell culture, which was much higher than previous reports, thus providing a basis for large-scale production of EK and for further applications in biotechnology. [Copyright &y& Elsevier]

Abstract: NAD+-dependent malic enzyme (NAD-ME) gene from Escherichia coli K12 was inserted into an expression vector pET24b(+) and transformed into E. coli BL21 (DE3). Recombinant NAD-ME was expressed upon IPTG induction, purified with affinity chromatography, and biochemically characterized. The results showed that recombinant NAD-ME could be produced mainly in a soluble form. The monomeric molecular weight of recombinant NAD-ME was about 65kDa, whereas monomer, homotetramer, and homooctamer were formed in solution as revealed by nondenaturing polyacrylamide gel electrophoresis analysis. Finally, the K m values of NAD-ME for l-malate and NAD were determined as 0.420±0.174 and 0.097±0.038mM, respectively, at pH 7.2. By using this over-expression and purification system, recombinant E. coli K12 NAD-ME can now be obtained in large quantity necessary for further biochemical characterization and applications. [Copyright &y& Elsevier]

In this study, we isolated a new psychrophilic bacterium, Pseudoalteromonas sp. DL-6 from marine sediments, which grew well on chitin-containing plates at 4 °C. One endo-type chitinase gene, chiA , was cloned from the genomic DNA of this bacterium and heterologously expressed in Escherichia coli BL21 (DE3). ChiA showed very high catalytic activity, even at 4 °C, and exhibited maximal activity on a chitinous substrate at pH 8.0 and 20 °C. Kinetic studies indicated that ChiA has a greater catalytic efficiency on 4-methylumbelliferyl-β- d - N , N ′, N ″-triacetylchitotriose[4-MU(GlcNAc) 3 ] than on 4-methylumbelliferyl-β- d - N , N ′-diacetylchitobioside[4-MU(GlcNAc) 2 ]. Electrospray ionisation mass spectrometry (ESI-MS) analysis showed that the hydrolysis products of powdered chitin after ChiA digestion consisted of a series of chitin oligomers with different degrees of polymerisation. The ChiA mode of action was also examined using (GlcNAc) 2–6 as a substrate, and the results suggested that ChiA is a non-processive endo-type chitinase. [ABSTRACT FROM AUTHOR]

A new oligoalginate lyase encoding gene, designed oal17A , was cloned from marine bacterium Vibrio sp. W13, and then expressed in Escherichia coli . The recombinant Oal17A was purified by NTA-Ni resin with maximal activity at 30 °C and pH 7.0. Oal17A exhibited broad substrate specificity, and preferred to degrade alginate than polyM or polyG into monosaccharide acid. The specific activity of Oal17A toward alginate, polyM and polyG was 21.14 U/mg, 12.31 U/mg and 7.43 U/mg, respectively. With features of high-level expression and broad substrate specificity, Oal17A would be a potential tool for alginate monomer production process of alginate utilizing for biofuels and bioethanol production. [ABSTRACT FROM AUTHOR]