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

Abstract

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...

Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases

“…Moreover, a thermal activation effect was observed at that temperature, not unlike what has been observed with other proteins in the family [ 41 ]. Activity was heavily affected by the presence of metallic ions, which also has been reported for members of the GDSL family [ 43 ], although not to the same extent and with as many metal species as reported here. Lastly, detergents also impacted the activity of the enzyme: CHAPS and most notably Tween 80 enhanced the activity whereas Triton X100 decreased it.…”

“…GDSL family hydrolases are known for their broad substrate specificity and stereoselectivity, associated with a highly flexible active site environment [ 37 ]; thus, activity towards many structurally varied nitrophenyl esters was expected, and constitutes a desirable trait for biotechnologically relevant esterases. Regarding its thermostability, the enzyme was highly thermostable at temperature 60 °C with no significant loss of activity after a 3 h incubation period, similarly to other reported thermostable GDSL family esterases [ 43 ]. Moreover, a thermal activation effect was observed at that temperature, not unlike what has been observed with other proteins in the family [ 41 ].…”

“…The biochemical characterization of CelE is not documented but assays had been performed at temperatures up to 60 °C using CMC and Xylan as substrates at pH 7.0 [ 39 ], and at 37 °C using substrates 4-nitrophenyl acetate and acetylated glucomannan at pH 6.5 [ 40 ]. Although not included in the reviewed UniProt database, other thermostable GDSL family proteins have been reported, including the GDSL family esterase from Geobacillus thermodenitrificans T2 EstL5 with temperature and pH optima at 60 °C and 8.0, respectively, with pNP-butyrate as the substrate [ 41 ]; an acetyl xylan esterase from Caldicellulosiruptor bescii Cbes-AcXE2 with an optimum temperature of 70 °C and optimum pH of 7.0 using pNP-acetate as the substrate [ 42 ]; and a GDSL-type lipase from Geobacillus thermocatenulatus Lip29 that had optimal activity at 50 °C and pH 9.5 using pNP-palmitate as the substrate [ 43 ]. Our results show that the enzyme LipB12_A11 found in the functional screening of the metagenomic libraries from hot springs possesses comparable temperature optimum to other identified thermostable enzymes belonging to the SGNH/GDSL hydrolase family with an optimum of 60 °C and pH 9 using pNP-laurate as the substrate.…”

“…Activity in a broad pH range is observed and in agreement with other enzymes of the family [ 38 ]. Both substrate preference and presence of an interfacial activation mechanism are instrumental for classification of lipolytic enzymes as either lipases or esterases [ 43 , 44 , 45 ]. The preference for short substrates such as pNP-octanoate (C8) and pNP-laurate (C12) and lack of activity towards longer chain substrates such as pNP-estearate (C18), coupled with the enzyme kinetics not showing an interfacial activation mechanism typical of true lipases indicates that the SGNH/GDSL hydrolase found in this study is an esterase.…”

Endophytic Bacterium Serratia plymuthica From Chinese Leek Suppressed Apple Ring Rot on Postharvest Apple Fruit

“…Cytochrome P450 gene is required for the biosynthesis of the trichothecene toxin harzianum A in Trichoderma ( Cardoza et al, 2019 ) and promotes cell survival of the sugarcane smut fungus Sporisorium scitamineum under oxidative stress ( Cai et al, 2021 ). The GDSL lipase exhibits high tolerance against detergents and metal ions in Geobacillus thermocatenulatus ( Jo et al, 2021 ). MFS gene mfs1 or mfs2 reduce the susceptibility to aminoglycosides, quinolones, and paraquat in Pseudomonas aeruginosa ( Dulyayangkul et al, 2021 ) and play an essential role in resistance to toxicity, oxidants, and fungicides in Alternaria alternata ( Lin et al, 2018 ).…”