A thermostable and inhibitor resistant ß-glucosidase from Rasamsonia emersonii for efficient hydrolysis of lignocellulosics biomass.

The present study reports a highly thermostable ß-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular ß-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110 kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified ß-glucosidase revealed the optimal activity at 80 °C, pH 5.0 and displayed high thermostability over broad range of temperature 50-70 °C with maximum half-life of ~ 60 h at 50 °C, pH 5.0. The putative transglycosylation activity of ß-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of ß-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal ß-glucosidase titres of 134,660 units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant ß-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted ß-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme.

Solvent-free synthesis of hydrophobic and amphiphilic esters using a chemically modified lipase from Thermomyces lanuginosus: a comparative study with native and immobilized forms.

Using lipases to catalyze the synthesis of the most differentiated type of compounds remains one of the major challenges among scientists. Seeking more economic and advantageous catalysts is a current goal of green chemistry. In this work, we demonstrate the potential of a chemically modified form of lipase from Thermomyces lanuginosus (cmLTL) for the synthesis of both hydrophobic (heptyl heptanoate, heptyl octanoate, heptyl decanoate, decyl heptanoate, decyl octanoate and decyl decanoate) and amphiphilic (2-(2-ethoxyethoxy)ethyl oleate and 2-(2-ethoxyethoxy)ethyl linoleate) esters, in bulk. The results were compared with its native (LTL) and immobilized (imLTL) forms. The data revealed that LTL showed poor activity for all reactions performed with n-heptane (η<20 %). ImLTL was able to synthesize all hydrophobic esters (η>60 %), with exception of the short ester, heptyl heptanoate. cmLTL was the only form of LTL capable of producing hydrophobic and amphiphilic esters, without compromising the yield when the reactions were performed under solvent-free conditions (>50 %). Molecular modeling showed that the active pocket of cmLTL is able to deeply internalize transcutol, with stronger interactions, justifying the outstanding results obtained. Furthermore, owing to the possibility of cmLTL filtration, the reusability of the catalyst is ensured for at least 6 cycles, without compromising the reaction yields.

Improving the Thermostability of Thermomyces lanuginosus Lipase by Restricting the Flexibility of N-Terminus and C-Terminus Simultaneously via the 25-Loop Substitutions.

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24-30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27…E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability.

Two new species of Trichocomaceae (Eurotiales), accommodated in Rasamsonia and Talaromyces section Bacillispori, from the Czech Republic.

During a previous study on microfungi associated with clematis roots, Penicillium-like fungi were isolated and identified based on morphology. In this study, we subjected those strains to a detailed examination which led to the proposal of two taxonomic novelties, named Rasamsonia chlamydospora and Talaromyces clematidis. The first taxon is characterized by rough-walled mycelium, acerose to flask shaped phialides, cylindrical conidia and by production of chlamydospore-like structures. The four-loci-based phylogeny analysis delineated the taxon as a taxonomic novelty in Rasamsonia. Talaromyces clematidis is characterized by restricted growth on Czapek yeast extract agar, dichloran 18% glycerol agar and yeast extract sucrose agar, and production of yellow ascomata on oatmeal agar. Phylogenetic analyses placed this taxon as a taxonomic novelty in Talaromyces sect. Bacillispori. Both taxa are introduced here with detailed descriptions, photoplates and information on their phylogenetic relationship with related species.

Disseminated Rasamsonia argillacea infection in a dog.

CASE HISTORY: A 4-year-old, male neutered Borzoi presented for unlocalised pain and frequent episodes of vocalisation. CLINICAL FINDINGS: Pain was localised to the lumbar spine and radiographs revealed a L3-L4 lesion consistent with discospondylitis. The dog was treated for presumptive bacterial discospondylitis with surgical debridement, spinal stabilisation, and cephalexin. Samples collected from the affected intervertebral disc at the time of surgery revealed lymphoplasmacytic inflammation with no causative agent identified on histopathology or bacterial culture. After an initial period of improvement, signs recurred despite an 8-week antibiotic course, with the development of inappetence, weight loss, polydipsia, and polyuria. Repeat radiographs revealed a new cervical intervertebral lesion, and concurrent pyelonephritis was diagnosed based on blood and urine results. Fungal culture of urine resulted in growth of Rasamsonia argillacea species complex and disseminated fungal disease was clinically diagnosed. Antifungal treatment was commenced, however the dog deteriorated, and euthanasia was performed. PATHOLOGICAL FINDINGS: Multifocal white plaques were grossly visualised in the spleen, mesenteric lymph nodes, cervical vertebrae, and kidneys. Periodic acid-Schiff-positive, fine, parallel-walled, occasionally branching, septate hyphae 5-10 µm in diameter, and conidia 5-7 µm in diameter were found on sectioning all organs. R. argillacea species complex was identified by fungal culture of urine and was considered the species of fungal organism seen histologically. The isolate was subsequently confirmed as R. argillacea by DNA sequencing. DIAGNOSIS: Disseminated Rasamsonia argillacea infection. CLINICAL RELEVANCE: Rasamsonia argillacea species complex is a recognised invasive mycosis in veterinary medicine, with disseminated disease causing significant clinical complications and death. This is believed to be the first report of infection caused by R. argillacea in a dog in Australasia and highlights the importance of awareness of a potential fungal aetiology in dogs with discospondylitis.Abbreviations: CLSI: Clinical and Laboratory Standards Institute; CRI: Constant rate infusion; MEC: Minimum effective concentration; MIC: Minimum inhibitory concentration; PAS: Periodic acid-Schiff.

Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion.

The function of most lipases is controlled by the lid, which undergoes conformational changes at a water-lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases' function is important for designing improved variants. Lipases' function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes' diffusional behavior, to study Thermomyces lanuginosus lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent.

Studies on the morphology, phylogeny, and bioremediation potential of Penicillium citrinum and Paecilomyces variotii (Eurotiales) from oil-contaminated areas.

Crude oil pollution is one of the most arduous issues to address, as it is hazardous to both public health and the environment. The discovery of novel biosurfactants-producing fungi and bacteria is in high demand due to their excellent properties and wide range of applications. The aim of this research is to isolate a powerful biosurfactant-producing fungus from the crude oil site near Barauni oil refinery in Bihar, India. Standard protocols were used to collect samples from the site. An integrative taxonomic approach was used, which included morphological, molecular, and phylogenetic analysis. The use of plating samples on Bushnell-Hass (BH) media aided in the isolation of a fungal strain from an enrichment culture. Two fungal strains isolated from contaminated soils, Penicillium citrinum and Paecilomyces variotti, showed potent oil degrading activity in a single culture. For preliminary biosurfactants screening, drop collapse assays, oil spreading, and emulsification activity tests were used. The results showed that the cultures performed well in the screening test and were further evaluated for degradation capacity. Different treatment periods (0, 3, 6, 9, 12, and 15 days) were used to observe degradation in single cultures. A steady drop in pH, an alteration in optical density and an increase in carbon dioxide release showed the ability of fungal strain to degrade the crude oil in a single culture. Fungi mycelia provide a larger surface area for absorption and degradation of the pollutants in contaminated environment. They produce extracellular enzymes to degrade the oil, and at the same time absorb and utilise carbon, allowing them to remove toxic substances from the oil. Thus, they could be candidates for bioremediation of a hydrocarbon-contaminated site.

Anisotropic protein-protein interactions in dilute and concentrated solutions.

Interactions between biomolecules are ubiquitous in nature and crucial to many applications including vaccine development; environmentally friendly textile detergents; and food formulation. Using small angle X-ray scattering and structure-based molecular simulations, we explore protein-protein interactions in dilute to semi-concentrated protein solutions. We address the pertinent question, whether interaction models developed at infinite dilution can be extrapolated to concentrated regimes? Our analysis is based on measured and simulated osmotic second virial coefficients and solution structure factors at varying protein concentration and for different variants of the protein Thermomyces Lanuginosus Lipase (TLL). We show that in order to span the dilute and semi-concentrated regime, any model must carefully capture the balance between spatial and orientational correlations as the protein concentration is elevated. This requires consideration of the protein surface morphology, including possible patch interactions. Experimental data for TLL is most accurately described when assuming a patchy interaction, leading to dimer formation. Our analysis supports that the dimeric proteins predominantly exist in their open conformation where the active site is exposed, thereby maximising hydrophobic attractions that promote inter-protein alignment.

Fabrication of immobilized lipases for efficient preparation of 1,3-dioleoyl-2-palmitoylglycerol.

This study aims to fabricate immobilized lipases for efficient preparation of 1,3-dioleoyl-2-palmitoyl-glycerol (OPO) through acidolysis of glycerol tripalmitate (PPP). Twelve (three types) supports and five lipases were studied carefully. Among them, the immobilized Thermomyces lanuginosa lipase (TLL) samples exhibited overall better performance than that of other immobilized lipases. Particularly, organic groups functionalized SBA-15 (R-SBA-15) supported TLL (TLL@R-SBA-15) samples gave PPP conversion from 97.70 to 99.00 % and OPO content from 59.52 to 64.73 %. After optimization, PPP conversion up to 99.07 %, OPO content 73.15 % and sn-2 palmitic acid content 90.09 % were obtained with TLL@C18H37-SBA-15 as catalyst. Moreover, TLL@C18H37-SBA-15 exhibited better acidolysis performance from 50 °C than that from 60 to 80 °C, which helped inhibit acyl migration. In addition, after 5 cycles of reuse, TLL@C18H37-SBA-15 retained 81.04 % (based on OPO content) and 98.88 % (based on sn-2 palmitic acid content) of its initial activity, indicating it had an attractive prospect in future applications.

Immobilization of Thermomyces lanuginosus lipase on a new hydrophobic support (Streamline phenyl™): Strategies to improve stability and reusability.

This paper establishes an efficient protocol for the immobilization of Thermomyces lanuginosus lipase (TLL) on a hydrophobic resin, Streamline phenyl. The biocatalyst produced by TLL immobilization on Streamline phenyl resin was named iTLL. In addition, strategies to improve stability and reusability of iTLL were performed using polyethylenimine (PEI) or/and glutaraldehyde (GA), producing iTLL-GA, iTLL-PEI, iTLL-PEI-GA biocatalysts. The immobilization yield was about 50%, using 1 mg/g of enzyme loading, and the immobilized enzyme activity was about 77 U/g, achieving about 100% of recovered activity. Desorption assays of the enzyme from the support using 0.6% cetyltrimethylammonium bromide (CTAB) and thermal and operational stability assays were performed. Although iTLL-PEI-GA lost about 50% of its initial activity after PEI and GA modifications, it was the most thermally and operationally stable (increases its stability about 66% if comparing with soluble enzyme at 65 ºC and maintenance 90% of its initial activity after 5 cycles of pNPB hydrolysis at 25 °C and pH 7.0). Furthermore, it showed almost no desorption of enzyme molecules with 24 h of CTAB incubation. Moreover, the streamline phenyl demonstrated a high TLL loading potential, with no diffusion limitations up to 14 mg/g. These characteristics allow future application of the iTLL-PEI-GA biocatalyst in fluidized bed reactors.

Biogenesis and characterization of proficient silver nanoparticles employing marine procured fungi Hamigera pallida and assessment of their antioxidative, antimicrobial and anticancer potency.

OBJECTIVE: To assess the anticancer potential of biosynthesized silver nanoparticles using marine derived fungi Hamigera pallida with their antibacterial and antioxidant activities. RESULTS: The biosynthesis of silver nanoparticles (AgNPs) was assessed by the change in color from bright yellow to dark brown. UV-Visible spectroscopy revealed its stability at 429 nm; ATR-FTIR spectroscopy revealed the functional group responsible for its production; X-Ray Diffraction revealed its crystalline FCC structure resembling the peaks in the XRD pattern, corresponding to (110), (111), (200), and (311) planes; TEM imaging revealed its spherical morphology with an average particle size of 5.85 ± 0.84 nm ranging from 3.69 to 16.11 nm and Tauc's plot analysis revealed a band gap energy of 2.22 eV, revealing aptitude of AgNPs as a semiconductors. The subsequent characterization results revealed the effective synthesis of silver nanoparticles. The biosynthesized AgNPs were found to have significant antimicrobial effect against three Gram-positive and three Gram-negative bacteria. They also demonstrated higher antioxidative potential by demonstrating strong radical scavenging activity against DPPH (2, 2-diphenyl-1-picrylhydrazyl). AgNPs showed highest anticancer activity (62.69 ± 1.73%) against human breast cancer (MCF-7) cell line at 100 µg/mL with the IC50 value of 66.07 ± 2.17 µg/mL. CONCLUSIONS: This study revealed the prospect for further utilization of AgNPs by Cell free filtrate of Hamigera pallida as an antibacterial, antioxidative and anticancer agents.

Discovery of the Key Mutation Site Influencing the Thermostability of Thermomyces lanuginosus Lipase by Rosetta Design Programs.

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications.

Prebiotic effect of sorghum biomass xylooligosaccharides employing immobilized endoxylanase from Thermomyces lanuginosus PC7S1T.

Purified endoxylanase from Thermomyces lanuginosus PC7S1T was immobilized in calcium alginate, resulting in a yield of 78.5% and a reusability for 11 cycles. The stability of the immobilized enzyme was given for a pH range of 4 to 9 for 96 h. Endoxylanase immobilized in calcium alginate at 65 °C exhibited thermal stability equal to the soluble enzyme for 5 h, and at high temperatures of 75 °C and 85 °C showed half-lives of 4 and 3 h, respectively. Both soluble endoxylanase and immobilized forms were able to hydrolyze hemicellulose, obtained from low-lignin sorghum biomass pretreated with 5% H2O2 and 2% NaOH, after 1 h of incubation at 65 °C, releasing a mixture of short-chain xylooligosaccharides (X2-X6). The highest amounts of XOS generated were those for X5 (24 to 40%), X4 (33 to 39%), and X3 (11 to 22%). These XOS acted as prebiotics, promoting the growth of the probiotic L. acidophilus, similar to glucose in the MRS broth. These results show the potential of low-lignin sorghum to generate XOS with prebiotic activity, suggesting the application of these compounds in the food industry.

Characteristics of Crosslinking Polymers Play Major Roles in Improving the Stability and Catalytic Properties of Immobilized Thermomyces lanuginosus Lipase.

This study aimed to improve the stability and catalytic properties of Thermomyces lanuginosus lipase (TLL) adsorbed on a hydrophobic support. At the optimized conditions (pH 5 and 25 °C without any additions), the Sips isotherm model effectively fitted the equilibrium adsorption data, indicating a monolayer and the homogenous distribution of immobilized lipase molecules. To preserve the high specific activity of adsorbed lipase, the immobilized lipase (IL) with a moderate loading amount (approximately 40% surface coverage) was selected. Polyethylenimine (PEI) and chitosan (CS) were successfully applied as bridging units to in situ crosslink the immobilized lipase molecules in IL. At the low polymer concentration (0.5%, w/w) and with 1 h incubation, insignificant changes in average pore size were detected. Short-chain PEI and CS (MW ≤ 2 kDa) efficiently improved the lipase stability, i.e., the lipase loss decreased from 40% to <2%. Notably, CS performed much better than PEI in maintaining lipase activity. IL crosslinked with CS-2 kDa showed a two- to three-fold higher rate when hydrolyzing p-nitrophenyl butyrate and a two-fold increase in the catalytic efficiency in the esterification of hexanoic acid with butanol. These in situ crosslinking strategies offer good potential for modulating the catalytic properties of TLL for a specific reaction.

Combination of system biology and classical approaches for developing biorefinery relevant lignocellulolytic Rasamsonia emersonii strain.

The objective of this study was to develop thermophilic fungus Rasamsonia emersonii using integrated system biology tools (genomics, proteomics and transcriptional analysis) in combination with classical strain breeding approaches. Developed hyper cellulolytic mutant strain M36 showed endoglucanase (476.35 U/ml), ß-glucosidase (70.54 U/ml), cellobiohydrolase (15.17 U/ml), FPase (4.89 U/ml) and xylanase (485.21 U/ml) on cellulose/gram flour based production medium. Comparison of the expression profile at proteome and transcriptional level of the developed strain and wild type parent gave detailed insight into the up-regulation of different CAZymes including glycosyl hydrolases (GH5, GH6, GH7, GH3, GH10) and auxiliary enzymes (lytic polysaccharide monooxygenase, swollenin) at system level. Furthermore, the potential of lignocellulolytic enzyme produced by the developed strain and custom designed cocktail spiked with heterologously expressed lytic polysaccharide monooxygenase from Mycothermus thermophiloides were analyzed for the hydrolysis of biorefinery relevant unwashed pretreated rice straw slurry (PRAJ and IOCL) @17% substrate loading rate.

Two-in-One Surfactant Disinfectant Potential of Xylitol Dicaprylate and Dilaurate Esters Synthesized by Talaromyces thermophilus galactolipase for Cleaning Industries.

Talaromyces thermophilus galactolipase (TTL) was found to produce alcohol sugar fatty acid diesters. The modulation of the solvent composition was used for the esterification reaction screening of diesters from xylitol and various fatty acids using the immobilized Talaromyces thermophilus galactolipase. The reactions were assessed by LC-MS analysis. The antimicrobial activity assay showed that both xylitol dicaprylate and xylitol dilaurate esters had more ability to inhibit the growth of several bacteria involved in surface contamination in the food industry. The xylitol dilaurate ester has the highest activity against Gram-positive strains with the lowest MIC values of 0.0016 and 0.005 mg mL-1 against Bacillus licheniformis and Staphylococcus aureus, respectively. Xylitol dicaprylate ester is more active against Gram-negative ones with significantly low MIC values of 0.25 and 0.4 mg mL-1 against Escherichia coli and Pseudomonas aeruginosa, respectively. The highest antifungal activity of the xylitol dicaprylate ester has been also proven, with a MIC value of 0.02 mg mL-1 against Penicillium occitanis and Fusarium solani. A better reduction in critical micelle concentrations and air-water surface tension were observed with these diesters compared to their corresponding monoesters in addition to their efficient emulsifying properties. The stability of these diesters in a liquid detergent formula after one year of storage was tested by a positive oil spreading assay and a retained antimicrobial activity. They exhibit a typical surfactant behavior with a two-in-one effect that can act as a detergent and a disinfectant with potential use in different cleaning processes.

Improved deinking and biobleaching efficiency of enzyme consortium from Thermomyces lanuginosus VAPS25 using genetic Algorithm-Artificial neural network based tools.

The present study reports the combined enzymatic production efficiency of thermophilic fungus Thermomyces lanuginosus VAPS25 using a combinatory artificial intelligence-based tool, resulting in 2.7 IU/ml, 5.2 IU/ml, and 18.85 U/ml activity of endoglucanase, amylase, and lipase, respectively with good thermostability at 90 °C (pH 8-10). Interestingly, the metal ions viz. Cu2+ and Mg2+ increased the endoglucanase activity to 5 folds, i.e.,5.6 IU/ml compared to control. Further, the amylase and lipase activity was also enhanced by Fe2+ and Co2+ to 5.4 IU/ml and 19.57 U/ml, respectively. Additionally, the deinking efficiency was improved by 68.9%, 42.7%, and 52.8% by endoglucanase, amylase, and lipase, respectively, while the consortium increased the deinking efficiency to 72.7%. The bio-bleached paper strength parameters such as burst index, breaking length, tear index, and tensile index of sheets were significantly improved by 1.38%, 13.54%, 7.54%, and 20.88%, respectively. These enzymes at an industrial scale would help develop an economical paper recycling process.

Purification and characterization of chitinase produced by thermophilic fungi Thermomyces lanuginosus.

BACKGROUND: In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed. RESULT: The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of ß-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa. CONCLUSION: The characterization study concludes that Cu2+, Hg2+, and EDTA have an inhibitory effect on chitinase activity, whereas ß-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste.

Hydroxyapatite/Glycyrrhizin/Lithium-Based Metal-Organic Framework (HA/GL/Li-MOF) Nanocomposite as Support for Immobilization of Thermomyces lanuginosus Lipase.

The hydroxyapatite/glycyrrhizin/lithium-based metal-organic framework (HA/GL/Li-MOF) nanocomposites were synthesized via the hydrothermal method in the presence of lecithin and glycyrrhizin. Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS) were applied for characterization of the fabricated nanocomposites. The HA/GL/Li-MOF and Li-MOF nanocomposites were employed as support for immobilization of Thermomyces lanuginosus lipase (TLL). The Plackett-Burman and Box-Behnken designs were used for screening and optimizing of variables affecting the immobilization conditions, respectively. The optimum specific activity of immobilized TLL on HA/GL/Li-MOF and Li-MOF nanocomposites (41.8 ± 1.2 U/mg and 39.4 ± 3.1 U/mg, respectively) was predictably determined at support concentration of 0.5 mg/mL, glutaraldehyde concentration of 5 mM, and enzyme activity of 20 U/mg, while the specific activities of TLL@ HA/GL/Li-MOF and TLL@Li-MOF were experimentally found to be 39.5 ± 3.7 U/mg and 38.5 ± 2.3 U/mg, respectively. The stability results showed that the TLL@ HA/GL/Li-MOF has suitable stability against pH and thermal denaturation. However, the immobilized TLL on Li-MOF represented lower stability compared with that of the HA/GL/Li-MOF. The immobilized TLL on HA/GL/Li-MOF maintained near 70% of its original activity after 15 days' storage and during 5 runs of application. In addition, TLL@HA/GL/Li-MOF exhibited higher enzyme-substrate affinity (Km, 10.1 mM) compared to that of TLL@Li-MOF (Km, 23.4 mM). Therefore, these findings demonstrated the potential use of HA/GL/Li-MOF nanocomposites for enzyme immobilization.

The changing face of SDS denaturation: Complexes of Thermomyces lanuginosus lipase with SDS at pH 4.0, 6.0 and 8.0.

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.