Indentification And Expression Of The Gene PelB, AmyX And YvdF From Bacillus Licheniformis

B. licheniformis was thought an industrial organism which has such good characters as anti-heat, nonpathogenic, abundant enzyme system. The genome sequence of B. licheniformis was published in 2004, which provided an important material to the development of its potential applications. In this paper, cloning and expression of the gene pelB, amyX and yvdF in different hosts were investigated and ecombinant enzymes were characterized, respectively.A complete structure gene pelB which probably encoded pectinase was cloned from B. licheniformis CICIM B1699 by polymerase chain reaction. The recombinant plasmids pLa-PQ-pelB and pHY-PQ-pelB were constructed with pelB under the control of PQ promoter. The pectinase was successfully expressed in Escherichia coli JM109 via the mediation of plasmid pLa-PQ-pelB. The optimal pH and temperature of recombinant pectinase were pH 10.5 and 45℃. The activity of enzyme was enhanced by Ca2+ and strongly inhibited by ions of Mg2+, Zn2+ and Cu2+. Under the different concentration of Ca2+, the optimum concentration was 6 mmol/L. The products from polygalacturonic acid degraded by enzyme were analyzed by electrospray ionization mass spectrometry (ESI-MS). Unsaturated bigalacturonic acid and unsaturated trigalacturonic acid were found. B. licheniformis CICIM B1699 carrying pHY-PQ-pelB produced 4.2 U/mL pectinase, which was about 60% higher than that of parent strain.Analysis of the complete genome sequence of B. licheniformis 14580 revealed that there were two genes amyX and yvdF, which probably encoded pullulanase. The recombinant plasmids of pHY-P43-amyX and pHY-P43-yvdF were constructed and expressed in Bacillus subtilis 1A717. The AmyX as intracellular enzyme were detected and the YvdF showed the enzyme activity of 4.6 U/mL in B. subtilis 1A717. The hydrolytic characters of YvdF were observed that the recombinant belonged to type I pullulan hydrolases. The enzymatic properties of the recombinant enzymes were analyzed systemically in the study. Pullulan hydrolysis activity was optimal at pH 6.5 and 45℃. Metal ions were needless in the enzymatic reaction, however, the pullulanase activity was improved more or less by the addition of some ions such as Mg2+,Mn2+,Li+ and Na+, and was inhibited by the addition of Zn2+,Cu2+,Co2+,EDTA and SDS. The recombinant B. licheniformis CICIM B1699 with pHY-P43-yvdF produced 6.8 U/mL, which was 2-fold higher than that of the control strain.