Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure.

Clostridium sp. G0005 glucoamylase (CGA) is composed of a ß-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.

Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification.

The biochemical properties of the maltodextrin-hydrolyzing enzymes of cold-tolerant proteobacterium Caulobacter crescentus CB15 remain to be elucidated, although whose maltodextrin transport systems were well investigated. We cloned the putative glucoamylase of C. crescentus CB15 (CauloGA) gene. The CauloGA gene product that was expressed in E. coli was prone to forming inclusion bodies; however, most of the gene product was expressed in a soluble and active form when it was expressed as a fusion protein with Staphylococcus Protein A. The fusion protein was purified using an IgG Sepharose column and was identified as the active GA. The optimum temperature and pH for the activity of this GA toward maltotriose as a substrate were approximately 40°C and 5.0, respectively, and a differential scanning fluorimetry (DSF) analysis revealed that the melting temperature (Tm) of CauloGA was 42.9°C. The kinetic analyses with maltotriose and other maltodextrins as the substrates indicated that CauloGA has higher kcat and smaller Km values at 30°C with both substrates compared with other GAs at lower substrate concentration. However, the enzyme activities toward the substrates decreased as the substrate concentrations increased at concentrations higher than approximately 10-fold the Km. The function-based identification of thermolabile Caulobacter GA contributes to the understanding of the maltodextrin-degradation system of C. crescentus as well as the bacterial GA's function-structure relationship.

Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein.

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.