Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus.

Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 ± 0.6 U mL(-1) of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 °C and an optimum pH of 4.5. It had a K m of 0.322 mM and a V max of 4317 µM min(-1) mg(-1) protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 °C for 1 h or at pH ranging from 3.0 to 6.0 at 4 °C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase.

Characterization of a new endo-type polyM-specific alginate lyase from Pseudomonas sp.

An alginate lyase gene, algA, encoding a new poly ß-D-mannuronate (polyM)-specific alginate lyase AlgA, was cloned from Pseudomonas sp. E03. The recombinant AlgA with (His)6-tag, consisting of 364 amino acids (40.4 kDa),was purified using Ni-NTA Sepharose. The purified lyase had maximal activity (222 EU/mg) at pH 8 and 30 °C and also maintained activity between pH 7-9 and below 45 °C. It exclusively and endolytically depolymerized polyM by ß-elimination into oligosaccharides with degrees of polymerization (DP) of 2-5. Due to its high substrate specificity, AlgA could be a valuable tool for production of polyM oligosaccharides with low DP and for determining the fine structure of alginate.

[Sequence analysis of 16S rDNA and genes of soluble methane monooxygenase from Methylomonas sp. GYJ3].

Soluble methane monooxygenase (MMO) from methanotrophs is a member of binuclear iron-containing multicomponent oxygenases, which can catalyze bioconversion of methane to methanol at ambient temperature and regulate methane recycle in nature. The research focused mainly on the sequence analysis of 16S rDNA and sMMO genes from Methylomonas sp. GYJ3. With the aid of the information from GenBank, the PCR primers and the sequence primers were designed, obtained a 5690bp of sMMO fragment and a 1280bp of 16S rDNA. Sequence comparison for MMOX with counterpart of other five strains showed that from 78% to 99% identity in protein level and from 71 % to 97% identity in gene level, in the separate comparison of six components, only orfY component had a lower identical. The multiple alignment of MMOX amino acid sequence with other four strains showed that there is a high conservation, especially in two Fe binding regions. 16S rDNA phylogenetic analysis demonstrated that Methylomonas sp. GYJ3 is relative with gamma proteobacteria. Phylogenetic analysis of MMOX amino acid sequence showed that Methylomonas sp. GYJ3 is closer to Methylomonas sp. KSW III of type I methanotrophs. It was concluded that Methylomonas sp. GYJ3 is belong to the genus of type I methanotroph Methylomonas, and the result was a direct evidence for the sMMO can be expressed in type I methanotrophs. The theoretical pI of hydroxylase was 6.28 and the theoretical MW of hydroxylase was 248874.41Da.