Dongming Lan

A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86–34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH4)2SO4 precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15–35 °C and pH 5–9, with the optimal conditions being 15–25 °C and pH 5–6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold–active lipase. Its activity was found to increase in the presence of Zn2+, but it was strongly inhibited by Fe2+, Fe3+, Hg2+ and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short-and medium-chain length p-nitrophenyl (C4 and C8 acyl group) esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group) with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil.

Site-directed mutagenesis studies of the aromatic residues at the active site of a lipase from Malassezia globosa

The lipase from Malassezia globosa (SMG1) has specific activity on mono- and diacylglycerol but not on triacylglycerol. The structural analysis of SMG1 structure shows that two bulky aromatic residues, W116 and W229, lie at the entrance of the active site. To study the functions of these two residues in the substrate recognition and the catalytic reaction, they were mutated to a series of amino acids. Subsequently, biochemical properties of these mutants were investigated. Although the activities decrease, W229L and W116A show a significant shift in substrate preference. W229L has an increased preference for short-chain substrates whereas W116A has preference for long-chain substrates. Besides, the half-lives of W116A and W116H at 45 °C are 346.6 min and 115.5 min respectively, which improve significantly compared to that of native enzyme. Moreover, the optimum substrate of W116A, W116F and W229F mutants shifted from p-nitrophenyl caprylate to p-nitrophenyl myristate. These findings not only shed light onto the lipase structure/function relationship but also lay the framework for the potential industrial applications.

Molecular basis for substrate selectivity of a mono- and diacylglycerol lipase from Malassezia globosa

The lipase from Malassezia globosa (SMG1) was identified to be strictly specific for mono- and diacylglycerol but not triacylglycerol. The crystal structures of SMG1 were solved in the closed conformation, but they failed to provide direct evidence of factors responsible for this unique selectivity. To address this problem, we constructed a structure in the open, active conformation and modeled a diacylglycerol analogue into the active site. Molecular dynamics simulations were performed on this enzyme-analogue complex to relax steric clashes. This bound diacylglycerol analogue unambiguously identified the position of two pockets which accommodated two alkyl chains of substrate. The structure of SMG1-analogue complex revealed that Leu103 and Phe278 divided the catalytic pocket into two separated moieties, an exposed groove and a narrow tunnel. Analysis of the binding model suggested that the unique selectivity of this lipase mainly resulted from the shape and size of this narrow tunnel, in which there was no space for the settlement of the third chain of triacylglycerol. These results expand our understanding on the mechanism underlying substrate selectivity of enzyme, and could pave the way for site-directed mutagenesis experiments to improve the enzyme for application.

The Lid Domain in Lipases: Structural and Functional Determinant of Enzymatic Properties

Lipases are important industrial enzymes. Most of the lipases operate at lipid–water interfaces enabled by a mobile lid domain located over the active site. Lid protects the active site and hence responsible for catalytic activity. In pure aqueous media, the lid is predominantly closed, whereas in the presence of a hydrophobic layer, it is partially opened. Hence, the lid controls the enzyme activity. In the present review, we have classified lipases into different groups based on the structure of lid domains. It has been observed that thermostable lipases contain larger lid domains with two or more helices, whereas mesophilic lipases tend to have smaller lids in the form of a loop or a helix. Recent developments in lipase engineering addressing the lid regions are critically reviewed here. After on, the dramatic changes in substrate selectivity, activity, and thermostability have been reported. Furthermore, improved computational models can now rationalize these observations by relating it to the mobility of the lid domain. In this contribution, we summarized and critically evaluated the most recent developments in experimental and computational research on lipase lids.

Fatty acid specificity of T1 lipase and its potential in acylglycerol synthesis.

BACKGROUND T1 lipase has received considerable attention due to its thermostability. Fatty acid specificity of T1 lipase (crude and purified) was investigated, and its potential in the synthesis of acylglycerols was also evaluated. RESULTS Fatty acid specificity of T1 lipase (crude and purified) was investigated in the esterification of fatty acids (C6:0 to C18:3), suggesting that crude and purified T1 lipase had the lowest preference for C18:0 [specificity constant (1/α) = 0.08] followed by C18:1 (1/α = 0.12) and showed the highest preference for C8:0 (1/α = 1). A structural model was constructed to briefly explore interactions between the lipase and its substrate. Furthermore, crude T1 lipase-catalysed synthesis of diacylglycerols (DAGs) and monoacylglycerols (MAGs) by esterification of glycerol with C18:1 was studied for evaluating its potential in acylglycerols synthesis. The optimal conditions were glycerol/oleic acid molar ratio 5:1, the lipase concentration 9.7 U g(-1) of substrates, water content 50 g kg(-1) of substrates and temperature 50 °C, which yielded 42.25% DAGs, 26.34% MAGs and 9.18% triacylglycerols at 2 h. CONCLUSION DAGs and MAGs were synthesised in good yields although C18:1 (a much poorer substrate) was used. Our work demonstrates that T1 lipase, which was discovered to show 1,3-regio-selectivity, is a promising biocatalyst for lipids modification.

Screening and characterization of a thermostable lipase from marine Streptomyces sp. strain W007

A screening method along with the combination of genome sequence of microorganism, pairwise alignment, and lipase classification was used to search the thermostable lipase. Then, a potential thermostable lipase (named MAS1) from marine Streptomyces sp. strain W007 was expressed in Pichia pastoris X‐33, and the biochemical properties were characterized. Lipase MAS1 belongs to the subfamily I.7, and it has 38% identity to the well‐characterized Bacillus subtilis thermostable lipases in the subfamily I.4. The purified enzyme was estimated to be 29 kDa. The enzyme showed optimal temperature at 40 °C, and retained more than 80% of initial activity after 1 H incubation at 60 °C, suggesting that MAS1 was a thermostable lipase. MAS1 was an alkaline enzyme with optimal pH value at 7.0 and had stable activity for 12 H of incubation at pH 6.0–9.0. It was stable and retained about 90% of initial activity in the presence of Cu2+, Ca2+, Ni2+, and Mg2+, whereas 89.05% of the initial activity was retained when ethylene diamine tetraacetic acid was added. MAS1 showed the tolerance to organic solvents, but was inhibited by various surfactants. MAS1 was verified to be a triglyceride lipase and could hydrolyze triacylglycerol and diacylglycerol. The result represents a good example for researchers to discover thermostable lipase for industrial application.

Biochemical properties of a new cold-active mono- and diacylglycerol lipase from marine member Janibacter sp. strain HTCC2649.

Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1) from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1), Penicillium camembertii lipase U-150 (PCL), and Aspergillus oryzae lipase (AOL). Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity.

Structure of product-bound SMG1 lipase: active site gating implications.

Monoacylglycerol and diacylglycerol lipases are industrially interesting enzymes, due to the health benefits that arise from the consumption of diglycerides compared to the traditional triglyceride oils. Most lipases possess an α‐helix (lid) directly over the catalytic pocket which regulates the activity of the enzyme. Generally, lipases exist in active and inactive conformations, depending on the positioning of this lid subdomain. However, lipase SMG1, a monoacylglycerol and diacylglycerol specific lipase, has an atypical activation mechanism. In the present study we were able to prove by crystallography, in silico analysis and activity tests that only two positions, residues 102 and 278, are responsible for a gating mechanism that regulates the active and inactive states of the lipase, and that no significant structural changes take place during activation except for oxyanion hole formation. The elucidation of the gating effect provided data enabling the rational design of improved lipases with 6‐fold increase in the hydrolytic activity toward diacylglycerols, just by providing additional substrate stabilization with a single mutation (F278N or F278T). Due to the conservation of F278 among the monoacylglycerol and diacylglycerol lipases in the Rhizomucor miehei lipase‐like family, the gating mechanism described herein might represent a general mechanism applicable to other monoacylglycerol and diacylglycerol lipases as well.

Biochemical Properties of Recombinant Leucine Aminopeptidase II from Bacillus stearothermophilus and Potential Applications in the Hydrolysis of Chinese Anchovy (Engraulis japonicus) Proteins

The effects of various factors on the activity and conformation of recombinant leucine aminopeptidase II (rLAP II) from Bacillus stearothermophilus and its potential utilization in the hydrolysis of anchovy proteins were investigated. The optimal temperature and pH of rLAP II were 55 °C and 8.0 in phosphate buffer, and its activity was strongly stimulated by Co(2+). Conformational studies indicated that maintaining the α-helical structure had a critical effect on rLAP II activity. rLAP II was used to hydrolyze anchovy proteins, and it exhibited high specificity for peptides with molecular weight between 6000 and 1000 Da and positive coordination with endogenous enzymes and commercial Flavourzyme. Its use will enhance protein hydrolysis in species of aquatic animals. rLAP II could potentially be used to remove bitterness in the protein hydrolysis industry.

Conversion of a Mono‐ and Diacylglycerol Lipase into a Triacylglycerol Lipase by Protein Engineering

Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono‐ and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By protein engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild‐type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity.