In vitro lipolysis experiments are used to assess digestion of lipid-based formulations, and probe solubilisation by colloidal phases during digestion. However, proteins and other biological components in the pancreatin often used as the lipase result in high-background scattering when interrogating structures using scattering approaches, complicating the resolution of colloidal structures. In this study, to circumvent this problem, a modified in vitro digestion model employing lipase immobilised on polymer beads, which allows for separation of the lipid digestion components during lipolysis, was investigated. Titration of the fatty acids released during digestion of medium chain triglycerides using pancreatin compared with immobilised lipase, combined with HPLC was used to follow the digestion, and small-angle X-ray scattering was used to determine colloidal structure formation. Digestion of medium chain triglycerides at the same nominal activity revealed that for the immobilised lipase, a longer digestion time was required to achieve the same extent of digestion. However, the same structural endpoint was observed, indicating that structure formation was not affected by the choice of lipase used. Lipolysis with immobilised lipase led to the reduction of parasitic scattering, resulting in clearer and more defined scattering from the structures generated by the lipolysis products. (c) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1311-1318, 2015.
Lipases from Bacillus thermocatenulatus are a member of superfamily of alpha/beta hydrolase, but there are structural differences between them. In this work, we focused on the alpha5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical alpha/beta hydrolase fold. In silico study showed that the alpha5 helix is a region that causes disorder in BTL2 protein. So, the alpha5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Deltaalpha5-BTL2) has been expressed in Pichia pastoris yeast. The alpha5 deletion results in increase of enzyme-specific activity in the presence of tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and olive oil (C18) substrates by 1.4-, 1.7-, 2.0-, 1.2-, 1.75-, and 1.95-fold, respectively. Also, deletion leads to increase in enzyme activity in different temperatures and pHs, whereas it did not significantly affect on enzyme activity in the presence of organic solvents, metal ions, and detergents.
        
Title: A cold-adapted and organic solvent-tolerant lipase from a psychrotrophic bacterium Pseudomonas sp. strain YY31: identification, cloning, and characterization Yamashiro Y, Sakatoku A, Tanaka D, Nakamura S Ref: Appl Biochem Biotechnol, 171:989, 2013 : PubMed
A novel cold-adapted lipase (designated as LipYY31) was obtained from a psychrotrophic Pseudomonas sp. YY31. The strain YY31 was gram-negative, rod shaped, motile by means of one polar flagellum, and exhibited chemotaxis toward oil droplets under a microscope. The strain displayed remarkable degradation of edible oil and fat even at 5 degrees C. The LipYY31 DNA fragment contains an open reading frame of 1,410 bp which encoded a protein of 470 amino acids with an estimated molecular mass of 49,584 Da. LipYY31 showed high sequence similarity to those of subfamily Iota.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 25-30 degrees C, and it retained 20-40 % of its activity at 0-5 degrees C. The optimal pH value was 8.0. The activity was strongly inhibited by Cd(2+), Zn(2+), EDTA and was highly dependent on Ca(2+). Tricaprin and p-nitrophenyl caprate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipYY31 also had high activity towards natural substrates including edible vegetable oils and animal fat. Furthermore, LipYY31 was very active and stable in the presence of several detergents and organic solvents. In particular, the lipase exhibited high stability against organic solvents such as methanol, ethanol, and isopropanol.
In vitro lipolysis experiments are used to assess digestion of lipid-based formulations, and probe solubilisation by colloidal phases during digestion. However, proteins and other biological components in the pancreatin often used as the lipase result in high-background scattering when interrogating structures using scattering approaches, complicating the resolution of colloidal structures. In this study, to circumvent this problem, a modified in vitro digestion model employing lipase immobilised on polymer beads, which allows for separation of the lipid digestion components during lipolysis, was investigated. Titration of the fatty acids released during digestion of medium chain triglycerides using pancreatin compared with immobilised lipase, combined with HPLC was used to follow the digestion, and small-angle X-ray scattering was used to determine colloidal structure formation. Digestion of medium chain triglycerides at the same nominal activity revealed that for the immobilised lipase, a longer digestion time was required to achieve the same extent of digestion. However, the same structural endpoint was observed, indicating that structure formation was not affected by the choice of lipase used. Lipolysis with immobilised lipase led to the reduction of parasitic scattering, resulting in clearer and more defined scattering from the structures generated by the lipolysis products. (c) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1311-1318, 2015.
Lipases from Bacillus thermocatenulatus are a member of superfamily of alpha/beta hydrolase, but there are structural differences between them. In this work, we focused on the alpha5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical alpha/beta hydrolase fold. In silico study showed that the alpha5 helix is a region that causes disorder in BTL2 protein. So, the alpha5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Deltaalpha5-BTL2) has been expressed in Pichia pastoris yeast. The alpha5 deletion results in increase of enzyme-specific activity in the presence of tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and olive oil (C18) substrates by 1.4-, 1.7-, 2.0-, 1.2-, 1.75-, and 1.95-fold, respectively. Also, deletion leads to increase in enzyme activity in different temperatures and pHs, whereas it did not significantly affect on enzyme activity in the presence of organic solvents, metal ions, and detergents.
        
Title: A cold-adapted and organic solvent-tolerant lipase from a psychrotrophic bacterium Pseudomonas sp. strain YY31: identification, cloning, and characterization Yamashiro Y, Sakatoku A, Tanaka D, Nakamura S Ref: Appl Biochem Biotechnol, 171:989, 2013 : PubMed
A novel cold-adapted lipase (designated as LipYY31) was obtained from a psychrotrophic Pseudomonas sp. YY31. The strain YY31 was gram-negative, rod shaped, motile by means of one polar flagellum, and exhibited chemotaxis toward oil droplets under a microscope. The strain displayed remarkable degradation of edible oil and fat even at 5 degrees C. The LipYY31 DNA fragment contains an open reading frame of 1,410 bp which encoded a protein of 470 amino acids with an estimated molecular mass of 49,584 Da. LipYY31 showed high sequence similarity to those of subfamily Iota.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 25-30 degrees C, and it retained 20-40 % of its activity at 0-5 degrees C. The optimal pH value was 8.0. The activity was strongly inhibited by Cd(2+), Zn(2+), EDTA and was highly dependent on Ca(2+). Tricaprin and p-nitrophenyl caprate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipYY31 also had high activity towards natural substrates including edible vegetable oils and animal fat. Furthermore, LipYY31 was very active and stable in the presence of several detergents and organic solvents. In particular, the lipase exhibited high stability against organic solvents such as methanol, ethanol, and isopropanol.
        
Title: A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene Sulong MR, Rahman RNZRA, Salleh AB, Basri M Ref: Protein Expr Purif, 49:190, 2006 : PubMed
UNLABELLED: An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity. KEYWORDS: Bacter