Gene Cloning and Characterization of an α-Amylase from Alteromonas macleodii B7 for Enteromorpha Polysaccharide Degradation

Abstract

Enteromorpha polysaccharides (EP) extracted from green algae have displayed a wide variety of biological activities. However, their high molecular weight leads to a high viscosity and low solubility, and therefore, greatly restrains their application. To solve this problem, bacteria from the surface of Enteromorpha were screened, and an Alteromonas macleodii strain B7 was found to be able to decrease the molecular weight of EP in culture media. Proteins harvested from the supernatant of the A. macleodii B7 culture were subjected to native gel electrophoresis, and a band corresponding to the Enteromorpha polysaccharide lyase (EPL) was detected by activity staining. The enzyme identity was subsequently confirmed by MALDI-TOF/TOF mass spectrometry as the putative α-amylase reported in A. macleodii ATCC 27126. The amylase gene (amySTU) from A. macleodii B7 was cloned into Escherichia coli, resulting in highlevel expression of the recombinant enzyme with EP-degrading activity. AmySTU was found to be cold-adapted; however, its optimal enzyme activity was detected at 40°C. The α-amylase was highly stable over a broad pH range (5.5-10) with the optimal pH at 7.5-8.0. The highest enzyme activity was detected when NaCl concentration was 2%, which dropped by 50% when the NaCl concentration was increased to 16%, showing an excellent nature of halotolerance. Furthermore, the amylase activity was not significantly affected by tested surfactants or the presence of some organic solvents. Therefore, the A. macleodii strain B7 and its α-amylase can be useful in lowering EP molecular weight and in starch processing.

Identification of Cultivable Bacteria from Tropical Marine Sponges and Their Biotechnological Potentials

“…Among the identified species, the most common biotechnological function was associated with bioremediation, while subsequent common capabilities were plant growth promotion, plant systemic resistance, and biological syntheses of biocompatible nanometal and biodegradable polymers. We would like to note several interesting species that harbour unique abilities or products, namely lead, cadmium and zinc biosorption mechanism in A. macleodii ( Loaëc et al 1997 ); halotolerant and psychrotolerant α-amylase with broad pH tolerance in A. macleodii ( Han et al 2014 ); skeletal muscle relaxation properties and central nervous system (CNS) depressants in B. cereus ( Kumar et al 2014 ); antileukaemic and antineoplastic L-asparaginase in B. aryabhattai ( Singh & Srivastava 2014 ); polyhydroxyalkanoate (PHA) synthesis in P. putida ( Wang & Nomura 2010 ); biodegradation of highly explosive pentaerythritol tetranitrate (PETN) by E. cloacae ( Binks et al 1996 ); as well as antibacterial, immunosuppressive and anticancer prodigiosin in P. denitrificans ( Sertan-de Guzman et al 2007 ).…”

Enhanced Fermentable Sugar Production from <b><i>Enteromorpha</i></b> Polysaccharides by the Crude Enzymes of <b><i>Vibrio</i></b> sp. H11

“…A321 [Li et al, 2013] was recently reported. A. macleodii B7 from the surface of E. prolifera decreased the molecular weight of EP in culture media [Han et al, 2014]. Alteromonas sp.…”

“…The process of hydrolyzing EP requires a different set of enzymes. In this regard, the microbial hydrolysis of EP by Alteromonas macleodii B7 [Han et al, 2014] and Alteromonas sp. A321 [Li et al, 2013] was recently reported.…”

“…Extraction of EP from E. prolifera EP was separated from E. prolifera collected from Zhoushan (China), according to the methods of Han et al [2014] with minor modifications. Dried E. prolifera samples were ground into pow-der and filtered through 40-mesh sieves.…”

“…The hydrolytic capability of EP was examined through the analysis of enzymatic activity using the 3,5-dinitrosalicylic acid (DNS) method [Han et al, 2014]. Centrifuged supernatants (1 mL) from each culture condition were incubated with 10 g/L of EP (1 mL) at 40 ° C for 30 min, and incubated at 100 ° C for 15 min, following the addition of 2 mL of DNS reagent.…”