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Production, purification, and characterization of cold-active lipase from the psychrotroph Pseudomonas sp. A6.

ABSTRACT: Cold-active lipases are presently employed extensively in the detergent, chemical intermediate, fine chemical, food, and pharmaceutical industries. Seven cold-adaptive bacteria were isolated from the Mediterranean Sea near Alexandria, Egypt, and tested for their ability to produce cold-active lipase, with the highest activity at 10 °C. The most potent isolate was Pseudomonas sp. A6. To determine the most important variables, the bacterium was exposed to a necessary medium component and environmental factor screening using a single factor-at-a-time approach, followed by a multifactorial Plackett-Burman design strategy. After purification and characterization, the optimal activity levels for the cold-active lipase were figured out. Inoculation of Pseudomonas A6 under near optimum conditions using medium consisting of (g/L) peptone 7.14; soybean oil 7.5% (v/v); K₂HPO₄, 0.4; MgSO₄, 0.1; glucose 2; pH 8; and temperature 10 °C led to a maximum lipase activity anticipated to be 23.36 U/mL. Purified lipase showed the best activity and thermal stability at a pH of 8 and a temperature of 10 °C. The Pseudomonas A6 lipase tolerated the monovalent ions, while greater valence ions did not.

SUBMITTER: Abdella B

PROVIDER: S-EPMC10484855 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Production, purification, and characterization of cold-active lipase from the psychrotroph Pseudomonas sp. A6.

Abdella Bahaa B Youssif Asmaa Mohamed AM Sabry Soraya A SA Ghozlan Hanan A HA

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20230802 3

Cold-active lipases are presently employed extensively in the detergent, chemical intermediate, fine chemical, food, and pharmaceutical industries. Seven cold-adaptive bacteria were isolated from the Mediterranean Sea near Alexandria, Egypt, and tested for their ability to produce cold-active lipase, with the highest activity at 10 °C. The most potent isolate was Pseudomonas sp. A6. To determine the most important variables, the bacterium was exposed to a necessary medium component and environme ...[more]

PMID: 37531003

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Project description:Backgroundβ-D-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Cold-active β-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis.ResultsA genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the β-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-β-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C.ConclusionsA novel bgaL gene was isolated from Paracoccus sp. 32d encoded a novel cold-active β-D-galactosidase. An E. coli expression system has enabled efficient production of soluble form of BgaL Paracoccus sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active β-D-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of Paracoccus sp. 32d β-D-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.

Project description:AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.

Dataset Information

Production, purification, and characterization of cold-active lipase from the psychrotroph Pseudomonas sp. A6.

Publications

Production, purification, and characterization of cold-active lipase from the psychrotroph Pseudomonas sp. A6.

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