Unmodified Enzyme Research Articles

Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase.

Methyl-coenzyme M reductase (MCR), the key catalyst in the anoxic production and consumption of methane, contains an unusual 2-methylglutamine residue within its active site. In vitro data show that a B12-dependent radical SAM (rSAM) enzyme, designated MgmA, is responsible for this post-translational modification (PTM). Here, we show that two different MgmA homologs are able to methylate MCR in vivo when expressed in Methanosarcina acetivorans, an organism that does not normally possess this PTM. M. acetivorans strains expressing MgmA showed small, but significant, reductions in growth rates and yields on methylotrophic substrates. Structural characterization of the Ni(II) form of Gln-methylated M. acetivorans MCR revealed no significant differences in the protein fold between the modified and unmodified enzyme; however, the purified enzyme contained the heterodisulfide reaction product, as opposed to the free cofactors found in eight prior M. acetivorans MCR structures, suggesting that substrate/product binding is altered in the modified enzyme. Structural characterization of MgmA revealed a fold similar to other B12-dependent rSAMs, with a wide active site cleft capable of binding an McrA peptide in an extended, linear conformation.IMPORTANCEMethane plays a key role in the global carbon cycle and is an important driver of climate change. Because MCR is responsible for nearly all biological methane production and most anoxic methane consumption, it plays a major role in setting the atmospheric levels of this important greenhouse gas. Thus, a detailed understanding of this enzyme is critical for the development of methane mitigation strategies.

Screening of novel β-carotene hydroxylases for the production of β-cryptoxanthin and zeaxanthin and the impact of enzyme localization and crowding on their production in Yarrowia lipolytica

Zeaxanthin, a vital dietary carotenoid, is naturally synthesized by plants, microalgae, and certain microorganisms. Large-scale zeaxanthin production can be achieved through plant extraction, chemical synthesis, or microbial fermentation. The environmental and health implications of the first two methods have made microbial fermentation an appealing alternative for natural zeaxanthin production despite the challenges in scaling up the bioprocess. An intermediate between β-carotene and zeaxanthin, β-cryptoxanthin, is found only in specific fruits and vegetables and has several important functions for human health. The low concentration of β-cryptoxanthin in these sources results in low extraction yields, making biotechnological production a promising alternative for achieving higher yields. Currently, there is no industrially relevant microbial fermentation process for β-cryptoxanthin production, primarily due to the lack of identified enzymes that specifically convert β-carotene to β-cryptoxanthin without further conversion to zeaxanthin. In this study, we used genetic engineering to leverage the oleaginous yeast Yarrowia lipolytica as a bio-factory for zeaxanthin and β-cryptoxanthin production. We screened 22 β-carotene hydroxylases and identified eight novel enzymes with β-carotene hydroxylating activity: six producing zeaxanthin and two producing only β-cryptoxanthin. By introducing the β-carotene hydroxylase from the bacterium Chondromyces crocatus (CcBCH), a β-cryptoxanthin titer of 24 ± 6 mg/L was achieved, representing the highest reported titer of sole β-cryptoxanthin in Y. lipolytica to date. By targeting zeaxanthin-producing β-carotene hydroxylase to the endoplasmic reticulum and peroxisomes, we increased the production of zeaxanthin by 54% and 66%, respectively, compared to untargeted enzyme. The highest zeaxanthin titer of 412 ± 34 mg/L was achieved by targeting β-carotene hydroxylases to peroxisomes. In addition, by constructing multienzyme scaffold-free complexes with short peptide tags RIDD and RIAD, we observed a 39% increase in the zeaxanthin titer and a 28% increase in the conversion rate compared to the strain expressing unmodified enzyme. The zeaxanthin titers obtained in this study are not the highest reported; however, our goal was to demonstrate that specific approaches can enhance both titer and conversion rate, rather than to achieve the maximum titer. These findings underscore the potential of Y. lipolytica as a promising platform for carotenoid production and provide a foundation for future research, where further optimization is required to maximize production.

Effect of chemical modification on the distribution of electrophoretic mobilities of individual molecules of E. coli β-galactosidase

Escherichia coli β-galactosidase was labelled with 1 mmol/L fluorescein 5-carbamoylmethylthiopropanoic N-hydroxysuccinimidyl ester for 1 and 3 min. The samples were separated by capillary electrophoresis and peak areas compared to that of labelled BSA for the purpose of quantification of the concentration of attached label. Enzyme concentration in the samples was determined by single molecule counting. The average number of labels attached to each molecule of enzyme was found to be 3.1 and 4.5 when labelled for 1 and 3 min, respectively. The distribution of single enzyme molecule electrophoretic mobilities for the unlabelled enzyme and that labelled for 1 and 3 min were measured using capillary electrophoresis. The average mobilities were found determined to be −(1.99 ± 0.13) × 10–8 m2 V−1 s−1 ( N = 39), −(2.16 ± 0.19) × 10–8 m2 V−1 s−1 ( N = 46), and –(2.18 ± 0.21) × 10–8 m2 V−1 s−1 ( N = 39), respectively. A protein electrophoresis model was applied and predicted that the differences in average mobilities could be explained through relatively minor changes in overall charge, Stokes radius, and shape. This difference was similar to the range in mobilities observed in the unlabelled protein. This is consistent with the electrophoretic heterogeneity of the unmodified enzyme being caused by relatively small differences in charge, size, and shape of the individual molecules in the population.

Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the β-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and β-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and β-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.

The nature of the buffer alters the effects of the chemical modification on the stability of immobilized lipases

The objective of this paper was to analyze whether an interaction between the effects of the buffer nature and chemical modification on enzyme stability exists. For this, the lipase B from Candida antarctica (CALB) and the lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose beads and modified with picryl sulfonic acid (TNBS) and ethylenediamine via the carbodiimide route. The obtained biocatalysts were them inactivated in different buffers (2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris)-HCl, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) or phosphate). A significant interaction was found between the chemical modification of the enzyme surfaces and their stabilities in these different buffers. While phosphate was the buffer where the lowest enzyme stabilities were found for both unmodified immobilized enzymes, the differences on enzyme stabilities become much smaller after some chemical modifications. In many instances, chemical modification improves enzyme stability when using a buffer and was negative when using the other one (e.g., TNBS modification of TLL was positive using Tris-HCl or negative using the other buffers, amination of CALB decreased its stability when inactivated in Tris-HCl or HEPES while it almost had no effect in phosphate). Thus, clear co-interactions of the effects on enzyme stability between chemical modification and buffer nature were established.

Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.

Investigating the Impact of Polymer Length, Attachment Site, and Charge on Enzymatic Activity and Stability of Cellulase.

The thermophilic cellulase Cel5a from Fervidobacterium nodosum (FnCel5a) was conjugated with neutral, cationic, and anionic polymers of increasing molecular weights. The enzymatic activity toward an anionic soluble cellulose derivative, thermal stability, and functional chemical stability of these bioconjugates were investigated. The results suggest that increasing polymer chain length for polymers compatible with the substrate enhances the positive impact of polymer conjugation on enzymatic activity. Activity enhancements of nearly 100% were observed for bioconjugates with N,N-dimethyl acrylamide (DMAm) and N,N-dimethyl acrylamide-2-(N,N-dimethylamino)ethyl methacrylate (DMAm/DMAEMA) due to proposed polymer-substrate compatibility enabled by potential noncovalent interactions. Double conjugation of two functionally distinct polymers to wild-type and mutated FnCel5a using two conjugation methods was achieved. These doubly conjugated bioconjugates exhibited similar thermal stability to the unmodified wild-type enzyme, although enzymatic activity initially gained from conjugation was lost, suggesting that chain length may be a better tool for bioconjugate activity modulation than double conjugation.

Modelling conformational dynamics of enzymes trapped in a plasmonc hotspot

Recently we demonstrated that trapping single proteins from solution in a plasmonic double nanohole (DNH) and monitoring the intensity changes of the transmitted light makes it possible to resolve their conformational changes. The power of this technique lies in the possibility of studying unmodified enzymes at work for several minutes up to hours with a temporal resolution of at least 40 μs. Here, we show that for active enzymes that undergo conformational changes, a combination of automated signal analysis and kinetic modelling allows to take a step further: reconstructing the reaction cycle from the detected sequence of conformations.Two llustrative examples will be discussed: adenylate kinase (AdK) and citrate synthase (CS).

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

Purification, characterization, and chemical modification of Bacillus velezensis SN-14 fibrinolytic enzyme

Fermented bean foods are a crucial source of fibrinolytic enzymes. The presented study aimed to purify, characterize, and chemically modify Bacillus velezensis SN-14 fibrinolytic enzyme. The fibrinolytic enzyme was purified using CTAB/isooctane/hexyl alcohol/n-butyl alcohol reverse micellar system, and the purified enzyme was chemically modified to improve its enzymatic activity and stability. Enzyme activity recovery and the purification fold for this enzyme were 44.5 ± 1.9% and 4.93 ± 0.05 fold, respectively. SDS-PAGE results showed that the molecular weight of the purified fibrinolytic enzyme was around 28 kDa. Besides, the optimum temperature and pH of the purified fibrinolytic enzyme were 37 °C and 8–9, respectively. Fe2+, mPEG5000, and pepsin were used for chemical modification and for improving the activity and stability of the purified enzyme. Thermal and acid-base stability of chemically modified enzymes increased significantly, whereas enzymatic activity increased by 7.3 times. After 30 d of frozen storage, the modified enzyme's activity was remarkably lower (33.2%) than the unmodified enzyme (60.6%). The current study on B. velezensis SN-14 fibrinolytic enzyme and chemical modification method using Fe2+, mPEG5000, and pepsin provide a reference for developing fibrinolytic drugs and foods.

Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast

Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme's substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH production was further improved, resulting in a titer of 252 mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae.

Stabilization of Glycosylated β-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues.

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts.

Summary Metschnikowia reukaufii is a widespread yeast able to grow in the plants’ floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l−1, which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload‐sucrose reactions. These products were purified and structurally characterized by MS‐ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α‐(1→1), α‐(1→3) and α‐(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l−1, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l−1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.

Addition of new catalytic sites on the surface of versatile peroxidase for enhancement of LRET catalysis

Versatile peroxidase (VP) from Bjerkandera adusta is an enzyme able to oxidize bulky and high-redox substrates trough a Long-Range Electron Transfer (LRET) pathway. In this study, the introduction of radical-forming aromatic amino acids by chemical modification of the protein surface was performed, and the catalytic implications of these additional surface active-sites on the oxidation of 2,6-dimethylphenol, Mn2+ and Remazol Brilliant Blue R (RBBR) were determined. These three different substrates are oxidized in different active-sites of enzyme molecule, of which the high redox RBBR the only one that is transformed by an external radical formed on the protein surface. Both catalytic constants kcat and KM were significantly affected by the chemical modifications. Tryptophan- and tyrosine-modified VP showed higher catalytic transformation than the unmodified enzyme for RBBR, while the Mn2+ oxidation was significantly reduced by all chemical modifications. Electron Paramagnetic Resonance studies demonstrated the formation of additional protein-based radicals after the chemical modification with radical-forming amino acids. In addition, the catalytic rate of the LRET-mediated transformation showed a good correlation with the ionization energy of the additional amino acid on the protein surface.

Manipulating intradiol dioxygenases by C-terminus truncation

Intradiol dioxygenases (EC 1.13.11.1) are bacterial enzymes that catalyze the ring cleavage of catechols which is a central step in the aerobic degradation of aromatic compounds. Some members of this enzyme group have a C-terminus which is 4–5% longer (an additional 13–18 amino acids) compared to the majority of known sequences. The longer C-terminus itself is not highly conserved and appears to be poorly integrated in the protein structural models developed for representative intradiol dioxygenases. Using a protein engineering approach variant intradiol dioxygenases were produced by truncating the C-terminus to a size comparable to the shorter versions of the enzyme. Three enzymes were selected and were originally described from the model organisms; Burkholderia xenovorans LB400, Pseudomonas putida KT2440 and Acinetobacter baylyi ADP1. The activity of the truncated enzymes were compared to the unmodified enzymes which revealed that truncation of the C-terminus could alter the enzyme activity; increasing the LB400 enzyme activity by as much as five fold, but reducing the activity of the intradiol dioxygenases from KT2440 and ADP1. The difference in effect is explained by the presence of a greater number of amino acid residues that can contribute to forming stable protein structures in the KT2440 and ADP1 enzymes. It is hypothesized that C-terminal truncation could in some cases provide a useful strategy for increasing intradiol dioxygenase activity for biotechnological production of muconic and adipic acids.

Solid phase chemical modification of agarose glyoxyl-ficin: Improving activity and stability properties by amination and modification with glutaraldehyde

Abstract Ficin extract immobilized on glyoxyl-agarose was chemically modified using carbodiimide and ethylenediamine (full or partial amination of the carboxylic groups of the enzyme) or glutaraldehyde (modifying primary amino groups) under different conditions. Aminated enzyme altered its activity versus casein and benzoyl-arginine-p-nitroanilide (BANA), e.g., the activity versus casein even slightly increased while versus BANA it decreased, being this more significant at pH 9 (the fully aminated biocatalyst increased the activity versus casein by 10% but it decreased more than 5 folds versus BANA), greatly altering the enzyme specificity. The amination improved the enzyme stability at pH 5 while stability was impoverished at pH 9. Glutaraldehyde treatment usually decreased the activity versus BANA while it improved the activity versus casein (by around a factor of 2 when using 1% glutaraldehyde). Enzyme thermostability was enhanced, mainly at pH 7. This permitted to have more linear casein hydrolysis courses at pH 7 and 66 °C compared to the unmodified enzyme. Amination followed by glutaraldehyde treatment drove to the almost full enzyme inactivation. Thus, these treatments may be considered interesting for preparing an improved biocatalyst of ficin, mainly in proteolytic applications, and may be considered (mainly the amination) a possibility to further improve enzyme immobilization.

Enzyme-Polymer Conjugates to Enhance Enzyme Shelf Life in a Liquid Detergent Formulation.

Herein, the synthesis of enzyme-polymer conjugates is reported. Four different activated polymers (mPEG-aldehyde, mPEG-NHS, maltodextrin-aldehyde, carboxymethyl cellulose aldehyde) are conjugated to the surface of protease, α-amylase, and lipase using two different strategies (reductive amination and alkylation with NHS-activated acid). Although the chemical modification of the enzymes is accompanied by losses in enzyme activity (maximum loss 40%), the covalent attachment of polymers increases the thermal stability and the stability in a standard detergent formulation compared to the unmodified enzymes. The enzyme-polymer conjugates are characterized by asymmetrical-flow field-flow fractionation and differential scanning microcalorimetry. Furthermore, it is demonstrated that conjugated enzymes still show performance in a real washing process. Enzyme-polymer conjugates show a potential as a stabilizing system for enzymes in detergents.

Monobody-Mediated Alteration of Lipase Substrate Specificity.

Controlling the catalytic properties of enzymes remain an important challenge in chemistry and biotechnology. We have recently established a strategy for altering enzyme specificity in which the addition of proxy monobodies, synthetic binding proteins, modulates the specificity of an otherwise unmodified enzyme. Here, in order to examine its broader applicability, we employed the strategy on Candida rugosa lipase 1 (CRL1), an enzyme with a tunnel-like substrate binding site. We successfully identified proxy monobodies that restricted the substrate specificity of CRL1 toward short-chain fatty acids. The successes with this enzyme system and a β-galactosidase used in the previous work suggest that our strategy can be applied to diverse enzymes with distinct architectures of substrate binding sites.

Recyclable Thermoresponsive Polymer-β-Glucosidase Conjugate with Intact Hydrolysis Activity.

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose and is a rate-limiting enzyme in the conversion of lignocellulosic biomass to sugars toward biofuels. Since the cost of enzyme is a major contributor to biofuel economics, we report the bioconjugation of a temperature-responsive polymer with the highly active thermophilic β-glucosidase (B8CYA8) from Halothermothrix orenii toward improving enzyme recyclability. The bioconjugate, with a lower critical solution temperature (LCST) of 33 °C withstands high temperatures up to 70 °C. Though the secondary structure of the enzyme in the conjugate is slightly distorted with a higher percentage of β-sheet like structure, the stability and specific activity of B8CYA8 in the conjugate remains unaltered up to 30 °C and retains more than 70% specific activity of the unmodified enzyme at 70 °C. The conjugate can be reused for β-glucosidic bond cleavage of cellobiose for at least four cycles without any significant loss in specific activity.

Immobilization of Lipase by Ionic Liquid-Modified Mesoporous SiO2 Adsorption and Calcium Alginate-Embedding Method.

Porcine pancreatic lipase (PPL) was immobilized onto functionalized ionic liquid-modified silica carrier using gelatinization and physical adsorption. The immobilized lipase was characterized with N2 adsorption-desorption, X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR) before and after modification and immobilization. The results showed that the modification of the ionic liquid and the introduction of lipase had been successfully approved. The rate of enzymatic reaction and its influencing factors was primarily studied by enzymatic reaction kinetics. K m values of PPL-SiO2@CA and PPL-IM/BF4-SiO2@CA were 4.9 and 3.7mg/ml, respectively. It indicated that the modification of the functionalized ionic liquid enhanced the affinity between the immobilized enzyme and the substrate. The immobilization efficiency, specific activity, optimum temperature, optimum pH, thermal stability, reusability, and storage stability of the immobilized enzyme were investigated. We found that the stability of the immobilized enzyme was significantly higher than that of the unmodified immobilized enzyme. Specially, PPL-IM/BF4-SiO2@CA maintained good thermal stability and retained more than 92% of its activity at 65°C after preheating 3h. Graphical Abstract Above, the immobilized lipase maintained more than 92% of its initial activity after incubating at 65°C for 3h.