Purification and characterization of the α‐agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B

Abstract

The phenotypic features of strain GJlB, an unidentified marine bacterium that degrades agar [Young, K. S., Bhattacharjee, S. S. & Yaphe, W. (1978) Curbohydx Res. 66, 207-2121, were investigated and its agarolytic system was characterized using I3C-NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonus and the new combination A. ugurlyzicus (Cataldi) is proposed. An a-agarase, i.e specific for the a(l+3) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CLdB) and by anion-exchange chromatograpy (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDSPAGE, the purified a-agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0-9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45 "C or by removing calcium. In addition, a 8-galactosidase specific for the end products of the a-agarase was present in the a-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues.Enzymes capable of degrading agar polysaccharides have been isolated from bacteria [l-121 and marine molluscs [13]. These enzymes behave as glycosyl hydrolases, which hydroly se the P-D-( 1 +4) linkages between D-galactopyranose and 3,6 anhydro-L-galactopyranose residues in agarose and related polysaccharides, resulting in the release of neoagaro-oligosaccharides, with a D-galactose residue at the reducing end and 3, 6-anhydro-~-galactose at the non-reducing end [14-181.Another lytic mechanism, cleavage of the a-~-(1+3) linkages in agarose, was also reported [19-201, which yielded oligosaccharides of the agarobiose series, i.e. a 3,6-anhydro-L-galactose at the reducing end and a D-galactose residue at the non-reducing end. Agaro-oligosaccharides, however, have not yet been completely characterized by NMR. 13C-NMR spectra have been reported for unfractionated mixtures of agaro-oligosaccharides produced by a partially purified a-agarase from strain GJlB [17, 211. Only the anomeric carbons of the agarotetraose and agarohexaose aldito1 forms were assigned chemical shifts [20].As a prerequisite for the complete NMR characterization of the agaro-oligosaccharide series, we have further characterized the bacterial strain GJlB and purified its a-...

Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

“…Compared with families GH16, GH50, GH86, GH117, and GH118, enzymes in family GH96 are extremely rare with only four sequences in CAZy (8). Both endo-acting ␣-and ␤-glycoside hydrolases are identified by agar lysis around agarolytic bacteria, which has been the classical method for functional screening and agarase discovery (44,45), identifying both ␣-and ␤-activities. Thus, screening bias does not account for the discrepancy in total numbers of highly abundant endo-␤-agarases versus extremely rare endo-␣-agarases, strongly suggesting that the latter are actually rare in nature.…”

Enzymatic Properties and Nucleotide and Amino Acid Sequences of a Thermostable β-Agarase from the Novel Marine Isolate, JAMB-A94

“…14) Most agarases that have been purified and characterized belong to the -agarase group except for anagarase from Alteromonas agarlyticus GJ1B. 6) Biochemical and genetic studies of -agarases show high degrees of heterogeneity in terms of their amino acid (aa) sequences, molecular masses, substrate specificities, and catalytic properties even though the enzymes are functionally similar. To understand the catalytic mechanisms and diversity of agarases, we have isolated a number of deep-sea, agar-degrading microorganisms and sequenced the genes for agarases and characterized the enzymes.…”

Optimization of Anion-exchange Chromatography for the Separation of Agarase from Culture Broth of Pseudoalteromonas sp.

“…아가레이즈는 아가로오스를 가수분해하는 효소 로 그 분해 특성에 따라 두 그룹으로 나뉜다 [1][2][3]. β-아가레이즈는 α-1,3 결합을 끊어 agarooligosaccharide를 생성하는 반면 [4][5][6], β-아 가레이즈는 β-1,4 결합을 끊어 neooligosaccharide를 생성한다 [7][8][9][10].…”

“…최근에는 해조류가 바이오에 탄올 생산을 위한 발효용 기질로 각광을 받으면서 아가레이즈에 대 한 관심도 증가하고 있다 [13,14]. [6,8,16,17]. 순도를 향상시키기 위하여 친화성 크로마 토그래피(affinity chromatography)를 병행하여 사용하기도 한다 [6].…”