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Lipase, a type of enzyme that decomposes and synthesizes triglycerides, plays an important role in lipid processing. In this study, a heat-resisting lipase gene ( lip 4) from Thermomyces lanuginosus was subcloned into the pPICZαA vector and then transformed into Pichia pastoris X33. The recombinant yeast cell concentration reached the maximum (119.5 g/L) at 144 h, and the lipase (Lip4) activity reached the maximum (3900 U/mL) at 168 h in 10 L bioreactor. Through bioinformatics analysis, S168, as the key site of Lip4, participated in the formation of the catalytic triads S168-D223-H280 and G166-H167-S168-L169-G170. Furthermore, S168 and seven conserved amino acids of G104/288, S105, A195, P196, V225 and I287 constitute the active center of Lip4. Specifically, the structure modeling showed two α-helices of the lid domain, outside the active pocket domain, controlling the entry of the substrate on Lip4. The potential glycosylation of Asn-33 may be involved in exhibiting the high stable temperature for lipase activity. Therefore, the eukaryotic system was constructed to express Lip4 efficiently, and the amino acid sites related to the catalytic efficiency of Lip4 were clarified, providing a new way for its subsequent property research and industrial application.

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.

Conventional bulk protein structure determination methods are not suitable for understanding the distinct and diverse interactions of proteins with interfaces. Notably, interfacial activation is a feature common to many lipases involving movement of a helical "lid" region upon contact with a hydrophobic surface to expose the catalytic site. Here we use the surface specificity of vibrational sum frequency generation spectroscopy (VSFG) spectroscopy to directly probe the conformation of Thermomyces lanuginosus lipase (TLL) at hydrophobic interfaces. The TLL-catalyzed reaction at the air/water interface is monitored by VSFG spectroscopy, showing loss of ester carbonyl modes and appearance of carboxylate stretching modes of the fatty acid products. Furthermore, comparison of experimental and calculated VSFG spectra of the amide I band of TLL allows us to discern the subtle structural changes involved with lid-opening at a hydrophobic surface. Finally, we report a likely orientation of this lid-open state, which interacts with the surface through a loop region away from the lid and active site. This experimental framework for probing protein structure and function at interfaces addresses a significant problem in protein science that is not only impeding the design of better enzymes for biotechnology applications but also drug discovery targeting membrane associated proteins.

Here, we report a study of the effect of the blocking agent on the properties of the lipase from Thermomyces lanuginosus (TLL) immobilized on a heterofunctional support (Purolite C18-ethylnediamina (EDA)- vinyl sulfone (VS)-TLL-blocking agent) in different reactions. The performance of the biocatalysts was compared to those immobilized on standard hydrophobic support (Purolite C18-TLL) and the commercial one (TLL-IM). The nature of the blocking agent (Cys, Gly and Asp) altered the enzyme features. TLL-IM always gave a comparatively worse performance, with its specificity for the oil being very different to the Purolite biocatalysts. Under optimized conditions, Purolite C18-TLL yielded 97 % of hydrolysis conversion after 4 h using a water/waste cooking soybean oil (WCSO) mass ratio of 4.3, biocatalyst load of 6.5 wt% and a temperature of 44.2 °C (without buffer or emulsification agent). In esterification reactions of the purified free fatty acids (FFAs) obtained from WCSO, the best TLL biocatalysts depended on the utilized alcohol: linear amyl alcohol was preferred by Purolite C18-TLL and Purolite C18-EDA-VS-TLL-Gly, while higher activity was achieved utilizing isoamyl alcohol as nucleophile by Purolite C18-EDA-VS-TLL-Cys, Purolite C18-EDA-VS-TLL-Asp and IM-TLL as catalysts. All the results indicate the influence of the blocking step on the final biocatalyst features., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Heterogeneous biocatalysts were prepared by adsorbing T. lanuginosus lipase (TLL) onto uncalcined (SBAUC-TLL) and calcined (SBAC-TLL) SBA-15, using ammonium fluoride as a pore expander to facilitate TLL immobilization. At an enzyme load of 1 mg/g, high immobilization yields (>90 %) and recovered activities (>80 % for SBAUC-TLL and 70 % for SBAC-TLL) were achieved. When increasing the enzyme load to 5 mg/g, the immobilization yield of SBAUC-TLL was 80 %, and the recovered activity was 50 %, while SBAC-TLL had a yield of 100 % and a recovered activity of 36 %. Crosslinking with glutaraldehyde (GA) was conducted to improve stability (SBAUC-TLL-GA and SBAC-TLL-GA). Although SBAC-TLL-GA lost 25 % of initial activity after GA modifications, it exhibited the highest thermal (t 1/2  = 5.7 h at 65 °C), when compared to SBAC-TLL (t 1/2  = 12 min) and the soluble enzyme (t 1/2  = 36 min), and operational stability (retained 100 % activity after 5 cycles). Both biocatalysts presented high storage stability since they retained 100 % of initial activity for 30 days. These results highlight SBA-15's potential as an enzyme support and the protocol's efficacy in enhancing stability, with implications for industrial applications in the food, chemical, and pharmaceutical sectors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to explore the capacity of immobilized lipases on the acetylation of six aglycon flavonoids, namely myricetin, quercetin, luteolin, naringenin, fisetin and morin. For this purpose, lipase B from Candida antarctica (CaLB) and lipase from Thermomyces lanuginosus (TLL) were immobilized onto the surface of ZnOFe nanoparticles derived from an aqueous olive leaf extract. Various factors affecting the conversion of substrates and the formation of monoesterified and diesterified products, such as the amount of biocatalyst and the molar ratio of the substrates and reaction solvents were investigated. Both CaLB and TLL-ZnOFe achieved 100% conversion yield of naringenin to naringenin acetate after 72 h of reaction time, while TLL-ZnOFe achieved higher conversion yields of quercetin, morin and fisetin (73, 85 and 72% respectively). Notably, CaLB-ZnOFe displayed significantly lower conversion yields for morin compared with TLL-ZnOFe. Molecular docking analysis was used to elucidate this discrepancy, and it was revealed that the position of the hydroxyl groups of the B ring on morin introduced hindrances on the active site of CaLB. Finally, selected flavonoid esters showed significantly higher antimicrobial activity compared with the original compound. This work indicated that these lipase-based nanobiocatalysts can be successfully applied to produce lipophilic derivatives of aglycon flavonoids with improved antimicrobial activity.

Glycoside hydrolase (GH) 30 family xylanases are enzymes of biotechnological interest due to their capacity to degrade recalcitrant hemicelluloses, such as glucuronoxylan (GX). This study focuses on a subfamily 7 GH30, TtXyn30A from Thermothelomyces thermophilus, which acts on GX in an "endo" and "exo" mode, releasing methyl-glucuronic acid branched xylooligosaccharides (XOs) and xylobiose, respectively. The crystal structure of inactive TtXyn30A in complex with 2 3 -(4-O-methyl-α-D-glucuronosyl)-xylotriose (UXX), along with biochemical analyses, corroborate the implication of E233, previously identified as alternative catalytic residue, in the hydrolysis of decorated xylan. At the -1 subsite, the xylose adopts a distorted conformation, indicative of the Michaelis complex of TtXyn30AEE with UXX trapped in the semi-functional active site. The most significant structural rearrangements upon substrate binding are observed at residues W127 and E233. The structures with neutral XOs, representing the "exo" function, clearly show the nonspecific binding at aglycon subsites, contrary to glycon sites, where the xylose molecules are accommodated via multiple interactions. Last, an unproductive ligand binding site is found at the interface between the catalytic and the secondary β-domain which is present in all GH30 enzymes. These findings improve current understanding of the mechanism of bifunctional GH30s, with potential applications in the field of enzyme engineering., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Biocatalytic engineering was carried out by varying monotonically the binary CNTs-silica composition and, accordingly, the physicochemical characteristics of adsorbents developed for immobilization of recombinant T. lanuginosus lipase (rPichia/lip). The adsorbents based on composite carbon-silica materials (CCSMs) were produced by impregnating finely dispersed multi-walled carbon nanotubes with silica hydrosol followed by calcination in argon at 350°C; the mass ratio of the hydrophobic and the hydrophilic components varied over a wide range. Biocatalysts (BCs) for green low-temperature synthesis of various esters in a non-aqueous medium of organic solvents were prepared by adsorption of rPichia/lip with subsequent drying under ambient conditions. The characteristics of the CCSMs and BCs were characterized by thermogravimetry, nitrogen porosimetry and electron microscopy. The catalytic properties of BCs, such as enzymatic activity, substrate conversion and specificity, as well we their operational stability depending on the chemical composition of CCSMs were extensively studied in the esterification of saturated monocarboxylic acids (C 4 , C 7 , C 18 ) and primary aliphatic alcohols (C 2 , C 4 , C 16 ) in hexane at 20°C. It was found that the esterifying activity manyfold decreased with increasing the silica content primarily due to a decrease in adsorption ability of CCSMs toward rPichia/lip. The substrate specificity and operational stability of the lipase-active BCs did not greatly depend on the composition of CCSMs. Biocatalysts retained more than half of their initial esterifying activity after 10 reaction cycles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cross-linked enzyme (CLEs) of Thermomyces lanuginosa lipase (TLL) was prepared in an isocyanide-based multi-component reactions (ICMRs) platform by applying three di-acidic cross-linkers to unveil more factors contributing to the functional properties of CLEs. The linkers were 1,11-undecanedicarboxylic acid, azelaic acid, and adipic acid with 11, 7, and 4 carbon lengths, respectively, providing a proper tool to investigate the effect of linker length on the activity, stability, and selectivity of the resulting CLEs. The immobilization yields of 60-90 % and the specific activities of 168, 88.4 and 49 U/mg were obtained for the CLEs of 1,11-undecanedicarboxylic acid, azelaic acid, adipic acid, respectively. The lower activity of azelaic and adipic acid-mediated CLEs compared to the soluble TLL (110 U/mg) was explained by in silico calculations. The results revealed that as opposed to 1,11-undecanedicarboxylic acid, both linkers tended to penetrate the enzyme active site, thus resulting in a major inhibitory effect on the enzyme functionality. The thermal and co-solvent stability of the immobilized derivatives improved compared to those of free TLL. The selectivity of CLEs was also examined by catalytic release of main omega-3 fatty acids from fish oil, presenting the highest selectivity of 22 for the CLEs of azelaic acid., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 μmol/minute for K m and V max , respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hypothesis: Lipases are widely used in the detergent industry and must withstand harsh conditions involving both anionic and zwitterionic surfactants at alkaline pH. Thermomyces lanuginosus lipase (TlL) is often used and stays active at high concentrations of the anionic surfactant sodium dodecyl sulfate (SDS) at pH 8.0, but is sensitive to SDS at pH 6.0 and below. We propose that enhanced stability at pH 8.0 results from a structurally distinct complex formation with SDS., Experiments: We use small-angle X-ray scattering (SAXS) to elucidate structures of TlL:SDS at pH 4.0, 6.0, and 8.0 and further investigate the complexes at pH 8.0 using hydrogen/deuterium exchange mass spectrometry (HDX-MS)., Findings: At pH 4.0, large dense aggregates are formed at low [SDS], which become gradually less dense at higher [SDS], resulting in a core-shell structure. At pH 6.0, SDS induces a TlL dimer and forms a hemi-micelle along the side of the dimer. At higher [SDS], TlL adopts a core-shell structure. At pH 8.0, TlL forms a dimer with a SDS hemi-micelle but avoids a core-shell structure and maintains activity. Three helices are identified as SDS anchor points. This study provides important structural insight into the stability of TlL towards SDS under alkaline conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Arabinan in plant cell wall constitutes a major source of arabinose and arabino-oligosaccharides in nature. Exo-α-l-1,5-arabinanases release arabinose or arabino-oligosaccharides from arabinan in an exo-acting manner and therefore contribute to arabinan degradation. In this study, an exo-α-l-1,5-arabinanase belonging to GH93 family was identified from the thermophilic filamentous fungus Rasamsonia emersonii. The corresponding encoding gene (Reabn93) was cloned from the R. emersonii genome and heterologously expressed in Pichia pastoris. The purified recombinant ReAbn93 exhibited the maximum activity at 70 °C and retained 70% of its activity after incubation at 70 °C for 3 h ReAbn93 had an acidic pH optimum (pH 4.0) but remained stable over a broad pH range (pH 3-9). The specific activity of ReAbn93 toward linear arabinan under optimal conditions was 466.08 U mg -1 . Similar to the few other reported GH93 members, ReAbn93 degrades linear arabinan or arabino-oligosaccharides in an exo-acting manner with arabinobiose as the only hydrolytic product. Of note, ReAbn93 possessed remarkably better thermostability and higher specific activity compared to the only reported thermophilic counterpart in GH93, and therefore holds potential in relevant biotechnological applications., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Levoglucosenone (LGO) is a cellulose-derived molecule that is present commercially on a multi-ton/year scale. Taking advantage of the α,β-conjugated ketone of LGO, a new citronellol-containing 5-membered lactone (HBO-citro) was synthesized through a one-pot two-step pathway involving oxa-Michael addition and Baeyer-Villiger oxidation. The solvent-free treatment of HBO-citro with NaBH 4 at room temperature led to the full reduction of the lactone moiety which gave a novel fully renewable triol monomer having a citronellol side chain (Triol-citro). Noticeably, by simply changing the reducing agent, temperature and reaction duration, the partial reduction of HBO-citro can be achieved to yield a mixture of 5- and 6-membered Lactol-citro molecules. Triol-citro was chosen to prepare functional renewable polyesters having citronellol pendant chains via polycondensation reactions with diacyl chlorides having different chain lengths. Good thermal stability ( T d5% up to 170 °C) and low glass transition temperatures (as low as -42 °C) were registered for the polyesters obtained. The polymers were then hydrolyzed using a commercial lipase from Thermomyces lanuginosus (Lipopan ® 50 BG) to assess their biodegradability. A higher degradation profile was found for the polyesters prepared using co-monomers (acyl chlorides) having longer chain lengths. This is likely due to the decreased steric hindrance around the ester bonds which allowed enhanced accessibility of the enzyme.

The standalone metallo-β-lactamase-type thioesterase (MβL-TE), belongs to the group V nonreducing polyketide synthase agene cluster, catalyzes the rate-limiting step of product releasing. Our work first investigated on the orthologous MβL-TEs from different origins to determine which nonconserved amino acid residues are important to the hydrolysis efficiency. A series of chimeric MβL-TEs were constructed by fragment swapping and site-directed mutagenesis, in vivo enzymatic assay showed that two nonconserved residues A19 and E75 (numbering in HyTE) were critical to the catalytic performance. Protein structure modeling suggested that these two residues are located in different areas of HyTE. A19 is on the entrance to the active sites, whereas E75 resides in the linker between the two β strands which hold the metal-binding sites. Combining with computational simulations and comparative enzymatic assay, different screening criteria were set up for selecting the variants on the two noncatalytic and nonconserved key residues to improve the catalytic activity. The rational design on A19 and E75 gave five candidates in total, two (A19F and E75Q) of which were thus found significantly improved the enzymatic performance of HyTE. The double-point mutant was constructed to further improve the activity, which was increased by 28.4-fold on product accumulation comparing to the wild-type HyTE. This study provides a novel approach for engineering on nonconserved residues to optimize enzymatic performance., (© 2021 Wiley Periodicals LLC.)

Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Rasamsonia emersonii (previously Talaromyces emersonii) is a thermophilic filamentous fungus displaying optimum growth at 45 °C. It has a history of use in commercial food enzyme production. Its unfractionated chitinolytic secretome was partially characterised in the early 1990s; however, no individual chitinase from this source has been described in literature previously. This study describes two GH18 chitinases originating from the R. emersonii genome, expressed in the methylotrophic yeast P. pastoris. Chit1 comprises of a GH18 catalytic domain and Chit2 comprises of a GH18 catalytic domain and a chitin-binding motif at the C-terminal. The chitinases were expressed as glycoproteins. The apparent molecular weight of Chit1 was 35.8-42.1 kDa with a smearing tail associated with glyco-sidechains visible up to 72.2 kDa. This became two bands of 30.8 and 29.0 kDa upon de-glycosylation. The apparent molecular weight of Chit2 was 50.4 kDa, reducing to 48.2 kDa upon de-glycosylation. Both chitinases displayed endo-chitinase and chitobiosidase activity, temperature optima of 50-55 °C and low pH optima (pH 4.5 or lower); Chit1 displayed a pH optimum of 3.5, retaining > 60% maximum activity at pH 2.2, a pH range lower than most enzymes of fungal origin. Chit2 displayed the highest chitin-degrading ability at 3456 µmol/mg on 4-NP-triacetylchitotriose, but lost activity faster than Chit1, which displayed 403 µmol/mg on the same substrate. The predicted D values (time required to reduce the enzyme activity to 10% of its original value at 50 °C) were 19.2 and 2.3 days for Chit1 and Chit2, respectively. Thus, Chit1 can be considered one of few hyperthermostable chitinase enzymes described in literature to date. Their physicochemical properties render these chitinases likely suitable for shrimp chitin processing including one-step chitin hydrolysis and alternative sustainable protein processing and the attractive emerging application of mushroom food waste valorisation.Key points• Two GH18 chitinases originating from the industrially relevant thermophilic fungus R. emersonii were cloned and expressed in P. pastoris.• The purified recombinant chitinases showed low pH and high temperature optima and appreciable thermostability at industrially relevant temperatures.• The chitinases displayed characteristics that indicate their likely suitability to several industrial applications including sustainable alternative protein processing, food waste valorisation of commercial mushroom production and one-step shrimp chitin processing., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In the present study, we demonstrated the use of molecular docking as an efficient in silico screening tool for lipase-triglyceride interactions. Computational simulations using the crystal structures from Burkholderia cepacia lipase (BCL), Thermomyces lanuginosus lipase (TLL), and pancreatic porcine lipase (PPL) were performed to elucidate the catalytic behavior with the majority triglycerides present in Licuri oil, as follows: caprilyl-dilauryl-glycerol (CyLaLa), capryl-dilauryl-glycerol (CaLaLa), capryl-lauryl-myristoyl-glycerol (CaLaM), and dilauryl-myristoyl-glycerol (LaLaM). The computational simulation results showed that BCL has the potential to preferentially catalyze the major triglycerides present in Licuri oil, demonstrating that CyLaLa, (≈25.75% oil composition) interacts directly with two of the three amino acid residues in its catalytic triad (Ser87 and His286) with the lowest energy (-5.9 kcal/mol), while other triglycerides (CaLaLa, CaLaM, and LaLaM) interact with only one amino acid (His286). In one hard, TLL showed a preference for catalyzing the triglyceride CaLaLa also interacting with His286 residue, but, achieving higher binding energies (-5.3 kcal/mol) than found in BCL (-5.7 kcal/mol). On the other hand, PPL prefers to catalyze only with LaLaM triglyceride by His264 residue interaction. When comparing the computational simulations with the experimental results, it was possible to understand how BCL and TLL display more stable binding with the majority triglycerides present in the Licuri oil, achieving conversions of 50.86 and 49.01%, respectively. These results indicate the production of fatty acid concentrates from Licuri oil with high lauric acid content. Meanwhile, this study also demonstrates the application of molecular docking as an important tool for lipase screening to reach a more sustainable production of fatty acid concentrates from vegetable oils., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lipases comprise one of the major enzyme classes in biotechnology with applications within, e.g., baking, brewing, biocatalysis, and the detergent industry. Understanding the mechanisms of lipase function and regulation is therefore important to facilitate the optimization of their function by protein engineering. Advances in single-molecule studies in model systems have provided deep mechanistic insights on lipase function, such as the existence of functional states, their dependence on regulatory cues, and their correlation to activity. However, it is unclear how these observations translate to enzyme behavior in applied settings. Here, single-molecule tracking of individual Thermomyces lanuginosus lipase (TLL) enzymes in a detergency application system allowed real-time direct observation of spatiotemporal localization, and thus diffusional behavior, of TLL enzymes on a lard substrate. Parallelized imaging of thousands of individual enzymes allowed us to observe directly the existence and quantify the abundance and interconversion kinetics between three diffusional states that we recently provided evidence to correlate with function. We observe redistribution of the enzyme's diffusional pattern at the lipid-water interface as well as variations in binding efficiency in response to surfactants and calcium, demonstrating that detergency effectors can drive the sampling of lipase functional states. Our single-molecule results combined with ensemble activity assays and enzyme surface binding efficiency readouts allowed us to deconvolute how application conditions can significantly alter protein functional dynamics and/or surface binding, both of which underpin enzyme performance. We anticipate that our results will inspire further efforts to decipher and integrate the dynamic nature of lipases, and other enzymes, in the design of new biotechnological solutions.

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.

Rutin (3',4',5,7-tetrahydroxy-flavone-3-rutinoside) was enzymatically acylated with benzoic acid and its esters (methyl benzoate and vinyl benzoate) using Thermomyces lanuginosus lipase (Lipozyme TLIM). The acylation reaction was optimized by varying the reaction medium, reaction temperature, acyl donor, substrate molar ratio, and reaction time. The highest conversion yield (76%) was obtained in tert-amyl alcohol (60 °C, 72 hr) using vinyl benzoate (molar ratio of 1:10) as acyl donor. The acylation occurred at the 2'''-OH and 4'''-OH of the rhamnose unit and the 2''-OH position of the glucose moieties. Three novel rutin acylated derivatives (compounds 1-3) were purified and characterized by HR-MS and 1D and 2D NMR spectroscopy. We found that acylation significantly improved lipophilicity, capacity to inhibit lipid peroxidation, anticancer capacity and substantially maintained the antioxidant activity of rutin. This research provides important insights in the acylation of flavonoids with different glycosyl moieties. PRACTICAL APPLICATION: In this study, three novel rutin derivatives were successfully synthesized and the highest conversion yield (76%) was obtained by reacting the rutin and vinyl benzoate at molar ratio of 1:10 in tert-amyl alcohol for 72 hr at 60 °C. Introducing a benzoic acid substituent into rutin molecule significantly improved their lipophilicity and inhibition of lipid peroxidation in lipophilic system. Furthermore, this study demonstrated that acylation significantly improved anticancer capacity and substantially maintained the antioxidant activity., (© 2021 Institute of Food Technologists®.)

As a consequence of intense industrialization in the last few decades, the amount of agro-industrial wastes has increasing, where new forms of valorization are crucial. In this work, five residual biomasses from Maranhão (Brazil) were investigated as supports for immobilization of lipase from Thermomyces lanuginosus (TLL). The new biocatalysts BM-TLL (babaçu mesocarp) and RH-TLL (rice husk) showed immobilization efficiencies >98% and hydrolytic activities of 5.331 U g -1 and 4.608 U g -1 , respectively, against 142 U g -1 by Lipozyme ® TL IM. High esterification activities were also found, with 141.4 U g -1 and 396.4 U g -1 from BM-TLL and RH-TLL, respectively, against 113.5 U g -1 by TL IM. Results of porosimetry, SEM, and BET demonstrated BM and RH supports are mesoporous materials with large hydrophobic area, allowing a mixture of hydrophobic adsorption and confinement, resulting in hyperactivation of TLL. These biocatalysts were applied in the production of hexyl laurate, where RH-TLL was able to generate 94% conversion in 4 h. Desorption with Triton X-100 and NaCl confirmed that new biocatalysts were more efficient with 5 times less protein than commercial TL IM. All results demonstrated that residual biomass was able to produce robust and stable biocatalysts containing immobilized TLL with better results than commercial preparations.

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. Strikingly, the cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. However, its molecular mechanism of action is not clearly understood, because currently there is no structural information available on fungal cyanases. Here we report the crystal structure of Tl-Cyn in complex with inhibitors malonate and formate at 2.2 Å resolution. The structure reveals extensive interactions at the subunit interfaces in a dimer, and a decamer is formed by a pentamer of these dimers. Our biochemical, kinetic and mutagenesis studies confirm the structural observations on the complex and provide further insights into its catalytic mechanism and inhibition. The structure has also aided the creation of a mutant enzyme with enhanced catalytic activity, and such enzymes may have the potential for biotechnological applications, including biotransformation and bioremediation. Moreover, other fungal cyanases with potentially high catalytic activity could also be predicted based on the Tl-Cyn structure, as the active site region among fungal cyanases are highly conserved.

We investigated the properties of the low molecular weight thermo-alkali-stable and mercury ion-tolerant xylanase production from Thermomyces dupontii KKU-CLD-E2-3. The xylanase was purified to homogeneity by ammonium sulfate, Sephadex G-100 and DEAE-cellulose column chromatography which resulted 27.92-fold purification specific activity of 56.19 U/mg protein and a recovery yield of 2.01%. The purified xylanase showed a molecular weight of 25 kDa by SDS-PAGE and the partial peptide sequence showed maximum sequence homology to the endo-1,4-β-xylanase. The optimum temperature and pH for its activity were 80 °C and pH 9.0, respectively. Furthermore, the purified xylanase can maintain more than 75% of the original activity in pH range of 7.0-10.0 after incubation at 4 °C for 24 h, and can still maintain more than 70% of original activity after incubating at 70 °C for 90 min. Our purified xylanase was activated by Cu 2+ and Hg 2+ up to 277% and 235% of initial activity, respectively but inhibited by Co 2+ , Ag + and SDS at a concentration of 5 mM. The K m and V max values of beechwood xylan were 3.38 mg/mL and 625 µmol/min/mg, respectively. Furthermore, our xylanase had activity specifically to xylan-containing substrates and hydrolyzed beechwood xylan, and the end products mainly were xylotetraose and xylobiose. The results suggested that our purified xylanase has potential to use for pulp bleaching in the pulp and paper industry.

In this study, a new mixed heterofunctional support (Chit-GA-Gly) has been prepared by sequential activation of chitosan hydrogel (Chit) with glutaraldehyde (GA) and further functionalization with glycine (Gly). The immobilization of the lipase from Thermomyces lanuginosus (TLL) on this support was compared with that on GA-activated Chit hydrogel (Chit-GA). The supports have been characterized by FT-IR, zeta potential and TG analyses. A similar maximum lipase loading of 53-55 mg per gram of support has been obtained for both supports. Both biocatalysts retained ≈40% of their initial activity after 48 h of incubation at 50 °C in heptane, toluene or iso-octane. The immobilization of TLL on Chit-GA proceeded via preferential covalent attachment (95%) and a combined ion exchange (cationic and anionic) and hydrophobic adsorption was observed using Chit-GA-Gly. TLL immobilized on Chit-GA-Gly was ≈4-times more active than when immobilized on Chit-GA in both olive oil emulsion hydrolysis and alkyl palmitate synthesis via esterification. Isoamyl palmitate synthesis in iso-octane at 50 °C using this new biocatalyst gave a maximum acid conversion of 85% after 90 min of reaction. After nine consecutive esterification batches, the biocatalyst retained around 40% of its initial activity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study sought to prepare a cognitive enhancer l-α-glycerylphosphorylcholine (l-α-GPC) using an immobilized Lecitase Ultra (LU, phospholipase A 1 ) to catalyze the hydrolysis of soy phosphatidylcholine (PC). Immobilization of LU on Lewatit VP OC 1600 provided the highest fixation level (83.1 g/100 g) and greatest catalytic activity achieving 100 g/100 g l-α-GPC within 20 h and was therefore selected as the optimal system for biocatalysis. Immobilization of LU increased its positional specificity compared to free LU, as shown by a decrease in the production of the phosphocholine byproduct. Under the optimal conditions determined by response surface methodology, PC was completely hydrolyzed to l-α-GPC and required a simple purification via phase separation of the biphasic media to obtain a yield of ∼26.4 g l-α-GPC from 100 g PC, with a purity of 98.5 g/100 g. Our findings suggest a possibility of using the immobilized LU as a new biocatalyst for the l-α-GPC production.

D-Amino-acid oxidases (DAAOs) catalyze the oxidative deamination of neutral and basic D-amino acids. The DAAO from the thermophilic fungus Rasamsonia emersonii strain YA (ReDAAO) has a high thermal stability and a unique broad substrate specificity that includes the acidic D-amino acid D-Glu as well as various neutral and basic D-amino acids. In this study, ReDAAO was crystallized by the hanging-drop vapor-diffusion method and its crystal structure was determined at a resolution of 2.00 Å. The crystal structure of the enzyme revealed that unlike other DAAOs, ReDAAO forms a homotetramer and contains an intramolecular disulfide bond (Cys230-Cys285), suggesting that this disulfide bond is involved in the higher thermal stability of ReDAAO. Moreover, the structure of the active site and its vicinity in ReDAAO indicates that Arg97, Lys99, Lys114 and Ser231 are candidates for recognizing the side chain of D-Glu.

By using a isocyanide-based multi-component reaction for the immobilization of the soluble forms of Rhizomucor miehei lipase (RML) and Thermomyces lanuginosa lipase (TLL), the first step of enzyme aggregation or crystallization in the traditional methods of cross-linking was bypassed. High immobilization yields and specific activities were achieved for both lipases.

Lipase from Thermomyces lanuginosus (TLL) was immobilized onto a novel heterofunctional support, divinyl sulfone (DVS) superparamagnetic nanoparticles (SPMNs) functionalized with polyethyleneimine (PEI). Particle size and zeta potential measurements, elemental analysis, X-ray powder diffraction, magnetic measurements, and infrared spectroscopy analysis were used to characterize the TLL preparations. At pH 10, it was possible to achieve 100 % of immobilization yield in 1 h. The immobilization pH gives TLL preparations with different stabilities; indeed the TLL preparation immobilized at pH 5.0 was the most stable during the thermal inactivation at all pH values. For the hydrolysis of racemic methyl mandelate, the nanobiocatalysts immobilized at pH 5.0 and blocked with ethylenediamine (EDA) and ethanolamine (ETA) obtained good enantioselectivities (68 % and 72 %, respectively) with high catalytic activities in the reaction medium at pH 7.0. The operational stability of the systems was evaluated in the esterification reaction of benzyl alcohol, obtaining up to 61 % conversion after the seventh reaction cycle. These results show that SPMN@PEI-DVS support is a robust strategy for the easy and rapid recovery of the nanobiocatalyst by applying a magnetic field, showing great potential for industrial applications., Competing Interests: Declaration of Competing Interest The authors whose names are listed immediately below certify and/or informed consent that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or research involving human participants and/or animals, or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The characterization of the affinity and binding mechanism of specific molecules to a protein active site is scientifically and industrially relevant for many applications. In principle, this information can be obtained using molecular dynamics (MD) simulations by calculating the free energy profile of the process. However, this is a computationally demanding calculation. Currently, coarse-grained (CG) force fields are very well implemented for MD simulations of biomolecular systems. These computationally efficient force fields are a major advantage to the study of large model systems and/or those requiring long simulation times. The Martini model is currently one of the most popular CG force fields for these systems. For the specific case of protein simulations, to correctly maintain the macromolecular three-dimensional structure, the Martini model needs to include an elastic network (EN). In this work, the effect of protein flexibility, as induced by three EN models compatible with the Martini force field, was tested on the calculation of free energy profiles for protein-ligand binding. The EN models used were ElNeDyn, GoMartini, and GEN. The binding of triolein (TOG) and triacetin (TAG) to a lipase protein ( thermomyces lanuginosa lipase-TLL) was used as a case study. The results show that inclusion of greater flexibility in the CG parameterization of proteins is of high importance in the calculation of the free energy profiles of protein-ligand systems. However, care must be taken in order to avoid unjustified large protein deformations. In addition, due to molecular flexibility there may be no absolute need for the center of the ligand to reach the center of the protein-binding site. The calculation of the energy profile to a distance of about 0.5 nm from the active site center can be sufficient to differentiate the affinity of different ligands to a protein.

Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data are available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here, we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate-binding module (CBM). With a temperature optimum of 80 °C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with R work and R free factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate-binding cleft. An increased number of glycosylations were identified in ReCel7B, and we propose a stabilizing mechanism for one of the glycans. Global structure-function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding, and cellulose accessibility in GH7 endoglucanases. DATABASE: Structural data are available in RCSB Protein Data Bank database under the accession number 6SU8. ENZYMES: ReCel7B, endoglucanase (EC3.2.1.4) from Rasamsonia emersonii; ReCel7A, cellobiohydrolase (EC3.2.1.176) from Rasamsonia emersonii; TrCel7B, endoglucanase (EC3.2.1.4) from Trichoderma reesei; TrCel7A, cellobiohydrolase (EC3.2.1.176) from Trichoderma reesei., (© 2019 Federation of European Biochemical Societies.)

Physical adsorption of lipase from Thermomyces lanuginosus onto single-layer sheets of graphene oxide (GO) was studied using the response surface methodology to evaluate the physicochemical factors - temperature, pH, ionic strength, and concentration - affecting the enzymatic activity and the immobilization efficiency. The immobilization efficiency and the activity of the enzyme were inversely proportional to each other. Specifically, higher pH values increased the immobilization efficacy, but produced changes in the aggregation state and secondary structure of the enzyme, thus decreasing its activity. Lower pH values, in turn, reduced the immobilization efficacy, but increased the activity of the adsorbed lipase. The adsorbed and the free lipase were followed during 600 ns and 3.5 μs, respectively, in molecular dynamics (MD) simulations. MD trajectories showed that irreversible adsorption freezes the enzyme in a state with a correctly opened catalytic cavity, while the active site remains without a direct interaction with the GO adsorbent. In contrast to the interfacial activation of lipases in a hydrophobic environment, where the catalytic pocket attaches to the hydrophobic surface, the adsorption onto GO made the active site of the lipase accessible by altering the tertiary structure of the enzyme, leading to a higher catalytic efficiency. Experimental investigations confirmed that the physical adsorption onto GO induces tertiary structure changes in the lipase and protects it from H 2 O 2 by accepting the oxidative damage upon itself. In summary, the physical adsorption of the lipase onto GO is mainly affected by pH and could possibly provide a spreadable and robust catalytic interface for biotechnological applications., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This paper shows the step by step coimmobilization of up to five different enzymes following two different orders in the coimmobilization to alter the effect of substrate diffusion limitations. The enzymes were the lipases A and B from Candida antarctica, the lipases from Rhizomocur miehei and, Themomyces lanuginosus and the phospholipase Lecitase Ultra. The utilized strategy was a layer by layer immobilization, coating the immobilized enzymes with polyethylenimine followed by the crosslinking of the enzyme and PEI with glutaraldehyde to prevent enzyme release, and them adding a new lipase layer. The use of previously inactivated biocatalysts (using diethyl p-nitrophenylphosphate) permitted to visualize the immobilization of each enzyme layer, which was later confirmed by SDS-PAGE. This also confirmed the successful and complete covalent crosslinking of the glutaraldehyde treated enzyme layers. Activity of the combibiocatalysts was followed using diverse substrates. The protocol was successful and permitted to immobilize in an ordered way the 5 different enzymes in a down-up distribution., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Understanding the pH effect of cellulolytic enzymes is of great technological importance. In this study, we have examined the influence of pH on activity and stability for central cellulases (Cel7A, Cel7B, Cel6A from Trichoderma reesei, and Cel7A from Rasamsonia emersonii). We systematically changed pH from 2 to 7, temperature from 20°C to 70°C, and used both soluble (4-nitrophenyl β- d-lactopyranoside [pNPL]) and insoluble (Avicel) substrates at different concentrations. Collective interpretation of these data provided new insights. An unusual tolerance to acidic conditions was observed for both investigated Cel7As, but only on real insoluble cellulose. In contrast, pH profiles on pNPL were bell-shaped with a strong loss of activity both above and below the optimal pH for all four enzymes. On a practical level, these observations call for the caution of the common practice of using soluble substrates for the general characterization of pH effects on cellulase activity. Kinetic modeling of the experimental data suggested that the nucleophile of Cel7A experiences a strong downward shift in pK a upon complexation with an insoluble substrate. This shift was less pronounced for Cel7B, Cel6A, and for Cel7A acting on the soluble substrate, and we hypothesize that these differences are related to the accessibility of water to the binding region of the Michaelis complex., (© 2019 Wiley Periodicals, Inc.)

Adsorption of lipases on hydrophobic supports is a very easy immobilization protocol and it yields very interesting immobilized lipase derivatives. The open and active form of lipase molecules becomes stabilized by strong adsorption on the support surface. By using very rigid hydrophobic supports (e.g., methacrylate), lipase derivatives are very useful to catalyze biotransformations in fully anhydrous organic media (solvents, solvent-free systems, etc.) and design of continuous flow reactors. In addition to that, the design of different lipase derivatives allows the modulation of functional properties of the derivatives. In this chapter, methodology of immobilization into hydrophobic carriers is described using as case study the preparation of immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL), and the following particular features will be discussed: 1. Adsorption on hydrophobic supports yields lipase derivatives that are much more active and stable than other immobilized lipase derivatives. 2. Regioselectivity can be modulated, for example, TLL adsorbed on divinyl benzene hydrophobic supports retains a 1,3 regioselectivity during ethanolysis of oils. On the contrary, the enzyme adsorbed on octadecyl supports loses the regioselectivity and allows the complete ethanolysis of oils (e.g., biodiesel synthesis). 3. TLL adsorbed on octadecyl supports with large pore size (60 nm) is tenfold more active for ethanolysis in solvent-free systems than TLL derivatives adsorbed on supports with small pore size (10 nm).

Diisononyl adipate is a plasticizer which has excellent property of low temperature resistance and is permitted in food contact materials. In this study, diisononyl adipate was synthesized from adipic acid and isononyl alcohol in a solvent-free system via immobilized lipase-catalyzed esterification. Liquid Eversa lipase from Thermomyces lanuginosus was immobilized on Lewatit VP OC 1600 carrier. The effects of temperature, substrate molar ratio, water activity of enzyme, vacuum, and enzyme loading on the reaction efficiency were investigated. Application of vacuum played a key role to achieve a 100% conversion. The optimal temperature, molar ratio (adipic acid to isononyl alcohol), water activity of enzyme, vacuum were 50 °C, 1:3, 0.75, 13.3 kPa, and 10% (based on weight of total substrate). Under these conditions, 100% conversion was achieved within 6 h., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Current enzymatic methods for hemicellulosic biomass depolymerization are solution-based, typically require a harsh chemical pre-treatment of the material and large volumes of water, yet lack in efficiency. In our study, xylanase (E.C. 3.2.1.8) from Thermomyces lanuginosus is used to hydrolyze xylans from different sources. We report an innovative enzymatic process which avoids the use of bulk aqueous, organic or inorganic solvent, and enables hydrolysis of hemicellulose directly from chemically untreated biomass, to low-weight, soluble oligoxylosaccharides in >70% yields., Competing Interests: Some of the herein presented work is a part of the patent application US 62/465,443 filed on 1 March 2017. TF is a co-founder of Form-Tech Scientific, which has provided some of the equipment used in this study.

Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis. As a result, there is an incentive to understand the mechanisms underlying lipase activity at the molecular level, so as to be able to design new lipase variants with tailor-made functionalities. Our understanding of lipase function primarily relies on bulk assay averaging the behavior of a high number of enzymes masking structural dynamics and functional heterogeneities. Recent advances in single molecule techniques based on fluorogenic substrate analogues revealed the existence of lipase functional states, and furthermore so how they are remodeled by regulatory cues. Single particle studies of lipases on the other hand directly observed diffusional heterogeneities and suggested lipases to operate in two different modes. Here to decipher how mutations in the lid region controls Thermomyces lanuginosus lipase (TLL) diffusion and function we employed a Single Particle Tracking (SPT) assay to directly observe the spatiotemporal localization of TLL and rationally designed mutants on native substrate surfaces. Parallel imaging of thousands of individual TLL enzymes and HMM analysis allowed us to observe and quantify the diffusion, abundance and microscopic transition rates between three linearly interconverting diffusional states for each lipase. We proposed a model that correlate diffusion with function that allowed us to predict that lipase regulation, via mutations in lid region or product inhibition, primarily operates via biasing transitions to the active states.

Background: Structurally stable α-galactosidases are of great interest for various biotechnological applications. More thermophilic α-galactosidases with high activity and structural stability have therefore to be mined and characterized. On the other hand, few studies have been performed to prominently enhance the AOX1 promoter activity in the commonly used Pichia pastoris system, in which production of some heterologous proteins are insufficient for further study., Results: ReGal2 encoding a thermoactive α-galactosidase was identified from the thermophilic (hemi)cellulolytic fungus Rasamsonia emersonii. Significantly increased production of ReGal2 was achieved when ReGal2 was expressed in an engineered Pastoris pichia expression system with a modified AOX1 promoter and simultaneous fortified expression of Mxr1 that is involved in transcriptionally activating AOX1. Purified ReGal2 exists as an oligomer and has remarkable thermo-activity and thermo-tolerance, exhibiting maximum activity of 935 U/mg towards pNPGal at 80 °C and retaining full activity after incubation at 70 °C for 60 h. ReGal2 is insensitive to treatments by many metal ions and exhibits superior tolerance to protein denaturants. Moreover, ReGal2 efficiently hydrolyzed stachyose and raffinose in soybeans at 70 °C in 3 h and 24 h, respectively., Conclusion: A modified P. pichia expression system with significantly enhanced AOX1 promoter activity has been established, in which ReGal2 production is markedly elevated to facilitate downstream purification and characterization. Purified ReGal2 exhibited prominent features in thermostability, catalytic activity, and resistance to protein denaturants. ReGal2 thus holds great potential in relevant biotechnological applications.

Silica has been extracted from rice husks via a simple hydrothermal process and functionalized with triethoxy(octyl)silane -OCTES (Octyl-SiO 2 ) and (3-aminopropyl)triethoxysilane - 3-APTES (Amino-SiO 2 ), with the aim of using it as support to immobilize lipase from Thermomyces lanuginosus (TLL) via adsorption. The supports have been characterized by particle size distribution and elemental analyses, XRD, TGA, SEM, AFM and N 2 physisorption so as to confirm their functionalization. Effect of pH, temperature, initial protein loading and contact time on the adsorption process has been systematically evaluated. Maximum immobilized protein loading of 12.3 ± 0.1 mg/g for Amino-SiO 2 (5 mM buffer sodium acetate at pH 4.0, 25 °C and initial protein loading of 20 mg/g) and 21.9 ± 0.1 mg/g for Octyl-SiO 2 (5 mM buffer sodium acetate at pH 5.0, 25 °C and initial protein loading of 30 mg/g) was observed. However, these biocatalysts presented similar catalytic activity in olive oil emulsion hydrolysis (between 630 and 645 U/g). TLL adsorption was a spontaneous process involving physisorption. Experimental data on Octyl-SiO 2 and Amino-SiO 2 adsorption were well-fitted to the Langmuir isotherm model. It was also investigated whether these biocatalysts could synthesize cetyl esters via esterification reaction. Thus, it was found that cetyl stearate synthesis required 100-110 min of reaction time to attain maximum conversion percentage (around 94%). Ester productivity of immobilized TLL on Amino-SiO 2 was 1.3-3.1 times higher than Octyl-SiO 2 ., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study, the xylose reductase gene (XRTL) from Thermomyces lanuginosus SSBP was expressed in Pichia pastoris GS115 and Saccharomyces cerevisiae Y294. The purified 39.2 kDa monomeric enzyme was optimally active at pH 6.5 and 50 °C and showed activity over a wide range of temperatures (30-70 °C) and pH (4.0-9.0), with a half-life of 1386 min at 50 °C. The enzyme preferred NADPH as cofactor and showed broad substrate specificity. The enzyme was inhibited by Cu 2+ , Fe 2+ and Zn 2+ , while ferulic acid was found to be the most potent lignocellulosic inhibitor. Recombinant S. cerevisiae with the XRTL gene showed 34% higher xylitol production than the control strain. XRTL can therefore be used in a cell-free xylitol production process or as part of a pathway for utilization of xylose from lignocellulosic waste., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

D-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic D-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic D-amino acids and a neutral D-amino acid, D-Gln, with the highest activity toward D-Glu. The K m and k cat values for D-Glu were 2.16 mM and 217 s -1 , respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T 50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and D-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.

Enzymatic interesterification was carried out between high-oleic canola oil and fully hydrogenated soybean oil using indigenously immobilized Thermomyces lanuginosus lipas substrate concentration, moisture content of enzyme, and enzyme load. Interesterification resulted in a decrease in the concentration of tri-unsaturated and trisaturated TAG and an increase of mono- and di-saturated TAG as observed by reversed-phase HPLC. The alteration in TAG composition and the presence of new TAG species after interesterification was correlated with extended plasticity characterized by lower slip melting point with a significant change in functionality and consistency of the interesterified product. Thermal and structural properties of the blends before and after interesterification were assessed by differential scanning calorimetry (DSC), X-ray diffraction and polarized light microscopy. Trans- fat analysis indicated the absence of any trans fatty acid in the final interesterified product. The resultant interesterified products with varying slip melting points can be used in the formulation of healthier fat and oil products and address a critical industrial demand for trans free formulations for base-stocks of spreads, margarines, and confectionary fats.

Ion-exchange supports have been prepared via sequential functionalization of silica-based materials with (3‑Glycidyloxypropyl)trimethoxysilane (GPTMS) (Epx-SiO 2 ) and activation with glycine (Gly-Epx-SiO 2 ) in order to immobilize lipase from Thermomyces lanuginosus (TLL) via adsorption. Rice husk silica (RHS) was selected as support with the aim of comparing its performance with commercial silica (Immobead S60S). Sequential functionalization/activation of SiO 2 -based supports has been confirmed by AFM, SEM and N 2 adsorption-desorption analyses. Maximum TLL adsorption capacities of 14.8 ± 0.1 mg/g and 16.1 ± 0.6 mg/g using RHS and Immobead S60S as supports, respectively, have been reached. The Sips isotherm model has been used which was well fitted to experimental data on TLL adsorption. Catalytic activities of immobilized TLL were assayed by olive oil emulsion hydrolysis and butyl stearate synthesis via an esterification reaction. Hydrolytic activity of the biocatalyst prepared with a commercial support (357.6 ± 11.2 U/g) was slightly higher than that of Gly-Epx-SiO 2 prepared with RHS (307.4 ± 7.2 U/g). On the other hand, both biocatalysts presented similar activity (around 90% conversion within 9-10 h of reaction) and reusability after 6 consecutive cycles of butyl stearate synthesis in batch systems., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The lipase and Triton X-100 mixture is common for stabilization, immobilization and application processes of these kinds of enzymes. The objective of this article was to study the structural behavior and catalytic performance of Thermomyces lanuginose lipase in the presence of Triton X-100 at 25 °C and different pHs. The structural changes were followed by circular dichroism, correlating them with the catalytic performance, which is reported as the initial lipase activity in the hydrolysis of p‑nitro phenyl butyrate at zero time and residual activity after 48 h of incubation in the absence or presence of surfactant, at the selected pHs. Computational simulations allowed to explain the correlations between the physicochemical changes and the formation of surfactant protein complex, leading to the elucidation of the main interactions that drive activity and stability of this lipase in presence of the Triton X-100 surfactant. Main results showed the Triton X-100-enzyme complex modulates the site active geometry, favoring a better substrate-enzyme adjustment, which influences the activity and stability at evaluated pHs. This study contributes to understand the effect of some additives commonly used to improve the biocatalytic performance on several applications for different industrial fields., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Magnetite and magnetite core/shell (Fe 3 O 4 /SiO 2 ) nanoparticles were synthesized and functionalized with two different alkyl chain length linkers that were 3-Phosphonopropionic acid (3-PPA) and 16-Phosphonohexadecanoic acid (16-PHDA). Xylanase (EC 3.2.1.8, endo-1,4-xylanase, endo-1), was immobilized on as synthesized bare and silica coated magnetite nanoparticles via well-known EDC coupling. Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FESEM), X-Ray Diffraction Spectroscopy (XRD), X-ray Photoelectron Spectroscopy (XPS), Dynamic Light Scattering (DLS) and Thermogravimetric analysis (TGA) techniques were utilized to characterize all the modifications. The flexible linker chain length plays a vital role in the catalytic attributes of the immobilized enzyme. Result shows that long chain alkyl linker grafted magnetite and magnetite core/silica shell nanoparticles exhibited a superior performance in terms of lower K m , higher catalytic efficiency and better reusability. Furthermore, the immobilized xylanase shows improved tolerability performance at a wide range of pH and temperature. Silica-coated magnetite nanoparticles bound xylanase through 16-PHDA retained 90% of its initial activity after 10 consecutive cycles, further emphasize on the beneficial effect of linker chain length and inert silica coating., (Copyright © 2018. Published by Elsevier B.V.)

We present an efficient approach to the synthesis of a series of glycyrrhetinic acid derivatives. Six derivatives, five of them new compounds, were obtained through chemoenzymatic reactions in very good to excellent yield. In order to find the optimal reaction conditions, the influence of various parameters such as enzyme source, nucleophile:substrate ratio, enzyme:substrate ratio, solvent and temperature was studied. The excellent results obtained by lipase catalysis made the procedure very efficient considering their advantages such as mild reaction conditions and low environmental impact. Moreover, in order to explain the reactivity of glycyrrhetinic acid and the acetylated derivative to different nucleophiles in the enzymatic reactions, molecular docking studies were carried out. In addition, one of the synthesized compounds exhibited remarkable antiviral activity against TK + and TK- strains of Herpes simplex virus type 1 (HSV-1), sensitive and resistant to acyclovir (ACV) treatment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This paper takes a look on the effects of mass transport limitation occurring in hydrolysis of rape seed oil by means of an interfacial activated lipase from Thermomyces lanuginosus. In order to carry out investigations for process optimization, the slug flow reactor was chosen in which a large interfacial area can be generated and the mass transport can be investigated individually for each phase. The choice of the capillary material determines the dispersed and the continuous phase. As shown by computational fluid dynamics simulation, the continuous phase is well mixed due to wall effects. The mixing patterns in the dispersed phase differ due to viscous forces between the phases. It was found that, at the same fluid velocities, the conversion in the glass capillary is higher than in the PTFE capillary. The surface-specific hydrolysis rate is used for comparison purposes, since the properties of the capillary are different. Increasing the velocity, the hydrolysis rate can be considerably increased in comparison to stagnant conditions. Already at a fluid velocity of 1 mm s -1 , the hydrolysis rates increased to 2.3-fold in the glass capillary and moreover by a factor of 4 in the PTFE capillary.

In this study, the immobilization of xylanase using a protein-inorganic hybrid nanoflower system was assessed to improve the enzyme properties. The synthesis of hybrid xylanase nanoflowers was very effective at 4°C for 72 h, using 0.25 mg/ml protein, and efficient immobilization of xylanase was observed, with a maximum encapsulation yield and relative activity of 78.5% and 148%, respectively. Immobilized xylanase showed high residual activity at broad pH and temperature ranges. Using birchwood xylan as a substrate, the V max and K m values of xylanase nanoflowers were 1.60 mg/ml and 455 μmol/min/mg protein, compared with 1.42 mg/ml and 300 μmol/min/mg protein, respectively, for the free enzyme. After 5 and 10 cycles of reuse, the xylanase nanoflowers retained 87.5% and 75.8% residual activity, respectively. These results demonstrate that xylanase immobilization using a protein-inorganic hybrid nanoflower system is an effective approach for its potential biotechnological applications.

Lipases B from Candida antarctica (CALB), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL) were immobilized on octadecyl methacylate (OM) or octadecyl methacrylate (OMC) beads. Their specific activity and regioselectivity were studied in the synthesis of conjugated linoleic acid (CLA) partial glycerides, which presented nutraceutical properties. TLL derivatives were poor catalysts. Novozym® 435 was much better than Lipozyme® RM IM. RML activity (a GRAS enzyme) was modulated via immobilization. After only 3 h, OM-RML gave the highest CLA conversion (54% at 40 °C with 1:3 M ratio of glycerol to CLA). OM-RML reduced by a factor of 3.12 and 1.16 the activation energy of the reaction with Lipozyme® RM IM and Novozym® 435, respectively. The new GRAS preparation OM-RML brings forth an optimal regioselective preparation of sn-1 mono and sn-1,3 diacylglycerols rich in CLA, with a ratio of sn-1,3/sn-1,2 regioisomers of 21.8, compared to 2.3 for Novozym® 435., (Copyright © 2017 Elsevier Ltd. All rights reserved.)