Screening of pectinase producing bacteria and assessment of the effectiveness for biopulping of paper mulberry bark of the pectinase of the highest producer were carried out. Pectinolytic bacteria were initially screened from 6 identified and 118 unknown isolates. Twelve strains gave positive results, including 3 of Erwinia carotovora subsp. carotovora, 2 of Erwinia chrysanthemi and 7 of Bacillus sp.. Crude pectinases were prepared from the selected strains. Then, the activity of 3 pectinase types, namely polygalacturonase (PG), pectate lyase (PAL) and pectin lyase (PL), was investigated. The results showed the highest PG production from E. chrysanthemi strain N05 (342-11) isolated from onion and highest PAL and PL production from Bacillus sp. strain N10 isolated from paper mulberry bark. Both N05 and N10 possess similar optimum conditions at pH 10.0 and 35 o C, and were stable at pH 3-12 for 30 minutes and 20-40 o C for 24 h. Furthermore, the efficiency of Bacillus sp. strain N10 pectinase in biopulping of paper mulberry bark was studied. The bark was pretreated by soaking for 24 h in distilled water, 0.5% NaOH solution, or 1.0% NaOH solution or not pretreated. Then it was incubated with 20,000, 40,000, 100,000 and 200,000 units of enzyme solution and compared to water and NH 4 Cl-NH 4 OH buffer controls. Bark without any pretreatment, which was incubated with 100,000 unit of the enzyme had the maximum level of reducing sugar released, the softest pulp and fibers that were clearly separated when observed under the scanning electron microscope. Chemical composition analysis of the pulp showed slight increases in holocellulose 5%, alpha-cellulose (2%) and hemicellulose (3%) contents, whereas lignin content decreased by 50% when compared to the nontreated bark.

A novel PCR assay based on 16S-23S internal transcribed spacers (ITS) length polymorphism was developed for rapid differentiation and identification of the Bacillus subtilis group, especially B. subtilis, B. licheniformis and B. amyloliquefaciens, the most frequently isolated bacilli from fermented foods. A new group-specific conserved primer pair, CITS-F and CITS-R, was designed for specific amplification of the ITS region in B. subtilis and closely related species. The fingerprints of seven reference species, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. atrophaeus, B. vallismortis and B. mojavensis using CITS-F and CITS-R primers showed the same four signature bands of 227, 400, 542 and 650 bp. They are different from those of other genera and species tested. Therefore, these four signature bands could be used to differentiate and identify the B. subtilis group. Moreover, the sequence of the 227 bp signature band could also be used to distinguish closely related species of the B. subtilis group used in this study. A novel PCR assay based on ITS length polymorphism pattern using CITS-F and CITS-R could be considered as a rapid and easy method for the primary differentiation of the B. subtilis group.

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