Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst

The lipase from Thermomyces laguginosus (formerly Humicola laguginosa) (TLL) is a basophilic and noticeably thermostable enzyme, commercially available in both soluble and immobilized form. Although initially oriented toward the food industry, the enzyme has found applications in many different industrial areas, from biodiesel production to fine chemicals (mainly in enantio and regioselective or specific processes). This review intends to show some of the most relevant aspects of the use of this interesting enzyme. After checking the enzyme features, some of the most efficient methods of TLL immobilization will be commented. Finally, the main uses of the enzyme will be revised, with special emphasis in the modification of fats and oils, production of biodiesel, resolution of racemic mixtures, enantioselective hydrolysis of prochiral esters and regioselective process involving sugar preparations. In many instances, TLL has been compared to other lipases, the advantages or disadvantages of the enzyme will be discussed.     

Reactivation of covalently immobilized lipase from Thermomyces lanuginosus

Lipase from Thermomyces lanuginosus (TLL) immobilized on cyanogen bromide agarose (CNBr) may be fully inactivated when incubated in saturated solutions of guanidine. When this inactivated enzyme is re-incubated in aqueous medium, 20% of the activity may be recovered for several cycles. However, if the activity was determined in the presence of a detergent (CTAB, an activator of this enzyme), 100% of the initial activity in the presence of detergent was recovered. The enzyme was also inactivated in the presence of organic solvents and at high temperatures. Inactivations were more rapid when the activity was determined in absence of detergent. In both cases, some activity could be recovered just by incubation under mild conditions, and this increase was higher if the activity measurements were performed in the presence of CTAB. These results suggested that the opening of the lipase could be a critical step in the inactivation or reactivation of immobilized TLL. In inactivations in the presence of solvents, 100% of activity could be recovered during several cycles, while in thermal inactivations, the recovered activity decreased in each inactivation–reactivation cycle. The incubation of the enzyme inactivated by temperature in guanidine improved the results, but still 100% could not be achieved during several cycles even measured in the presence of CTAB.Thus, the simple incubation of the partially or fully inactivated enzyme under mild conditions permitted to recover some activity (enhancing the half life of the biocatalysts), even in thermal inactivations.     

Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10 mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.     

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning,expression and characterization

An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP₀₃80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca²⁺ and inhibited by Fe²⁺, Zn²⁺, K⁺, and Ag⁺. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.     

重组大肠杆菌产疏绵状嗜热丝孢菌脂肪酶分批补料发酵工艺

为了获得低成本的疏绵状嗜热丝孢菌脂肪酶(TLL),在5L发酵罐发酵中对工程菌E.coliBL21(DE3)/pET28b-TLL的分批补料发酵工艺进行研究。结果表明:以葡萄糖为碳源的补料培养基,采用溶氧(DO)反馈补料策略进行补料,当OD600=60时降温至28℃,分2次加入终质量浓度为30g/L乳糖进行诱导表达。优化后TLL的表达量提高到798.5U/L,是优化前的2.4倍。本研究为规模化发酵重组菌生产TLL奠定了基础。     

疏棉状嗜热丝孢菌脂肪酶基因在毕赤酵母工程菌中的遗传稳定性

【背景】重组工程菌的传代稳定性是保证外源蛋白高效稳定表达的前提,是决定工业化生产能力的关键因素之一。【目的】对异源的疏棉状嗜热丝孢菌脂肪酶基因在毕赤酵母重组工程菌中的遗传稳定性进行研究。【方法】将工程菌连续传代15次,取第1、5、10、15代作为受检代次,结合重组菌的菌落及菌体形态、水解酶活、目的基因片段、外源基因拷贝数等指标综合评价其遗传稳定性。【结果】传代过程中重组菌的菌落和菌体形态、目的蛋白分子量、目的基因序列均保持一致。目的基因的整合拷贝数经5次传代后发生一定损失,但随后稳定为7左右,而脂肪酶的相对酶活则提高至90%以上。【结论】适量的整合拷贝数更有利于该脂肪酶基因在毕赤酵母重组菌中的表达,经综合评价此工程菌的遗传稳定性良好,应用于工业化大规模生产是可行的。     

Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents

Lipases catalyze the hydrolysis of triglycerides and are activated at the water-lipid interface. Thus, their interaction with amphiphiles such as detergents is relevant for an understanding of their enzymatic mechanism. In this study, we have characterized the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and thermal stability of Thermomyces lanuginosus lipase (TlL). For all detergents, low concentrations enhance the activity of TlL toward p-nitrophenyl butyrate by more than an order of magnitude; at higher detergent concentrations, the activity declines, leveling off close to the value measured in the absence of detergent. Surprisingly, these phenomena mainly involve monomeric detergent, as activation and inhibition occur well below the cmc for the nonionic and zwitterionic detergents. For anionic and cationic detergents, activation straddles the monomer-micelle transition. The data can be fitted to a three state interaction model, comprising free TlL in the absence of detergent, an activated complex with TlL at low detergent concentrations, and an enzyme-inhibiting complex at higher concentrations. For detergents with the same headgroup, there is an excellent correspondence between carbon chain length and ability to activate and inhibit TlL. However, the headgroup and number of chains also modulate these effects, dividing the detergents overall into three broad groups with rising activation and inhibition ability, namely, anionic and cationic detergents, nonionic and single-chain zwitterionic detergents, and double-chain zwitterionic detergents. As expected, only anionic and cationic detergents lead to a significant decrease in lipase thermal stability. Since nonionic detergents activate TlL without destabilizing the protein, activation/inhibition and destabilization must be independent processes. We conclude that lipase-detergent interactions occur at many independent levels and are governed by a combination of general and structurally specific interactions. Furthermore, activation of TlL by detergents apparently does not involve the classical interfacial activation phenomenon as monomeric detergent molecules are in most cases responsible for the observed increase in activity.     

疏棉状嗜热丝孢菌脂肪酶在毕赤酵母中的表面展示及酶学性质

【目的】构建疏棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus lipase,TLL)在毕赤酵母GS115中的细胞表面展示体系,筛选展示成功且酶活力及展示率较高的重组子作为全细胞催化剂,并研究其酶学性质。【方法】克隆TLL基因tll,以酿酒酵母细胞壁蛋白Sed1p为锚定蛋白,构建表面展示载体pPICZαA-TLS。重组载体经SacⅠ线性化后转入毕赤酵母GS115中,经三丁酸甘油酯平板检测及摇甁发酵筛选获得高酶活力的毕赤酵母重组子,采用抗FLAG标签一抗和R-PE荧光素标记的二抗处理细胞后,进行荧光显微镜检测和流式细胞仪分析,并考察全细胞催化剂的最适反应温度和pH、金属离子耐受性等酶学性质。【结果】成功构建TLL毕赤酵母细胞表面展示体系,筛选到1株具有三丁酸甘油酯和橄榄油水解活力的克隆子,经1%的甲醇诱导发酵120 h后,水解橄榄油酶活力达257.8 U/g干细胞。经抗体处理后的重组菌发酵细胞在荧光显微镜下呈现强烈的红色荧光,流式细胞仪分析结果也证实脂肪酶被成功展示在酵母细胞表面,展示率达98.36%。展示的TLL作为全细胞催化剂水解对硝基苯酚丁酸酯(pNPB)的最适温度为30℃,最适pH为8.0,且具备良好的热稳定性和有机溶剂耐受性;K+、Ca2+、Mg2+对其有微弱的激活作用,Mn2+、Ni2+则有微弱的抑制作用,Cu2+的抑制作用较强,而EDTA、SDS、Tween 20对酶活力影响不明显。【结论】首次将TLL脂肪酶成功展示在毕赤酵母细胞表面,获得具有较高水解活力和良好酶学特性的全细胞催化剂,为表面展示TLL脂肪酶的规模化应用奠定了技术基础。     

Effect of solid-phase chemical modification on the features of the lipase from Thermomyces lanuginosus

The lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl Sepharose and further modified with ethylenediamine (EDA) after activation of the carboxylic groups with carbodiimide. Different degrees of modification of the carboxyl groups were carried out by controlling the concentration of carbodiimide (10%, 50% or 100%). Subsequently, the effect of incubation of the modified preparations on hydroxylamine to recover the modified tyrosine was also studied. The modified enzymes exhibited a mobility in native electrophoresis quite different from that of the unmodified lipase (as expected by the changes in charge), and required higher concentrations of cationic detergent to become desorbed from the support. Interestingly, the chemical modification of the immobilized TLL produced an improvement in its activity, proportional to the amination degree. This increase in activity was much more significant at pH 10, where the fully modified preparation increased the activity by a factor of 10 as compared to the unmodified preparation. Moreover, the incubation of the chemically aminated preparations in a hydroxylamine solution improved the activity by an additional factor of 1.2. The fully aminated and incubated in hydroxylamine preparation exhibited a thermostability higher than that of the unmodified preparation, mainly at pH 5 (almost a 30 fold factor). In the presence of tetrahydrofurane, some stabilization was observed at pH 7, while at pH 9 the stability of the modified enzyme decreased (under all the assayed amination degrees) when compared to that of the unmodified enzyme. Thus, this simple protocol may be a rapid and efficient way of preparing a TLL biocatalyst with higher activity and stability, although this will depend on the inactivation conditions.     

Surface charge engineering of Thermomyces lanuginosus lipase improves enzymatic activity and biodiesel synthesis

Objectives

This study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications.

Results

Site-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. One TLL mutant, D165E, was identified with increased specific activity (456.60 U/mg), catalytic efficiency (k_cat/K_m: 44.14 s^?1 mM^?1), the highest biodiesel conversion yield (93.56%), and comparable thermostability with that of the TLL.

Conclusions

Our study highlighted the importance of surface charge engineering in improving TLL activity and biodiesel production, and the resulting TLL mutant, D165E, is a promising candidate for biodiesel industry.

     

Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies

This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9 K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350 U/mL under the optimal culture conditions of a 500 mL shaking flask containing 20 mL culture medium with the addition of 1.2 % (w/v) methanol, cultivation for 144 h at pH 7.0 and 27 °C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600 U/mL, 6,000 U/mL and 4,800 U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000 U/mL after 130 h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500 U/mL after 145 h incubation when methanol was used as the sole carbon source.     

Thermomyces lanuginosus: properties of strains and their hemicellulases

The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus. Several strains of this fungus have been reported to produce high levels of cellulase-free beta-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of beta-xylanase production is apparent. Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis. Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50-80 degrees C and over a broad pH range (3-12). Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1. The gene encoding the T. lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases. The crystal structure of the xylanase indicates that the enzyme consists of two beta-sheets and one alpha-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted. The presence of an extra disulfide bridge between the beta-strand and the alpha-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry.     

Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system

Lipase from Thermomyces lanuginosus (TLL) was immobilized on mesoporous hydrophobic poly-methacrylate (PMA) particles via physical adsorption (interfacial activation of the enzyme on the support). The influence of initial protein loading (5–200 mg/g of support) on the catalytic properties of the biocatalysts was determined in the hydrolysis of olive oil emulsion and synthesis of isoamyl oleate (biolubricant) by esterification reaction. Maximum adsorbed protein loading and hydrolytic activity were respectively ≈100 mg/g and ≈650 IU/g using protein loading of 150 mg/g of support. The adsorption process followed the Langmuir isotherm model (R² = 0.9743). Maximum ester conversion around 85% was reached after 30 min of reaction under continuous agitation (200 rpm) using 2500 mM of each reactant in a solvent-free system, 45 °C, 20% m/v of the biocatalyst prepared using 100 mg of protein/g of support. Apparent thermodynamic parameters of the esterification reaction were also determined. Under optimal experimental conditions, reusability tests of the biocatalyst (TLL-PMA) after thirty successive cycles of reaction were performed. TLL-PMA fully retained its initial activity up to twenty two cycles of reaction, followed by a slight decrease around 8.6%. The nature of the product (isoamyl oleate) was confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR), proton (¹H NMR) and carbon (¹³C NMR) nuclear magnetic resonance spectroscopy analyses.     

Thermomyces lanuginosus lipase in the liquid-crystalline phases of aqueous phytantriol: X-ray diffraction and vibrational spectroscopic studies

The influence of Thermomyces lanuginosus lipase (TLL) on the phase behaviour of liquid-crystalline phases of aqueous phytantriol as well as conformational changes of TLL entrapped in the cubic Q230 phase have been studied by small angle X-ray diffraction (SAXD), FT-Raman, and FT-IR techniques. It was found that the lipidic Q230 phase is able to accommodate up to 10 wt.% of TLL, and the temperature of phase transition to the inverted hexagonal phase H(II) increases indicating stabilizing effect of the protein. FT-Raman analysis of Trp amino acid marker band W3 revealed that the average rotation angle around the C3-Cbeta bond of four Trp residues of TLL in the Q230 phase increases. Reasoning from available TLL crystallographic data, this result is explained by structural transition of entrapped protein to so-called "open" and more related to the enzymatically-active conformation. TLL secondary structure analysis by amide I and amide III vibrational bands showed that content of alpha-helixes does not change, while a part of beta-sheet structures transforms to less ordered elements upon incorporation of protein into the Q230 phase of aqueous phytantriol.     

A novel method for immobilization of invertase from the thermophilic fungus, Thermomyces lanuginosus

An invertase from the thermophilic fungus, Thermomyces lanuginosus was immobilized on phenyl-Sepharose and its properties were studied. Between the soluble and immobilized forms of the invertase, there were not much difference in their optimum pH, K _M and V _max for sucrose. In contrast, the K _M and V _max for raffinose changed significantly. The optimum temperature for the immobilized invertase was lower by 10 C. The immobilized invertase showed remarkable stability at 50 C and was less sensitive to inhibition by metal ions. There was no leaching of the enzyme for at least a month when stored in the refrigerator. The method is novel and specific for the thermophilic invertase as a mesophilic invertase (from yeast) did not bind to phenyl-Sepharose.     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

New insights into the molecular mechanism of methanol-induced inactivation of Thermomyces lanuginosus lipase: a molecular dynamics simulation study

Methanol intolerance of lipase is a major limitation in lipase-catalysed methanolysis reactions. In this study, to understand the molecular mechanism of methanol-induced inactivation of lipases, we performed molecular dynamics (MD) simulations of Thermomyces lanuginosus lipase (TLL) in water and methanol and compared the observed structural and dynamic properties. The solvent accessibility analysis showed that in methanol, polar residues tended to be buried away from the solvent while non-polar residues tended to be more solvent-exposed in comparison to those in water. Moreover, we observed that in methanol, the van der Waals packing of the core residues in two hydrophobic regions of TLL became weak. Additionally, the catalytically relevant hydrogen bond between Asp²⁰¹ OD2 and His²⁵⁸ ND1 in the active site was broken when enzyme was solvated in methanol. This may affect the stability of the tetrahedral intermediates in the catalytic cycle of TLL. Furthermore, compared to in water, some enzyme surface residues displayed enhanced movement in methanol with higher Cα root-mean-square atomic positional fluctuation values. One of such methanol-affecting surface residues (Ile²⁴¹) was chosen for mutation, and MD simulation of the I241E mutant in methanol was conducted. The structural analysis of the mutant showed that replacing a non-polar surface residue with an acidic one at position 241 contributed to the stabilisation of enzyme structure in methanol. Ultimately, these results, while providing molecular-level insights into the destabilising effect of methanol on TLL, highlight the importance of surface residue redesign to improve the stability of lipases in methanol environments.