Scale-up and genomics of alginase synthesis by Microbulbifer degradans, isolate 2-40.

摘要

Marine bacterium, Microbulbifer degradans strain 2-40 (2-40) was isolated from decaying salt marsh grass, Spartina alterniflora , in the Chesapeake Bay. 2-40 is capable of degrading numerous insoluble complex polysaccharides (ICP) including alginic acid, agar, cellulose, chitin, glucan, pectin, pullulan, starch and xylan.; Alginases are important in the marine carbon cycle and, commercially, in the bioremediation of algalculture wastes. The specific aims of this study were to: (A) optimize the growth medium and conditions for maximum alginase production; (B) scale up batch shake flask protocols to fermentor conditions; (C) concentrate and purify alginases; (D) characterize and define the best reaction conditions of the purified alginases; (E) carry out the genomics of alginases of 2-40; and (F) analyze functional genomics of 2-40 alginases.; The growth medium composition was optimized for maximum alginase production. The semidefined medium contained: 0.45% Alginate, 0.05% Glucose, 0.2% Yeast Extract, and 3.5% Instant Ocean. It induced the production of 1354 units of alginase activity after 39 hrs in batch culture, and 1690 units after 36 hrs in fermentors. This is a 143% increase over alginase production from the original 0.2% alginate minimal medium.; SDS-PAGE of 2-40 culture concentrated supernatant showed 6 alginases with molecular weight of 12, 56, 62, 81, 125, and 165 kDa. The 56kDa alginase had the greatest activity in gel zymograms. A Lineweaver-Buck plot was used to calculate the V_max, and K_m for the purified alginase preparation. These values were 90μg reducing sugar/min and 0.054mM, respectively.; Analysis of 2-40 sequenced genome showed that 2-40 has 11 alginase-coding genes; these were termed AlgA through AlgK. Their calculated molecular weights ranged from 32 to 163 kDa. AlgC, D, E and F depolymerized alginate in zymogram. With exception to AlgA and AlgK, all of alginase proteins had a secretion signal peptide. AlgD, E, G, H and J had known carbohydrate-binding-domains. AlgE and AlgJ had Threonine-Proline rich domains (22–39 amino acid residues) and AlgD had a Serine rich domain (24 residues). AlgB, AlgF and AlgH contained 4-9 repeats of parallel beta helix (21–35 residues). Two putative alginase operons were identified in the 2-40 genome. Each was comprised of 3 alginase genes. Cluster one was comprised of AlgA, AlgB and AlgC, and cluster two was comprised of AlgH, AlgI and AlgJ. The other alginases were scattered within the genome.