Thermomyces Lanuginosus Research Articles

Enzymatic Interesterification of Cold-Pressed Maqui (Aristotelia chilensis (Mol.) Stuntz) Seed Oil and Belly Oil from Rainbow Trout (Oncorhynchus mykiss) Through Supercritical CO2

A new antioxidant lipid (AL) was synthesized from rainbow trout (Oncorhynchus mykiss) belly oil and cold-pressed maqui (CPM) (Aristotelia chilensis (Mol.) Stuntz) seed oil via enzymatic interesterification using Thermomyces lanuginosus in supercritical CO2 medium. A Box–Behnken design with 15 experiments was employed, with the independent variables being the following: belly oil/CPM oil ratio (10/90, 50/50, and 90/10, w/w), supercritical CO2 temperature (40.0, 50.0, and 60.0 °C), and supercritical CO2 pressure (100.0, 200.0, and 300.0 bar) for enzymatic interesterification. A multiple optimization was conducted based on the response variables yield and eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and tocopherol contents. The optimized conditions for the AL synthesis were: 81.4/18.6 (w/w), 40.0 °C and 299.99 bar, respectively. The corresponding responses variables were: 77.10% for yield, 5.12 and 4.95 g·100 g−1 total fatty acids for EPA and DHA, respectively, and 217.96, 4.28, 3.48, 64.48, and 6.39 mg·kg−1 oil for α-tocopherol, α-tocotrienol, β-tocopherol, γ-tocopherol, and δ-tocopherol, respectively. A novel AL was successfully synthesized starting from two abundant natural resources commonly considered as by-products during industrial processing. In agreement with the high EPA, DHA, and tocopherol presence, this AL can be recommended to be employed in nutritional and therapeutic supplements, according to its health benefits, particularly concerning antioxidant and anti-inflammatory properties.

Continuous Flow Synthesis of Hexyl Laurate Using Immobilized Thermomyces Lanuginosus Lipase from Residual Babassu Mesocarp.

Brazil has one of the greatest biodiversities on the planet, where various crops play a strategic role in the country's economy. Among the highly appreciated biomasses is babassu, whose oil extraction generates residual babassu mesocarp (BM), which still needs new strategies for valorization. This work aimed to use BM as a support for the immobilization of Thermomyces lanuginosus lipase (TLL) in an 8.83 mL packed-bed reactor, followed by its application as a biocatalyst for the synthesis of hexyl laurate in an integrated process. Initially, the percolation of a solution containing 5 mg of TLL at 25 °C and flows ranging from 1.767 to 0.074 mL min-1 was investigated, where at the lowest flow rate tested (residence time of 2 h), it was possible to obtain an immobilized derivative with hydrolytic activity of 504.7 U g-1 and 31.7 % of recovered activity. Subsequent studies of treatment with n-hexane, as well as the effect of temperature on the immobilization process, were able to improve the activities of the final biocatalyst BM-TLLF, achieving a final hydrolysis activity of 7023 U g-1 and esterification activity of 430 U ⋅ g-1 against 142 U g-1 and 113.5 U g-1 respectively presented by the commercial TLIM biocatalyst. Desorption studies showed that the TL IM has 18 mg of protein per gram of support, compared to 4.92 mg presented by BM-TLL. Both biocatalysts were applied to synthesize hexyl laurate, achieving 98 % conversion at 40 °C within 2 h. Notably, BM-TLLF displayed exceptional recyclability, maintaining catalytic efficiency over 12 cycles. This reflects a productivity of 180 mg of product ⋅ h-1 U-1 of the enzyme, surpassing 46 mg h-1 U-1 obtained for TLIM. These results demonstrate the efficacy of continuous flow technology in creating a competitive and integrated process offering an exciting alternative for the valorization of residual lignocellulosic biomass.

Engineering lipase TLL to improve its acid tolerance and its biosynthesis in Trichoderma reesei for biodiesel production from acidified oil

Lipases catalyze the synthesis of biodiesel, which is an important renewable alternative energy source. Cost-efficient conversion of waste acidified oil to biodiesel entails acid-tolerant lipases which have not been extensively studied. This study showed that the commonly used Thermomyces lanuginosus lipase TLL displayed a weak acid tolerance and an unsatisfactory performance in biodiesel production from acidified oil. Directed evolution of TLL identified one TLL-T3 variant with three residue substitutions (A69S/V150P/N222G). TLL-T3 displayed significantly enhanced acid tolerance, and its application in acidified oil treatment led to a biodiesel yield up to 90 % (w/w). A scaled-up production of TLL-T3 in Trichoderma reesei was further achieved and the highest extracellular lipase activity reached 16,123 U/mL after fermentation optimization. These results provide new insights into structural adaptation to acid tolerance by lipases and show that TLL-T3 holds great potential in commercial biodiesel production from waste acidified oil.

Design of green DES-based aqueous two phase systems for lipolytic enzymes extraction

This study pioneers a novel green and cost-effective separation process using reline deep eutectic solvent (DES) for extracting Thermomyces lanuginosus lipase (TlL) from aqueous solutions. For the first time, it has been shown that various potassium organic salts combined with reline DES can induce phase separation in aqueous solutions of the biodegradable non-ionic surfactant Tergitol 15-S9, presenting a groundbreaking alternative to the banned surfactant Triton X-100. This work uniquely applies three different equations to describe the experimental solubility curves. The selection of reline DES is particularly innovative as it avoids significant deactivation effects with three commercial lipases: Candida antarctica lipases A and B, and TlL, with the latter undergoing a detailed study. This includes an unprecedented analysis of its thermodeactivation kinetics in reline DES aqueous solutions. Additionally, a novel investigation of immiscibility regions at different temperatures, involving comprehensive Tie-Line characterizations (slope and length), preceded the evaluation of lipase extraction. Remarkably, the study demonstrates that optimizing the feed composition to contain higher water concentrations can achieve exceptionally high extraction efficiencies (approximately 90%), suggesting a transformative potential for future applications in real aqueous cultures.

Cellulase gene expression in the thermophilic Thermomyces lanuginosus isolated from compost

Thermophilic fungi are superlative microorganisms for enzyme production, especially cellulase, and their using in biotechnological applications is due to their stability at utmost temperatures. In the current investigation, we isolated six genera encompassing six fungal species and one species variety from 30 samples of compost at 45 °C and 55 °C. Thermomyces lanuginosus was the most rampant species. The colony diameter of T. lanuginosus ranged from 2.8 to 4.3 cm at 45 °C on yeast-starch agar (YpSs) medium with white or greyish-brown mycelia. Fifteen isolates of T. lanuginosus were cellulase producers with variable competencies with a C/Z range of 1.09–1.38 cm. Fascinatingly, the clear zone diameter was much bigger when using Iodine than those obtained using Congo red. T. lanuginosus isolate no. 33 produced substantial amounts of cellulase on the four used media: Corncob (CC), Corncob treated with NaOH (C-NA), Yeast Peptone Dextrose (YPD), and CarboxyMethyl Cellulose (CMC) with the highest activity on CC; 143.9 μg/min, also cellulase gene expression levels of cel6Aq, cel7Aq, and bgl3Aq genes exhibited higher fold changes in the CC condition (7.26-fold, 11.51-fold, and 3.39-fold, respectively). X-ray fluorescence (XRF) analysis revealed the presence of 11 minerals with higher concentrations in CC than in C-NA. Supplementation of corncob medium with rosemary essential oil (CR) completely inhibited cellulase production. It adversely affected the growth, and changes in conidia, which were depicted using a scanning electron microscope (SEM). Interestingly, the conidia appeared much bigger than other media, and the large conidia diameter was 10.2–12.1 μm.

Enzymatic preparation of diacylglycerols: lipase screening, immobilization, characterization and glycerolysis performance.

Glycerolysis, with its advantages of readily available raw materials, simple operation, and mild reaction conditions, is a primary method for producing diacylglycerol (DAG). However, enzymatic glycerolysis faces challenges such as high enzyme costs, low reuse efficiency, and poor stability. The study aims to develop a cost-effective immobilized enzyme by covalently binding lipase to pre-activated carriers through the selection of suitable lipases, carriers, and activating agents. The optimization is intended to improve the glycerolysis reaction for efficient DAG production. Lipase CN-TL (from Thermomyces lanuginosus) was selected through glycerolysis reaction and molecular docking to catalyze the glycerolysis reaction. Optimizing the immobilization method by covalently binding CN-TL to poly(ethylene glycol) diglycidyl ether (PEGDGE)-preactivated resin LX-201A resulted in the preparation of the immobilized enzyme TL-PEGDGE-LX. The immobilized enzyme retained over 90% of its initial activity after five consecutive reactions, demonstrating excellent reusability. The DAG content in the product remained at 84.8% of its initial level, further highlighting the enzyme's potential for reusability and its promising applications in the food and oil industries. The immobilized lipase TL-PEGDGE-LX, created by covalently immobilizing lipase CN-TL on PEGDGE-preactivated carriers, demonstrated broad applicability and excellent reusability. This approach offers an economical and convenient immobilization strategy for the enzymatic glycerolysis production of DAG. © 2024 Society of Chemical Industry.

Immobilization of Lipase from Thermomyces Lanuginosus and Its Glycerolysis Ability in Diacylglycerol Preparation.

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.

Enzymatic Synthesis of Austroeupatol Esters with Enhanced Antiprotozoal Activity.

Austroeupatol, the principal diterpene isolated from the invasive shrub Austroeupatorium inulifolium, holds promise for structural diversification and biological assessment of its derivatives due to its abundant availability and high yield isolation. We propose an efficient enzymatic synthesis of a series of austroeupatol esters derived from aliphatic and heterocyclic carboxylic acids. Systematic optimization of reaction parameters, including enzyme type and quantity, acylating agent amount, solvent, and temperature, was conducted. Thermomyces lanuginosus lipase in cyclohexane at 55 °C, yielded esters with favorable conversion rates. Through enzymatic catalysis, mono- and diacylated derivatives were obtained, with a diacylation-monoacylation ratio influenced by temperature and acylating agent amount. The antiprotozoal activity of austroeupatol and all synthesized derivatives was evaluated, observing that acylation improved it. The 19-valeroyl, 19-indolylpropyl, and 19-octyl derivatives were the most potent compounds against Trypanosoma cruzi and Leishmania infantum, highlighting this approach as a valuable method for synthesizing austroeupatol derivatives as potential antiparasitic agents.

Obtaining a biodegradable biocatalyst – study on lipase immobilization on spent coffee grounds as potential carriers

Coffee waste can be reused as matrices for enzyme immobilization, as it contains various organic compounds able to adsorb catalytic proteins. In this study, spent coffee grounds were used as a support in the immobilization process in their native form and after being pretreated with hexane, ethanol, and sulphuric acid solution, respectively. Microbial lipases from Aspergillus oryzae, Thermomyces lanuginosus, and Rhizomucor miehei were adsorbed on the carrier, and as a reference, the abovementioned lipases were also immobilized on a synthetic matrix - Lewatit VP OC 1600. The research investigated the impact of the purification step on the immobilization process and the full characteristics of the obtained biocatalysts. The hydrolytic and synthetic activities of the immobilized enzyme preparations were tested, as well as substrate specificity, recovery, and temperature and pH activity profiles. SEM and FTIR analyses were also performed. The results showed that the chemical composition of coffee waste influenced the activity of the obtained biocatalysts, and the lack of hemicellulose caused a reduction in lipolytic activity. The study reveals that spent coffee grounds can be a potential support for enzyme immobilization, and lipases adsorbed on them have improved properties such as hydrolytic or synthetic activity, and stability in pH.

Diaminated Cellulose Beads as a Sustainable Support for Industrially Relevant Lipases.

Environmentally persistent polystyrene or polyacrylic beads are used as supports in enzyme large-scale bioprocesses, including conversion glucose isomerization for high-fructose corn syrup production, hydrolysis of lactose, and synthesis of active pharmaceutical ingredients. In this paper, we report the development of a novel sustainable and scalable method to produce diaminated cellulose beads (DAB) as highly efficient alternative supports for industrially relevant lipases. Regenerated cellulose beads were grafted with diaminated aliphatic hydrocarbons via periodate oxidation and reductive amination. The oxidation step indicated that aldehyde content can be easily tuned through the reaction time and concentration of reactants. Reductive amination of dialdehyde cellulose was more efficient as the length of the diaminated hydrocarbon compound increased. Morphological analysis of DAB showed that cellulose chemical grafting enabled the preservation of the bead shape and internal structure upon freeze-drying. Enzymatic degradability studies demonstrated that chemical functionalization did not undermine enzyme cellulose hydrolysis. The addition of aminated moieties on cellulose dramatically increased absorption efficiency for all industrially relevant lipases used, reaching 100% for Thermomyces lanuginosus lipase (TLL). Storage and recyclability experiments demonstrated that enzymes were retained and recyclable for at least nine cycles, although the activity gradually declined after each cycle. Medium chain triacylglycerol hydrolysis in a SpinChem reactor using TLL immobilized on 1,6 DAB exhibited higher activity compared to acrylic beads (588 vs 459 U/g) suggesting that biodegradable cellulose-based materials could be a valid and attractive alternative to plastics carriers.

The effect of carbon chain length of cross-linking agent on the functionality of carrier- free immobilized Thermomyces lanuginosa lipase particles

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.

One-Pot Synthesis of 1,3,5-Trisubstitued Pyrazoles via Immobilized Thermomyces lanuginosus Lipase (TLL) on a Metal-Organic Framework.

A regioselective enzyme-catalyzed system is selected for the synthesis of 1,3,5-trisubstituted pyrazole derivatives by adding phenyl hydrazines, nitroolefins, and benzaldehydes. The reaction is performed in a one-pot vessel with a yield ranging from 49 to 90%. TLL@MMI, immobilized Thermomyces lanuginosus lipase (TLL) on a multivariate of MOF-5/IRMOF-3 (MMI), showed good performance for the catalysis of this reaction. The prepared biocatalyst was characterized by FTIR, XRD, SEM, and EDX. The thermal and solvent stability of TLL@MMI was investigated in MeOH and EtOH after 24 h incubation. In the presence of 100% concentrations of EtOH, TLL@MMI has 80% activity.

Fabrication of Vascular Grafts Using Poly(ε-Caprolactone) and Collagen-Encapsuled ADSCs for Interposition Implantation of Abdominal Aorta in Rhesus Monkeys.

The production of small-diameter artificial vascular grafts continues to encounter numerous challenges, with concerns regarding the degradation rate and endothelialization being particularly critical. In this study, porous PCL scaffolds were prepared, and PCL vascular grafts were fabricated by 3D bioprinting of collagen materials containing adipose-derived mesenchymal stem cells (ADSCs) on the internal wall of the porous PCL scaffold. The PCL vascular grafts were then implanted in the abdominal aorta of Rhesus monkeys for up to 640 days to analyze the degradation of the scaffolds and regeneration of the aorta. Changes in surface morphology, mechanical properties, crystallization property, and molecular weight of porous PCL revealed a similar degradation process of PCL in PBS at pH 7.4 containing Thermomyces lanuginosus lipase and in situ in the abdominal aorta of rhesus monkeys. The contrast of in vitro and in vivo degradation provided valuable reference data for predicting in vivo degradation based on in vitro enzymatic degradation of PCL for further optimization of PCL vascular graft fabrication. Histological analysis through hematoxylin and eosin (HE) staining and fluorescence immunostaining demonstrated that the PCL vascular grafts successfully induced vascular regeneration in the abdominal aorta over the 640-day period. These findings provided valuable insights into the regeneration processes of the implanted vascular grafts. Overall, this study highlights the significant potential of PCL vascular grafts for the regeneration of small-diameter blood vessels.

Enzymatic production of xylose esters using degummed soybean oil fatty acids following a hydroesterification strategy

The enzymatic synthesis of xylose fatty acid esters was carried out in two steps: hydrolysis of degummed soybean oil (DSO) and esterification of the produced and purified free fatty acids (FFA) with xylose. Different lipases immobilized on a hydrophobic support were evaluated in the hydrolysis of DSO and an experimental design was used to optimize the reaction. Eversa® Transform 2.0 (EV-Purolite) yielded 93% conversion after 6 h using biocatalyst load of 6.53 wt%, water/oil mass ratio of 5.5 and temperature of 48.4 °C. High operational stability for EV-Purolite was found using a sequential batch strategy and the possibility of concentrating glycerol. In the esterification stage, lipase from Thermomyces lanuginosus (TLL-Purolite) and lipase B from Candida antarctica showed better performance than lipase from porcine pancreas and EV-Purolite. About 17% of FFAs (0.85 molecules of FAA per xylose molecule) was consumed using an enzyme load of 5 wt% of TLL-Purolite and FFA/xylose molar ratio of 5. Sequential esterification batches showed low operational stability of TTL-Purolite due to the desorption of TLL from the support. The final mixture of the reaction medium containing xylose fatty acid esters showed emulsifying properties similar to those of commercial surfactant.

Lipase-catalyzed synthesis of laurate esters from puerarin and its β-D-fructofuranosyl-(2→6)-puerarin derivative

This study investigates the transesterification of isoflavones puerarin and β-D-fructofuranosyl-(2→6)-puerarin to enhance their lipophilicity for specific pharmacological applications. The process achieved high yields (80-100%) in the isoflavones conversion when commercial lipases from Candida antarctica (CALB) and Thermomyces lanuginosus (TL IM) were used. The acylation patterns between CALB and TL IM varied depending on the substrate; both enzymes yielded principally monoacylated products from puerarin and β-D-fructofuranosyl-(2→6)-puerarin. However, TL IM also produced di- and triacylated derivatives of β-D-fructofuranosyl-(2→6)-puerarin in substantial amounts. Structural characterization by NMR identified puerarin-6ʹʹ-O-laurate and β-D-fructofuranosyl-(2→6)-puerarin-6ʹʹʹ-O-laurate as the primary products in CALB and TL IM reactions. These acyl derivatives presented improved lipophilicity (Log P from −1.0 − 0.16 to 3.26 − 3.58) and antioxidant activity (67.2-97.3 to 102.5-131.4 µM TE/mmol). Therefore, these compounds have potential pharmacological applications.

Enhancing methanol tolerance of Thermomyces lanuginosus lipase by rational design and biodiesel production through one-step feeding of methanol

The addition of a significant quantity of methanol solution during the biodiesel production results in the denaturation of lipase. To address this issue, this study proposed a novel approach to the preparation process by designing a methanol-tolerant Thermomyces lanuginosus lipase (TLL) using a computer-aided strategy. In silico prediction yielded seven mutants, out of which two mutants, E129Y and E134W, exhibited varying degrees of improved methanol tolerance and catalytic efficiency. The methanol tolerance and stability of E129Y and E134W were observed to increase by 10–30 % and 17–35 %, respectively. Additionally, E134W exhibited a 1.13-fold increase in catalytic efficiency compared to the wild type TLL, while E129Y showed a reduction of 12.05 %. Following a one-shot addition of methanol (molar ratio of 3:1 methanol to Cornus wilsoniana oil) for 72 h in the transesterification reaction, the yields of fatty acid methyl ester (FAME) for E129Y and E134W were 69 % and 85 %, respectively, with the yield of E134W increasing by 22 %. Structural analysis indicated that the E129Y mutation formed a single hydrogen bonding interaction, while E134W produced hydrophobic interactions with neighboring amino acids, thereby augmenting the structural stability of the mutations under high methanol concentration. This study has the potential to advance the commercialization of lipases in the biodiesel production industry.

Enzymatic kinetic resolution of an intermediate alcohol of the drug Tegoprazan using lipase from Thermomyces lanuginosus

An enzymatic approach for the synthesis of intermediate alcohol of the drug tegoprazan in 99% ee at 20 °C in hexane has been developed utilizing commercially available immobilized Thermomyces lanuginosus lipase (TL lipase) as a biocatalyst and vinyl acetate as a acyl donor. This method involves the enantioselective esterification of (RS)-5,7-difluorochroman-4-ol with vinyl acetate using TL Lipase. This enzymatic approach provides the (S)-chiral alcohol in 44% yield with 99% enantiomeric excess (ee) and the (R)-acetate in 43% yield and >99% ee. It is a sustainable approach for the synthesis of enantiopure tegoprazan intermediate, contributing to the advancement of green chemistry. The recovered TL lipase was reused in 13 subsequent runs without loss of activity.

Innovative approach for semi‐continuous production of puerarin palmitate via transesterification with novel immobilized lipase

BackgroundPuerarin, a flavonoid abundantly found in the roots of Pueraria lobata, exhibits antioxidant, anti-cancer, anti-inflammatory, and anti-aging properties. Despite its numerous benefits, the low bioavailability of puerarin hinders its widespread industrial application. To improve this, flavonoid acylation is required. Lipase is a biocatalyst primarily used for flavonoid acylation; immobilized lipase is preferred given the limited stability of free lipase. Although research on flavonoid acylation for structural modification is actively being conducted, there is a need to explore continuous production for enhancing the industrial application of flavonoid esters. Therefore, in this study, we aimed to produce puerarin palmitate innovatively by selecting an effective lipase, developing an immobilized lipase, characterizing the enzymatic reaction, and designing a semi-continuous reactor system.ResultsLipase from Thermomyces lanuginosus (TL) was found to be the most suitable for the synthesis of puerarin palmitate; it was successfully immobilized on glutaraldehyde-activated silica gel (GASG). TL GASG showed greater solvent, thermal, and operational stability than those of commercial immobilized lipases. Furthermore, TL GASG use resulted in a puerarin palmitate conversion rate of 97.17% within 3 h. Additionally, the reusability of TL GASG for this reaction was higher than that of commercially available immobilized lipases. Upon using a semi-continuous reactor, the cumulative conversion rate of puerarin palmitate remained above 85% after more than ten substrate injections.ConclusionsThe results support the feasibility of the continuous production of flavonoid esters, underscoring their diverse industrial applications. A biorefinery strategy is anticipated to be proposed to utilize P. lobata extract biomass for flavonoid ester production. Moreover, optimizing continuous reactors through statistical experimental design is expected to enhance flavonoid ester production efficiency; these aspects will be explored in future research.Graphical

Application of Thermomyces lanuginosus polygalacturonase produced in Komagataella phaffii in biomass hydrolysis and textile bioscouring

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 Km and Vmax, 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.

Exploring the contribution of temperature-adapted microbiota to enzyme profile of saccharification in Daqu using metagenomics and metaproteomics

Temperature is a critical factor that influences the microbiota in Daqu, which provides essential enzymes for the saccharification process in Baijiu (Chinese liquor) production. However, the relationship between temperature-adapted microbiota and saccharification properties of Daqu remains unclear. In this study, a profile of 23 major saccharifying enzymes and 40 responsible genera was obtained from the metagenomic data of medium- and high-temperature Daqu (MT- and HT-Daqu). The high-temperature pattern (65 °C) primarily affected the saccharification potential of Daqu by promoting Bacillales (fold-change: 0.25–0.75), whereas inhibiting Lactobacillales (fold-change: 3.80–5.60) and Mucorales (fold-change: 5.65–7.09). Eurotiales only exhibited a slight increase (fold-change: 1.01–1.24) to low-temperature pattern (56 °C). Metaproteomic analysis further revealed a notable contribution of Eurotiales in facilitating saccharification. Amylolytic enzymes were primarily derived from Eurotiales species (Byssochlamys spectabilis, Monascus purpureus, Rasamsonia emersonii, and Thermomyces lanuginosus) and one Mucorales species, Lichtheimia romasa. Cellulolytic enzymes were produced by Thermoascus aurantiacus and Paecilomyces sp. Among them, L. romasa was the principal contributor under the low-temperature pattern. Whereas, in HT-Daqu, B. spectabilis, M. purpureus, and T. lanuginosus were the major contributors, as L. romasa is susceptible to higher temperatures. This study offers valuable guidance for targeted optimization of industrial production.