Biochemical characterization of Fsa16295Glu from "Fervidibacter sacchari," the first hyperthermophilic GH50 with ß-1,3-endoglucanase activity and founding member of the subfamily GH50_3.

The aerobic hyperthermophile "Fervidibacter sacchari" catabolizes diverse polysaccharides and is the only cultivated member of the class "Fervidibacteria" within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a ß-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as ß-agarases. The purified enzyme has a wide temperature range of 4-95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa16295Glu possesses a relatively high kcat/KM of 1.82 × 107 s-1 M-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing ß-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from "Fervidibacteria." Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.

Agarose-Degrading Characteristics of a Deep-Sea Bacterium Vibrio Natriegens WPAGA4 and Its Cold-Adapted GH50 Agarase Aga3420.

Up until now, the characterizations of GH50 agarases from Vibrio species have rarely been reported compared to GH16 agarases. In this study, a deep-sea strain, WPAGA4, was isolated and identified as Vibrio natriegens due to the maximum similarity of its 16S rRNA gene sequence, the values of its average nucleotide identity, and through digital DNA-DNA hybridization. Two circular chromosomes in V. natriegens WPAGA4 were assembled. A total of 4561 coding genes, 37 rRNA, 131 tRNA, and 59 other non-coding RNA genes were predicted in the genome of V. natriegens WPAGA4. An agarase gene belonging to the GH50 family was annotated in the genome sequence and expressed in E. coli cells. The optimum temperature and pH of the recombinant Aga3420 (rAga3420) were 40 °C and 7.0, respectively. Neoagarobiose (NA2) was the only product during the degradation process of agarose by rAga3420. rAga3420 had a favorable stability following incubation at 10-30 °C for 50 min. The Km, Vmax, and kcat values of rAga3420 were 2.8 mg/mL, 78.1 U/mg, and 376.9 s-1, respectively. rAga3420 displayed cold-adapted properties as 59.7% and 41.2% of the relative activity remained at 10 3 °C and 0 °C, respectively. This property ensured V. natriegens WPAGA4 could degrade and metabolize the agarose in cold deep-sea environments and enables rAga3420 to be an appropriate industrial enzyme for NA2 production, with industrial potential in medical and cosmetic fields.

Characterization of a GH50 ß-Agarase: A Biotechnological Tool for Preparing Oligosaccharides from Agarose and Porphyran.

Agarase is of vital significance for functional agaro-oligosaccharides production from algal dived agarose. Especially, the exolytic agarases have the advantage of obtaining agaro-oligosaccharides with a specific degree of polymerization. Herein, we cloned and expressed a novel glycoside hydrolase (GH) 50 family ß-agarase OUC-PgJC50 from Photobacterium gaetbulicola. The degradation pattern analysis indicated that OUC-PgJC50 not only showed an exolytic activity with main products of neoagarotetraose from hydrolyzing agarose but also show a hydrolytic activity to transform neoagarotetraose into neoagarobiose. This is the first time that the discovery of a neoagarotetraose-producing exolytic GH50 ß-agarase possesses the activity to transform neoagarotetraose into neoagarobiose, which provided new insight into the recognition of the degradation mode of agarases. Molecular docking and sequence alignment analysis further revealed the His654 residue in OUC-PgJC50 may play a vital role in forming a strong force with l-AHG residue at -4 subsite that helps to produce neoagarotetraose from catalyzing agarose. Moreover, the catalytic ability of OUC-PgJC50 toward another agar polysaccharide porphyran was also described that could hydrolyze porphyran into sulfated oligosaccharides, in which the LA6S-d-Gal was the main products. This study is of vital significance for developing the application range of GH50 ß-agarases.

Characterization and Modeling of Thermostable GH50 Agarases from Microbulbifer elongatus PORT2.

Viewing the considerable potential of marine agar as a source for the sustainable production of energy as well as nature-derived pharmaceutics, this work investigated the catalytic activity of three novel GH50 agarases from the mesophilic marine bacterium Microbulbifer elongatus PORT2 isolated from Indonesian coastal seawaters. The GH50 agarases AgaA50, AgaB50, and AgaC50 were identified through genome analysis; the corresponding genes were cloned and expressed in Escherichia coli BL21 (DE3). All recombinant agarases hydrolyzed ß-p-nitrophenyl galactopyranoside, indicating ß-glycosidase characteristics. AgaA50 and AgaB50 were able to cleave diverse natural agar species derived from Indonesian agarophytes, indicating a promising tolerance of these enzymes for substrate modifications. All three GH50 agarases degraded agarose, albeit with remarkable diversity in their catalytic activity and mode of action. AgaA50 and AgaC50 exerted exolytic activity releasing differently sized neoagarobioses, while AgaB50 showed additional endolytic activity in dependence on the substrate size. Surprisingly, AgaA50 and AgaB50 revealed considerable thermostability, retaining over 75% activity after 1-h incubation at 50 °C. Considering the thermal properties of agar, this makes these enzymes promising candidates for industrial processing.

Characterization and activity enhancement of a novel exo-type agarase Aga575 from Aquimarina agarilytica ZC1.

The novel ß-agarase gene aga575 from the agarolytic bacterium Aquimarina agarilytica ZC1 is composed of 2142 bp, and the encoded protein Aga575 has the highest amino acid sequence homology of only 65.2% with known agarases. Though carrying a domain of glycoside hydrolase family 42 in the C-terminal, Aga575 should belong to glycoside hydrolase family 50 according to the phylogenetic analysis. Gene aga575 was successfully cloned and overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein had the maximal agarase activity at pH 8.0 and 37 °C. The values Km and Vmax toward agarose were 8.4 mg/mL and 52.2 U/mg, respectively. Aga575 hydrolyzed agarose and neoagarooligosaccharides to yield neoagarobiose as the sole product. The agarose hydrolysis pattern of Aga575 indicated that it was an exo-type ß-agarase. Random mutagenesis was carried out to obtain two beneficial mutants M1 (R534G) and M2 (S4R-R424G) with higher activities. The results showed that the agarase activity of mutant M1 and M2 reached 162% and 192% of the wild-type agarase Aga575, respectively. Moreover, the activity of the mixed mutant M1/M2 (S4R-R424G-R534G) increased to 227%. KEY POINTS: • Aga575 is a novel exo-type ß-agarase degrading agarose to yield neoagarobiose as the sole product. • Though owning a domain of glycoside hydrolase family GH42, Aga575 should belong to family GH50. • The agarase activity of one mutant increased to 227% of the wild-type Aga575.

Biochemical Characterization and Substrate Degradation Mode of a Novel Exotype ß-Agarase from Agarivorans gilvus WH0801.

Agarases are important hydrolytic enzymes for the biodegradation of agar. Understanding the degradation mode and hydrolysis products of agarases is essential for their utilization in oligosaccharide preparations. Herein, we cloned and expressed AgWH50B, a novel neoagarotetraose-forming ß-agarase from Agarivorans gilvus WH0801 that has high specific activity and a fast reaction rate. AgWH50B consists of a C-terminal glycoside hydrolase family 50 catalytic domain with two tandem noncatalytic carbohydrate-binding modules (CBMs) in the N-terminus (residues 45-214 and 236-442). AgWH50B exhibited good enzymatic properties with high specific activity and catalytic efficiency (1523.2 U/mg and a Vmax of 1700 µmol/min/mg) under optimal hydrolysis conditions of pH 7.0 and 40 °C. Analysis of the hydrolysis products revealed that this enzyme is an exotype ß-agarase and that the dominant product of agarose or oligosaccharide degradation was neoagarotetraose. These findings suggest that AgWH50B could be utilized to yield abundant neoagarotetraose.

Cloning and characterisation of a novel neoagarotetraose-forming-ß-agarase, AgWH50A from Agarivorans gilvus WH0801.

AgWH50A, a novel ß-agarase, was cloned from Agarivorans gilvus WH0801 by degenerate and nested PCR. It consists of 942 amino acids (105 kDa), including a 21-amino acid signal peptide. AgWH50A shares the highest amino acid sequence homology with AgaD02 from Agarivorans sp. QM38 (53%). The recombinant agarase gene was expressed in Escherichia coli and purified by affinity chromatography. Maximum enzymatic activity (Km 5.97 mg/mL and Vmax 0.781 U/mg) was observed at pH 6.0 and 30 °C. Using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography, we analysed the hydrolysis products and concluded that AgWH50A is a neoagarotetraose-forming ß-agarase, which can cleave agarose into neoagarotetraose. This novel agarase has potential applications in the industrial production of neoagarotetraose and provides a new agarose hydrolysis model for future research.

Agarase: review of major sources, categories, purification method, enzyme characteristics and applications.

Agarases are the enzymes which catalyze the hydrolysis of agar. They are classified into alpha-agarase (E.C. 3.2.1.158) and beta-agarase (E.C. 3.2.1.81) according to the cleavage pattern. Several agarases have been isolated from different genera of bacteria found in seawater and marine sediments, as well as engineered microorganisms. Agarases have wide applications in food industry, cosmetics, and medical fields because they produce oligosaccharides with remarkable activities. They are also used as a tool enzyme for biological, physiological, and cytological studies. The paper reviews the category, source, purification method, major characteristics, and application fields of these native and gene cloned agarases in the past, present, and future.