Biochemical characterization and key catalytic residue identification of a novel alpha-agarase with CBM2 domain.

Agarooligosaccharides have great potential in food industry because of their various bio-activities, while the limited availability and diversity of α-agarases hinder agarooligosaccharides' broader application. To overcome this limitation, a computer-assisted method was used to screen and identify novel agarases. Firstly, one novel α-agarase, AgaB, with an N-terminal CBM2 domain (the first report of this domain in agarases), was discovered. Purified agarases only exhibited activity against agarose, with optimum activity at 40℃ and pH 8.0. Analysis of hydrolysis products indicated that AgaB is an endo-type α-agarase, producing agarotetraose and agarohexaose. Secondly, AgaB truncated CBM2 showed increased Km values, suggesting that CBM2 aids in substrate binding. Thirdly, E468 and D333 are possibly catalytic amino acids, which was supported by molecular docking results and mutants. Biochemical characterization of first reported CBM2-containing agarase and catalytic mechanism study lay the foundation for the exploration and development of α-agarases in the future.

Antimicrobial high molecular weight pectin polysaccharides production from diverse citrus peels using a novel PL10 family pectate lyase.

The discovery of environmentally friendly enzymes that can convert inexpensive and abundant citrus peel pectin into high value-added product is a potential avenue for the citrus peel application. In this study, a novel PL10-family pectate lyase (pelA) was characterized from marine bacterium Echinicola pacifica. PelA was a Ca2+ dependent pectate lyase whose activity was highest at pH 8 and 40 °C. It was capable of degrading polygalacturonic acid (PGA) and citrus peel pectin (CPP), but not apple peel pectin. Notably, PelA hydrolyzed PGA to high molecular weight polysaccharide (average molecular weight 111.4 kDa). Moreover, PelA was also able to degrade CPP from nine distinct citrus species into polysaccharides (average molecular weight ranging from 84.7 to 539.2 kDa) that showed antimicrobial activity against Staphylococcus epidermidis (88.8 %), Bacillus subtilis (99.8 %), Staphylococcus aureus (92.1 %), Escherichia coli (100.0 %) and Klebsiella pneumoniae (86.4 %). Considering the high market value of pectin in the food industry, PelA's capacity to convert citrus pectin into high molecular weight polysaccharides lays a foundation for its applications.