Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region.

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.

Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence.

Phylogenetic analysis of archaea in the rumen ecosystem was analysed by PCR of 16S rDNA from the bovine rumen using archaea-specific primers. The libraries were constructed from rumen fluid (AF), rumen solid (AS), and rumen epithelium (AE) from a rumen-fistulated Korean cow (Hanwoo). The 45 AF clones could be divided into three groups and the largest group was affiliated with the Methanomicrobiaceae family (96% of clones). The AF clones contained a high proportion of unidentifiable clones (67%). The 39 AE clones could be divided into two groups and the largest group was also affiliated with the Methanomicrobiaceae family (95% of clones). The AE clones contained a low proportion of unidentifiable clones (5%). The 20 AS clones could be divided into two groups that were affiliated with either the Methanobacteriaceae family (55%) or the Methanomicrobiaceae family (45%). The AS clones contained a moderate proportion of unidentifiable clones (40%). The predominant family of whole rumen archaea was found to belong to the Methanomicrobiaceae (85%). Methanomicrobiaceae were predominant in the rumen epithelium and the rumen fluid while Methanobacteriaceae were predominant in the rumen solid. One clone from the rumen fluid and two clones from the rumen epithelium contained rDNA sequences of Non-Thermophilic-Crenarchaeota (NTC) and Thermophilic-Crenarchaeota (TC), respectively, which have not previously been described from the rumen.