Identification and Characterization of a New Alkaline SGNH Hydrolase from a Thermophilic Bacterium Bacillus sp. K91

Yu, Tingting; Ding, Junmei; Qingxia, Zheng; Han, Nanyu; Yu, Jia; Yang, Yunjuan; Li, Junjun; Mu, Yunxiang; Wu, Qian; Huang, Zunxi

doi:10.4014/jmb.1507.07101

Cited by 11 publications

(8 citation statements)

References 31 publications

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“…Most notably, FjoAcXE shows significant pH stability (Fig. 4 b) compared to other reported SGNH hydrolases, such as Est19 from Bacillus sp., which shows substantial loss of activity after 1-h pre-incubation at pH below 6.0 and above 10.0 [ 50 ]. None of the metal ions tested significantly affected FjoAcXE activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

Razeq

Jurak

Stogios

et al. 2018

Biotechnol Biofuels

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BackgroundAcetylated 4-O-(methyl)glucuronoxylan (GX) is the main hemicellulose in deciduous hardwood, and comprises a β-(1→4)-linked xylopyranosyl (Xylp) backbone substituted by both acetyl groups and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Whereas enzymes that target singly acetylated Xylp or doubly 2,3-O-acetyl-Xylp have been well characterized, those targeting (2-O-MeGlcpA)3-O-acetyl-Xylp structures in glucuronoxylan have remained elusive.ResultsAn unclassified carbohydrate esterase (FjoAcXE) was identified as a protein of unknown function from a polysaccharide utilization locus (PUL) otherwise comprising carbohydrate-active enzyme families known to target xylan. FjoAcXE was shown to efficiently release acetyl groups from internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, an activity that has been sought after but lacking in known carbohydrate esterases. FjoAcXE action boosted the activity of α-glucuronidases from families GH67 and GH115 by five and nine times, respectively. Moreover, FjoAcXE activity was not only restricted to GX, but also deacetylated (3-O-Araf)2-O-acetyl-Xylp of feruloylated xylooligomers, confirming the broad substrate range of this new carbohydrate esterase.ConclusionThis study reports the discovery and characterization of the novel carbohydrate esterase, FjoAcXE. In addition to cleaving singly acetylated Xylp, and doubly 2,3-O-acetyl-Xylp, FjoAcXE efficiently cleaves internal 3-O-acetyl-Xylp linkages in (2-O-MeGlcpA)3-O-acetyl-Xylp residues along with densely substituted and branched xylooligomers; activities that until now were missing from the arsenal of enzymes required for xylan conversion.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1074-3) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

Razeq

Jurak

Stogios

et al. 2018

Biotechnol Biofuels

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“…The thermophilic esterases from Fervidobacterium nodosum Rt17-B1 and Bacillus sp. K91 also preferred p NP-C2 as substrate [ 37 ]. However, Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of a Novel Thermostable GDSL-Type Lipase from Geobacillus thermocatenulatus

Kim

Lee

et al. 2021

J. Microbiol. Biotechnol.

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Lipases are glycerol ester hydrolases (E.C. 3.1.1.3) that catalyze the hydrolysis of triacylglycerols to free fatty acids and glycerol. They resemble esterases (E.C. 3.1.1.1) in catalytic activity but differ in that substrates. True lipases prefer water-insoluble fats containing medium-to long-chain fatty acids. Lipases are used extensively in the detergent, food, dairy, pulp, and pharmaceutical industries due to their high productivity and diversity, such as substrate specificity, stability in organic solvents, and high degree of regioselectivity [1].Bacterial lipases are classified into eight families based on amino acid sequence homology [2]. Family I lipases, called true lipases, are large group which is further divided into 6 subfamilies. They possess the pentapeptide Gly-Xaa-Ser-Xaa-Gly (GxSxG) motif with the active site serine situated near the center of the conserved sequence [2,3]. Most of the bacterial lipases from Bacillus and Staphyloccocus species belongs to this family. The enzymes grouped in family II do not exhibit the conventional GxSxG motif but rather display a Gly-Asp-Ser-Leu (GDSL) motif containing the active site serine residue. The GDSL motif localized in near N-terminus of amino acid sequence which is compared to GxSxG motif conserved in center of the sequence [4]. GDSL lipases represent the lipolytic activities with multifunctional properties and broad substrate specificity [5,6]. Furthermore, a subgroup of this GDSL family was classified as the SGNH hydrolase superfamily, with four conserved residues Ser, Gly, Asn and His in four conserved blocks I, II, III, and V [6]. While the SGNH hydrolases are well known in eukaryotic organisms, the isolation and characterization of SGNH hydrolases from bacteria remain to be limited [7]. In bacteria, GDSL motif enzymes are generally known as esterase type which has preference to short chain fatty acids [8][9][10][11]. To date, there have been few reports of GDSL family lipases in bacteria [12,13]. One example is a GDSL lipase from Mycobacterium tuberculosis and it was known to be actively involved in the intracellular survival during the nutritive stress conditions [12].Geobacillus species, which belongs to thermophilic Gram-positive spore-forming bacteria that can grow over a range of 45-75 o C, are of interest for biotechnology field as source of thermostable enzymes. Also, Geobacillus species are known to have potential availability for digesters of lignocellulose, hydrocarbons bioremediators, biofuel producers, cellular factories for heterologous expression of enzymes because of their structural and functional stability in extreme environments [14][15][16][17]. Several lipases which belong to family I have been reported from this species [18,19]. A number of family I and II esterases from this species have been characterized [20,21] Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est...

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“…The SGNH-hydrolases are involved in many biological processes such as bacterial virulence, plant development and morphogenesis, and in plant defense mechanisms. The SGNH enzymes have been detected in different compartments: extracellularly, , in the periplasm, − and in the cytoplasm . It has been known , that the lipolytic SGNH-superfamily is significantly different from α/β-hydrolases by the protein fold and by the location of the active-site serine.…”

Section: Introductionmentioning

confidence: 99%

“…In addition to important biological roles of SGNH enzymes, there is also a high potential in their biotechnological applications. Often detected substrate and enzyme promiscuities, optimal thermal stability, and wide pH-range have attracted attention because researchers want to explore and modify them to reach the maximal effect in biocatalysis. ,− SGNH esterases and lipases obtained from extremophiles are of importance due to their excellent performance under extreme physicochemical conditions being of potential use in laundry detergents, finishing fabrics, and pulp and paper industries …”

Section: Introductionmentioning

confidence: 99%

“…The SGNH enzymes have been detected in different compartments: extracellularly, 5,6 in the periplasm, 7−9 and in the cytoplasm. 10 It has been known 2,11 that the lipolytic SGNH-superfamily is significantly different from α/β-hydrolases by the protein fold and by the location of the active-site serine. In the 3-layered αβα sandwich-like fold, the active-site serine residue is positioned in a conserved Gly-Asp-Ser-Leu (GDSL) motif, close to the N-terminal end of an enzyme in contrast to the canonical α/β-fold, where a pentapeptide bearing the catalytic serine residue, usually Gly-Xaa-Ser-Xaa-Gly (where Xaa denotes any amino acid), is located in the middle of the amino acid sequence.…”

Section: ■ Introductionmentioning

confidence: 99%

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Catalytic Dyad in the SGNH Hydrolase Superfamily: In-depth Insight into Structural Parameters Tuning the Catalytic Process of Extracellular Lipase from Streptomyces rimosus

et al. 2017

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SrLip is an extracellular enzyme from Streptomyces rimosus (Q93MW7) exhibiting lipase, phospholipase, esterase, thioesterase, and tweenase activities. The structure of SrLip is one of a very few lipases, among the 3D-structures of the SGNH superfamily of hydrolases, structurally characterized by synchrotron diffraction data at 1.75 Å resolution (PDB: 5MAL ). Its crystal structure was determined by molecular replacement using a homology model based on the crystal structure of phospholipase A from Streptomyces albidoflavus (PDB: 4HYQ ). The structure reveals the Rossmann-like 3-layer αβα sandwich fold typical of the SGNH superfamily stabilized by three disulfide bonds. The active site shows a catalytic dyad involving Ser10 and His216 with Ser10-OγH···NεHis216, His216-NδH···O═C-Ser214, and Gly54-NH···Oγ-Ser10 hydrogen bonds essential for the catalysis; the carbonyl oxygen of the Ser214 main chain acts as a hydrogen bond acceptor ensuring the orientation of the His216 imidazole ring suitable for a proton transfer. Molecular dynamics simulations of the apoenzyme and its complex with p-nitrophenyl caprylate were used to probe the positioning of the substrate ester group within the active site and its aliphatic chain within the binding site. Quantum-mechanical calculations at the DFT level revealed the precise molecular mechanism of the SrLip catalytic activity, demonstrating that the overall hydrolysis is a two-step process with acylation as the rate-limiting step associated with the activation free energy of ΔG = 17.9 kcal mol, being in reasonable agreement with the experimental value of 14.5 kcal mol, thus providing strong support in favor of the proposed catalytic mechanism based on a dyad.

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Identification and Characterization of a New Alkaline SGNH Hydrolase from a Thermophilic Bacterium Bacillus sp. K91

Cited by 11 publications

References 31 publications

A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

Identification and Characterization of a Novel Thermostable GDSL-Type Lipase from Geobacillus thermocatenulatus

Catalytic Dyad in the SGNH Hydrolase Superfamily: In-depth Insight into Structural Parameters Tuning the Catalytic Process of Extracellular Lipase from Streptomyces rimosus

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