Purification, Characterization, Molecular Cloning, And Expression Of Agarase From Thalassomonas Sp. LD5

Agarose, an important marine polysaccharide present in the cell walls of some red algae, islimited to be used due to its high molecular weight, low solubility and absorption rate.Agaro-oligosaccharides can be prepared conveniently through acid hydrolysis of agar. However,the hydrolysis product is not homogenous and the hydrolyzing reaction is uncontrollable.Therefore, acid hydrolysis is not appropriate to large-scale preparation for oligosaccharides withhigh purity. So enzymatic degradation of polysaccharides should be a promising alternative to acidhydrolysis with high specificity and under mild conditions. Most known agarases are β-agarases,with the exception of two α-agarases found from Alteromonas agarlyticus GJ1B andThalassomonas sp. JAMB-A33. To date the research on α-agarase still remains vacant in ourcountry. Looking for new type of α-agarases can not only fill the blank of our country in this field,but will also enrich the resources of glycoside hydrolases and promote the high-value use ofagar-polysaccharides.In this report, we isolated a marine agar-degrading bacterium LD5. Judging from the16SrDNA sequence, it was classified into Thalassomonas sp. When cultured on plate with agar as thesole carbon source, a degrading hole which could reach the bottom of the plate was observed.Causing its oligosaccharides product formed continuously distributed on the TLC thin layer, wesupposed that both α-agarase and β-agarase could be secreted by Thalassomonas sp. LD5.An agarase was purified to homogeneity from the culture supernatant by hydrophobicchromatography （HiTrap Phenyl HP）, anion-exchange chromatograpy （HiTrap Q FF） andgel-filtration chromatography. SDS-PAGE analysis indicated that its molecular mass was75kDa,which was in accordance with the parameter of gel-filtration chromatography. Characterizing ofthe enzyme properties showed that the agarase degraded agarose at an optimum pH of7.5andoptimum temperature of40℃. The enzyme was inactivated by prolonged treatment at pH below 6.5, or by temperature over40℃. In addition, the enzyme activity was completely inhibited byMn²⁺, Fe³⁺and somewhat inhibited by Mg²⁺, Zn²⁺, Al³⁺, Cu²⁺, Fe²⁺, SDS and EDTA, whereassignificantly stimulated by Ca²⁺at1mM. But the analysis of the degradation product showed thatthe agarase was a β-agarase.According to the conserved regions of two α-agarases, an agarase gene named agaD wascloned from Thalassomonas sp. LD5genomic by degenerate PCR and site-finding PCR. Thefull-length of agaD consisted of a4401bp open reading frame, encoding1466amino acid residues,with a putative molecular mass of158.8kDa. The isolectric point was estimated to be3.9.Sequence analysis revealed that agarase AgaD was belonging to glycoside hydrolase96（GH96）,and it shared89%identity with the α-agarase of Alteromonas sp. GJ1B and65%with that ofThalassomonas sp. JAMB-A33.The agarase gene agaD gene is subcloned into pET24a （+）/E.coliBL21（DE3） system. After induction, the mature agarase secreted to the supernatant displayed amolecular weight of75kDa on SDS-PAGE, which may exist of post-translational modification.Due to the codon usage bias, agaD expressed inefficiently in any of the E.coli expression systems,likewise pET24a （+）/E.coli BL21（DE3）, pET22b （+）/E.coli BL21（DE3）, pET44b （+）/E.coliBL21（DE3） and pBAD/E.coliTOP10. Therefore, the codon corresponding to the catalyticdomain of agaD was optimized and synthesized artificially. The expression efficiency ofrecombinant protein improved significantly and part of the protein formed the inclusion bodies.However, recombinant protein with only the catalytic domain lost its activity, which provided usthat, the N-terminal domain might play an important role in the mature process of the AgaDagarase.Unexpected is that the analysis of the degradation product of the recombinant enzymeshowed the AgaD was a β-agarase. We presumed there might be some important changes of thegene, which resulted in the inversion of hydrolysis mechanism. In the future work, we will studyon this to find out the key sites which played a decisive role in α-and β-cleave pattern.