Candida Antarctica Research Articles

Highly efficient and recyclable monolithic bioreactor for interfacial enzyme catalysis

HypothesisBiocatalysts are key to the realization of all bioconversions in nature. However, the difficulty of combining the biocatalyst and other chemicals in one system limits their application in artificial reaction systems. Although some effort, such as Pickering interfacial catalysis and enzyme-immobilized microchannel reactors, have addressed this challenge an effective method to combine chemical substrates and biocatalysts in a highly efficient and re-usable monolith system is still to be developed. ExperimentsA repeated batch-type biphasic interfacial biocatalysis microreactor was developed using enzyme-loaded polymersomes in the void surface of porous monoliths. Polymersomes, loaded with Candida antarctica Lipase B (CALB), are fabricated by self-assembly of the copolymer PEO-b-P(St-co-TMI) and used to stabilize oil-in-water (o/w) Pickering emulsions as a template to prepare monoliths. By adding monomer and Tween 85 to the continuous phase, controllable open-cell monoliths are prepared to inlay CALB-loaded polymersomes in the pore walls. FindingsThe microreactor is proven to be highly effective and recyclable when a substrate flows through it, which offers superior benefits of absolute separation to a pure product and no enzyme loss. The relative enzyme activity is constantly maintained above 93% in 15 cycles. The enzyme is constantly present in the microenvironment of the PBS buffer ensuring its immunity to inactivation and facilitating its recycling.

Characterization of a Model Bioreactive Membrane for the Simultaneous Separation and Hydrolysis of Lipopolysaccharides

The design of functionalized selective layers to develop novel membranes is essential to provide potential solutions that meet the continuously growing demand of providing safe water. Herein, we present the interfacial response of an enzymatic thin-film composite (E-TFC) membrane displaying dual functionality and fabricated as a proof-of-concept for both efficient lipopolysaccharide (LPS) separation and ester bond hydrolysis. The enzymatic membrane model was constructed by employing lipase b from Candida antarctica (CALB) covalently coupled via chemically activated bisepoxide groups onto the surface of the di-block copolymer polystyrene-b-poly(4-vinyl pyridine) (PS-b-P4VP) layer. Our results show a complete rejection of size-excluded LPS molecules when using the fabricated E-TFC membrane in a forward osmosis (FO) application. Moreover, the immobilized enzyme was able to retain 97% of its enzymatic activity when using 4-nitrophenyl acetate (pNPA) and up to 74% to liberate free fatty acids from LPS molecules within the feed side of the FO system. This work provides fundamental insights into new emerging functional biomaterials that find applications in hybrid catalytic filtration processes that also selectively remove LPS molecules from water sources.

Controllable Water-Triggered Degradation of PCL Solution-Blown Nanofibrous Webs Made Possible by Lipase Enzyme Entrapment

Polymers in nanofibrous forms offer new opportunities for achieving triggered polymer degradation, which is important for functional and environmental reasons. The polycaprolactone (PCL) nanofibrous nonwoven polymer webs developed in this work by solution blow spinning with entrapped enzymes were completely, rapidly and controllably degraded when triggered by exposure to water. Lipase (CALB) from Candida antarctica was successfully entrapped in the PCL webs via an enzyme-compatible water-in-oil emulsion in the PCL–chloroform spinning solution with added surfactant. Protein (enzyme) in the nanofibrous webs was detected by Fourier Transform Infrared Spectroscopy (FTIR), while time of flight-secondary ion mass spectroscopy (ToF-SIMS) and laser confocal microscopy indicated that enzymes were immobilized within solid fibers as well as within microbead structures distributed throughout the webs. Degradation studies of CALB-enzyme functionalized solution-blown nonwoven (EFSBN)-PCL webs at 40 °C or ambient temperature showed that EFSBN-PCL webs degraded rapidly when exposed to aqueous pH 8 buffer. Scanning electron microscopy (SEM) images of partially degraded webs showed that thinner fibers disappeared first, thus, controlling fiber dimensions could control degradation rates. Rapid degradation was attributed to the combination of nanofibrous web structure and the distribution of enzymes throughout the webs. CALB immobilized in the solid dry webs exhibited long storage stability at room temperature or when refrigerated, with around 60% catalytic activity being retained after 120 days compared to the initial activity. Dry storage stability at ambient conditions and rapid degradation upon exposure to water demonstrated that EFSBN-PCL could be used as fibers or binders in degradable textile or paper products, as components in packaging, for tissue engineering and for controlled-release drug or controlled-release industrial and consumer product applications.

Biosynthesis of diisooctyl 2,5-furandicarboxylate by Candida antarctica lipase B (CALB) immobilized on a macroporous epoxy resin.

Diisooctyl 2,5-furandicarboxylate (DEF), an ester derivative of 2,5-furandicarboxylic acid (FDCA, a bio-based platform chemical), resembles the physical and chemical properties of phthalates. Due to its excellent biodegradability, DEF is considered a safer alternative to the hazardous phthalate plasticizers. Although FDCA esters are currently mainly produced by chemical synthesis, the enzymatic synthesis of DEF is a green, promising alternative. The current study investigated the biosynthesis of DEF by Candida antarctica lipase B (CALB) immobilized on macroporous resins. Out of five macroporous resins (NKA-9, LX-1000EP, LX-1000HA, XAD-7HP, and XAD-8) evaluated, the LX-1000EP epoxy resin was identified as the best carrier for CALB, and the XAD-7HP weakly polar resin was identified as the second best. The optimal immobilization conditions were as follows: CALB (500μL) and LX-1000EP (0.1g) were incubated in phosphate butter (20mM, pH 6.0) for 10h at 35°C. The resulting immobilized CALB (EP-CALB) showed an activity of 639U/g in the hydrolysis of p-nitrophenyl acetate, with an immobilization efficiency of 87.8% and an activity recovery rate of 56.4%. Using 0.02g EP-CALB as the catalyst in 10mL toluene, and the molar ratio of 2,5-dimethyl furanediformate (1mmol/mL) and isooctyl alcohol (4mmol/mL) that was 1:4, a DEF conversion rate of 91.3% was achieved after a 24-h incubation at 50°C. EP-CALB had similar thermal stability and organic solvent tolerance compared to Novozym 435, and both were superior to CALB immobilized on the XAD-7HP resin. EP-CALB also exhibited excellent operational stability, with a conversion rate of 52.6% after 10 repeated uses. EP-CALB could be a promising alternative to Novozym 435 in the biomanufacturing of green and safe plasticizers such as DEF.

Immobilized Candida antarctica lipase B as an sn-1,3 regiospecific biocatalyst for the interesterification of triacylglycerols with fatty acid ethyl esters.

Candida antarctica lipase B (CALB) is regarded as non-regiospecific. This study aimed to investigate the regiospecificity of CALB in the solvent-free interesterification of high-oleic sunflower oil with stearic acid ethyl ester for 1,3-distearoyl-2-oleoylglycerol (SOS)-rich fat preparation using a packed bed reactor. The content ratio of 1,2-distearoyl-3-oleoylglycerol (SSO) to SOS (denoted by SSO/SOS content) obtained using Lipozyme 435 (a commercially immobilized CALB; 0-4.1%), at residence times (1-32min) was similar to that obtained using Lipozyme RM IM (0-3.0%), but lower than that obtained using Lipozyme TL IM (6.0-39.4%). When immobilized on Lewatit VP OC 1600, Lipozyme CALB had an SSO/SOS content of 0-10.4%, which was greater than that of Palatase 20,000 L (0-1.1%) but was lower than that of Lipozyme TL 100 L (8.8-97.7%). Our findings suggest that immobilized CALB shows distinct sn-1,3 regiospecificity in the interesterification of triacylglycerol with fatty acid ethyl esters.

Enzymatic enrichment of acylglycerols rich in n-3 polyunsaturated fatty acids by selective methanolysis: Optimization and kinetic studies.

n-3 Polyunsaturated fatty acids (n-3 PUFA) have special physiological effect, but their contents in natural oils may not meet the growing demand. Lipase-catalyzed selective methanolysis could be used to produce acylglycerols rich in n-3 PUFA. To explore the kinetics of enzymatic methanolysis, factors affecting the reaction, including reaction system, water content, substrate molar ratio, temperature, lipase loading, and reaction time, were first investigated in the view of optimizing the reaction. Then the effects of triacylglycerol concentrations and methanol concentrations on initial reaction rate were studied. Finally, the key kinetic parameters of methanolysis were determined subsequently. The results showed that under optimal conditions, the n-3 PUFA content in acylglycerols increased from 39.88% to 71.41%, and the n-3 PUFA yield was 73.67%. The reaction followed a Ping-Pong Bi Bi mechanism with inhibition by methanol. The kinetic analysis indicated the lipase could selectively remove saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in acylglycerols. The inhibition constant of methanol to the n-3 PUFA (KiM , 0.30mmol/L) was lower than that to the SFA and MUFA (219.64 and 79.71mmol/L). The combined effects of the fatty acid selectivity of Candida antarctica lipase A and methanol inhibition resulted in an enrichment of n-3 PUFA in acylglycerols. Overall, the methanolysis reaction catalyzed by the lipase A is a prospective enrichment method. PRACTICAL APPLICATION: This study demonstrated that enzymatic selective methanolysis is a prospective enrichment method to produce acylglycerols rich in n-3 PUFA. This method is highly efficient, environment-friendly, and simple. n-3 PUFA concentrates have been widely applied in the food, health-care food, and pharmaceutical industries.

The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability

Thermal stability is one of the essential parameters characterizing biocatalysts with potential applications in the chemical and pharmaceutical industries. Therefore, it is extremely important to develop standardized procedures for enzyme stability studies. The paper attempts to assess the thermal stability of immobilized lipases in aqueous buffers: lipase B from Candida antarctica (CALB) and lipase from Candida rugosa (CRL-OF) immobilized on the Octyl-Sepharose CL-4B carrier. As part of the optimization conditions of the immobilization, the influence of time on the catalytic activity and lipase loading, as well as the effect of temperature on lipase activity (optimal incubation—14 h at 4 °C), was determined. The thermal stability test procedure was carried out for 7 days using a climatic chamber (65 °C) and a refrigerator (4 °C). The studies of immobilized lipases included the assessment of the impact of various solvents (water, citrate buffer, 1,2-dichloropropane—DCP), temperature, light in the visible spectral range (400–800 nm), and additions of calcium ions. The highest value of residual activity (564.5 ± 21.6%) was received by storing the immobilized CALB in citrate buffer (pH 4.0, 500 mM) with the addition of calcium ions (Ca2+). On the other hand, residual activity values for immobilized CRL-OF after storage in the climatic chamber were lower than 5%. A combining of techniques: immobilization onto the support in high ionic strength and low pH, with a technique of extremally high-temperature applied in a climatic chamber, with the addition of Ca2+ allowed to achieve of excellent thermal stability of the immobilized CALB, with increasing of catalytic activity more than five-fold. Additionally, performing studies on the thermal stability of the tested lipases using a climatic chamber seems to be particularly promising in the context of unifying and standardizing storage guidelines, enabling the comparison of results between different laboratories, as well as enhancing catalytic activity.

Lipase-catalysed changes in essential oils revealed by comprehensive two-dimensional gas chromatography

Candida antarctica lipase A (CALA) was applied for the chemo-selective enzymatic transesterification of terpene and phenyl alcohols in 35 different essential oil samples. Comprehensive two-dimensional gas chromatography with mass spectrometry (GC×GC‒MS) analysis enabled the separation and tentative identification of a cohort of 125 compounds, allowing the instant visualisation of the reaction process changes, amid the complex chemical background of the samples. The results indicate that 42 out of 79 alcohols so-identified were fully or partially esterified within 48 h of reaction, with primary alcohols being the substrates of preference of the enzyme (90–100% conversion), followed by secondary alcohols (mostly ~ 80–100% conversion). No significant conversion of tertiary alcohols and phenols was observed using the tested conditions. Overall, the enzyme’s performance was consistent for primary alcohol substrates identified in multiple samples of different compositions. The observed selectivity, efficiency, robustness, scalability (enzyme/substrate working concentration ratio > 1:160), potential reusability, mild reaction conditions, and other factors make this process a greener and more sustainable alternative for industry applications, particularly for the manufacture of novel flavours and fragrances.Graphical

A New Colorimetric Test for Accurate Determination of Plastic Biodegradation.

As plastic waste is accumulating in both controlled waste management settings and natural settings, much research is devoted to search for solutions, also in the field of biodegradation. However, determining the biodegradability of plastics in natural environments remains a big challenge due to the often very low biodegradation rates. Many standardised test methods for biodegradation in natural environments exist. These are often based on mineralisation rates in controlled conditions and are thus indirect measurements of biodegradation. It is of interest for both researchers and companies to have tests that are more rapid, easier, and more reliable to screen different ecosystems and/or niches for their plastic biodegradation potential. In this study, the goal is to validate a colorimetric test, based on carbon nanodots, to screen biodegradation of different types of plastics in natural environments. After introducing carbon nanodots into the matrix of the target plastic, a fluorescent signal is released upon plastic biodegradation. The in-house-made carbon nanodots were first confirmed regarding their biocompatibility and chemical and photostability. Subsequently, the effectivity of the developed method was evaluated positively by an enzymatic degradation test with polycaprolactone with Candida antarctica lipase B. Finally, validation experiments were performed with enriched microorganisms and real environmental samples (freshwater and seawater), of which the results were compared with parallel, frequently used biodegradation measures such as O2 and CO2, dissolved organic carbon, growth and pH, to assess the reliability of the test. Our results indicate that this colorimetric test is a good alternative to other methods, but a combination of different methods gives the most information. In conclusion, this colorimetric test is a good fit to screen, in high throughput, the depolymerisation of plastics in natural environments and under different conditions in the lab.

Tuning the motion velocity of enzyme-driven dextran/polyacrylamide Janus nanomotors by incorporating the thermo-switchable PNIPAM moieties in their structure

Micro/nanomotors are miniaturized human-made devices capable of converting various forms of energy into movement. Over the past decades, a variety of micro/nanomotors with unique morphologies and different propulsion mechanisms have been developed. Enzyme-powered nanomotors have emerged as novel platforms in the biomedical field due to biocompatibility, versatility, and fuel bioavailability. Herein, we synthesized Janus enzymatic nanomotors based on magnetic iron oxide nanoparticles as a core. The two distinct faces of the magnetite structure were covered by dextran and a thermo-responsive terpolymer (N-isopropyl acrylamide-co-acrylamide-co-allylamine (NIPAM-co-AAm-co-AA) terpolymer) using the masking method. The Candida Antarctica lipase B (CALB) was used as the catalytic fuel of these nanoparticles and physically immobilized in the nanoparticles in different amounts for about 5, 10, 15, and 20 wt% of the nanoparticles to provide multi-stimuli propelled Janus nanomotors. After the enzymatic degradation of the modified dextran's ester groups, the nanoparticles begin to move. Optical microscopy studies indicated that the nanomotors had only a Brownian motion without the fuel (CALB enzyme). We found that the velocity motion of Janus nanomotors changed by immobilizing different enzyme concentrations in the nanoparticles. Based on the results, the lowest enzyme concentration required for the optimal hydrolysis rate of the ester group was determined to be 5% by weight of nanoparticles, and the maximum speed of nanomotors was determined at 15% by weight of nanoparticles. The effect of near-infrared (NIR) light on the velocity of nanomotors containing 15 wt% of CALB enzyme was investigated. It was found that the NIR light decreases the movement speed of these particles.

Lipase enzyme immobilized over magnetic titanium graphene oxide as catalyst for biodiesel synthesis from waste cooking oil

This research used titanium oxide-coated magnetic Fe3O4 nanoparticles doped graphene oxide (MTiGO) and modified with Candida antarctica Lipase B (CALB) enzyme. The MTiGO@CALB nanocomposite is a novel biocatalyst for transesterifying triglycerides to fatty acid methyl esters (FAMEs) from waste cooking oil. tThe physicochemical characteristics of the proposed biocatalyst were determined via FTIR, VSM, XRD, SEM, and EDS techniques. The maximum immobilization efficiencies and activity recovery of immobilized enzymes were obtained at 89% and 75% in the following operating condition: lipase concentration of 2.0 mg/mL, pH of 7.0, immobilization temperature of 35 °C, and immobilization time of 4 h. Various effective parameters on biodiesel were stated, and according to the revealed data, the maximum yield of biodiesel via the transesterification process was achieved at 92%, at a reaction temperature of 45 °C, catalyst quantity of 4 wt%, aa reaction duration of 40 h, and methanol to molar oil ratio of 5:1 These findings indicated that the provided bio-nanocomposite has a high potential for producing biodiesel as an efficient heterogeneous biocatalyst.

Evaluation the selectivity of three lipases in the synthesis of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol, an asymmetric triacylglycerol

1-Oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) is the most abundant triacylglycerol (TAG) molecule in Chinese human milk. The selectivity of three lipases including Rhizomucor miehei lipase (RML), Thermomyces lanuginose lipase (TL), and Candida antarctica lipase (CALB) in OPL synthesis were evaluated. The effects of different reaction conditions on selectivity were first investigated. Unexpectedly, the three sn-1,3 regioselectivity lipases, also have selectivity for the structure of product TAGs. Compared with symmetric TAGs, lipases had poor selectivity toward asymmetric OPL. Furthermore, molecular simulations were used to deeply understand the molecular factors affecting lipase selectivity. The results showed that hydrophobic interactions, lipases lid domain flexibility, and the stability of hydrogen bonds between lipases and TAGs were the main molecular factors responsible for the selectivity of lipases.

Improvement of the operational stability of the Novozym 435® biocatalyst in the oxidation of 2,5-furandicarboxaldehyde to 2,5-furandicarboxylic acid in a continuous packed-bed microbioreactor

The present work reports the evaluation of the yield, productivity and operational stability of the chemoenzymatic oxidation of 2,5-furandicarboxaldehyde (DFF) to 2,5-furan dicarboxylic acid (FDCA) catalyzed by immobilized lipase B from Candida antarctica (Novozym® 435) in a continuous packed bed microbioreactor. The highest yields and productivities were achieved using a feed for the reaction medium at 36 µL/min flow. The yield and specific productivity in continuous packed bed microbioreactor were approximately 20% and 1000 times higher than that obtained by operating in millibioreactors. The operational stability of the biocatalyst in the microbioreactor was higher than that achieved in a millibioreactor in continuous operation for 24 h. Finally, the packed bed microbioreactor is an interesting and straightforward strategy to increase the stability of the biocatalyst in continuous mode.

Asymmetric Synthesis of trans-3-Alkoxyamino-4-Oxygenated-2-Piperidones Mediated by Transition-Metal-Free Dual C-H Oxidation and Its CAL-B-Assisted Enzymatic Resolution

A general chemo-enzymatic approach to synthesize both enantioenriched trans-3-alkoxyamino-4-oxy-2-piperidones, which are important scaffold for various naturally occurring alkaloids, is reported. To this end, a selective transition-metal-free dual C−H oxidation of piperidines mediated by the TEMPO oxoammonium cation (TEMPO+) was used, followed by enzymatic resolution of the corresponding alkoxyamino-2-piperidones with Candida antarctica lipase (CAL-B), to yield the title compounds in high enantiomeric excess (ee). The absolute configuration of both enantioenriched compounds was determined using chemical correlation and circular dichroism (CD) spectroscopy. The former method highlights the oxidative ring contraction of the trans-alkoxyamine-2-piperidone ring into its corresponding 2-pyrrolidinone.

Biosurfactant from Candida: sources, classification, and emerging applications

Biosurfactants are surface-active molecules that are synthesized by many microorganisms like fungi, bacteria, and yeast. These molecules are amphiphilic in nature, possessing emulsifying ability, detergency, foaming, and surface-activity like characteristics. Yeast species belongs to the genus Candida has gained globally enormous interest because of the diverse properties of biosurfactants produced by theme. In contrast to synthetic surfactants, biosurfactants are claimed to be biodegradable and non-toxic which labels them as a potent industrial compound. Biosurfactants produced by this genus are reported to possess certain biological activities, such as anticancer and antiviral activities. They also have potential industrial applications in bioremediation, oil recovery, agricultural, pharmaceutical, biomedical, food, and cosmetic industries. Various species of Candida have been recognized as biosurfactant producers, including Candida petrophilum, Candida bogoriensis, Candida antarctica, Candida lipolytica, Candida albicans, Candida batistae, Candida albicans, Candida sphaerica, etc. These species produce various forms of biosurfactants, such as glycolipids, lipopeptides, fatty acids, and polymeric biosurfactants, which are distinct according to their molecular weights. Herein, we provide a detailed overview of various types of biosurfactants produced by Candida sp., process optimization for better production, and the latest updates on the applications of these biosurfactants.

Synthesis and characterization of Four-functional fluoresceins

The synthesis of fluorescein-diacrylate (FL-DA) and four-functional fluoresceins (FL) by Michael addition of secondary amines to FL-DA was investigated. FL-DA was successfully synthesized at the ∼ 100 g level. Diallyl amine (DAA) reacted with FL-DA instantaneously. Diethanolamine (DEA) also reacted within 5 min but was unstable and hydrolyzed back to FL. Michael addition of aminodiols with longer carbon chains and diethyl-amino-diacetate (DIDA) was slower but could be accelerated using Candida antarctica lipase B (CALB) as a biocatalyst. The structure of tetrafunctional fluorosceins was confirmed by 13C and 1H NMR. High resolution Mass Spectrometry (HRMS) demonstrated the formation of very pure tetraallyl- and tetraethylacetate-fluoresceins. These multifuntional fluoresceins are good candidates for the synthesis of imaging agents for a wide variety of applications.

A new approach for the direct acylation of bio-oil enriched with levoglucosan: Kinetic study and lipase thermostability

Lignocellulosic biomass can be valorized by fast pyrolysis, generating a bio-oil with levoglucosan (BoE), anhydrous sugar applied as building block for several compounds of great industrial interest. In this work we demonstrated for the first time the direct transesterification of BoE with different acyl donors by free and immobilized lipases. Influence of acetonitrile (ACN) and cyrene (CYE) in the stability of lipases from Candida rugosa and Candida antarctica B by differential scanning Nano fluorimetry (NanoDSF), at substrate concentrations of 20–300 mM as well as the alkyl chain effect for synthesis of the carbohydrate fatty acid esters (CFAEs) were carried out at 55 °C. Best results were obtained with Novozym® 435 in ACN and ethyl octanoate (C8) with 74% of conversion against 58% in CYE and ethyl palmitate (C16). In general it was observed that increasing on hydrophobicity of acyl donor contributed to greater regioselectivity for the hydroxyl bound to carbon 4 with up to 87% for C16 in ACN. Thermostability profile of CalB and CR suggested higher stabilty in CYE with values of Tm = 61 °C for CalB and Tm = 53 °C for CR. Finally, the kinetic parameters were obtained through the Michaelis-Menten constants generated by the double-reciprocal plot and indicated an inhibition by the LG substrate. Moreover, catalytic efficiency data suggest higher enzyme-substrate affinity in CYE.

Effective lipase immobilization on crosslinked functional porous polypyrrole aggregates

Enzyme immobilization is an efficient and growing method for the stabilization, separation and reutilization of the expensive and hard-to-extract enzymes used in industrial biocatalysts. A 3D porous functionalized polypyrrole (PPy) material is designed with superior properties for improved performance of covalently immobilized model enzymes. This was uniquely achieved by choosing biodegradable carboxymethylcellulose (CMC) crosslinker of different molecular weights (Mw) to alter the strength of porous aggregates. The ensemble-averaged aggregates radius of gyration 〈Rg〉 increased monotonically almost three-fold with crosslinkers’ Mw along with an open structure formation compared to phytic acid crosslinked aggregates. This improvement was connected with more than a 20-fold increase in adsorbed N2 and a resulting increase in the specific surface area for aggregates crosslinked with CMC compared to phytic acid counterparts. A larger number of COOH groups on the CMC surface combined with optimal pore size achieved with its decreasing Mw facilitated the enzymes’ free diffusion to the functional groups and their retention. The phenomena further allowed a larger fraction of covalent bond formation of enzyme-substrate, resulting in higher specific activity and stability for Candida rugosa and Candida Antarctica, found in commercial biocatalysts, which will guide the formation of improved biocatalysts on porous polymer supports in the future.

Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter.

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (P AOX1 ) and/or the constitutive GAP promoter (P GAP ). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (PDH) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical P GAP was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under PDH outperformed P GAP controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that PDH is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for PDH under different feeding and osmolarity conditions. In all of them the PDH controlled process outperformed the P GAP one in regard to CalB titer. The best PDH approach reached 3.6-fold more specific productivity than P GAP fed-batch at low μ. Compared to the optimum approach for P GAP -based process, the best PDH fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, PDH is an inducible promoter that exhibited a non-coupled growth regulation showing high performance, which provides a methanol-free additional solution to the usual growth-coupled systems for RPP. Thus, this novel system emerges as a potential alternative for K. phaffii RPP bioprocess and for revaluing crude glycerol, promoting the transition towards a circular economy.

Rational Design of a Biocatalyst Based on Immobilized CALB onto Nanostructured SiO2

The adsorption of the lipase B from Candida antarctica (CALB) over nanostructured SiO2 (Ns SiO2 from now on) with and without the addition of polyols (sorbitol and glycerol) was investigated. The isotherms of adsorption made it possible to establish that the maximum dispersion limit was 0.029 µmol of protein per surface area unit of Ns SiO2 (29.4 mg per 100 mg of support), which was reached in 30 min of exposure. The studies through SDS-PAGE of the immobilization solutions and infrared spectroscopy of the prepared solids determined that CALB (from a commercial extract) is selectively adsorbed, and its secondary structure distribution is thus modified. Its biocatalytic activity was corroborated through the kinetic resolution of rac-ibuprofen. Conversions of up to 70% and 52% enantiomeric excess toward S-ibuprofen in 24 h of reaction at 45 °C were achieved. The biocatalytic performance increased with the increase in protein loading until it leveled off at 0.021 µmol.m−2, reaching 0.6 µmol.min−1. The biocatalyst containing the lipase at the maximum dispersion limit and co-adsorbed polyols presented the best catalytic performance in the kinetic resolution of rac-ibuprofen, an improved thermal resistance (up to 70 °C), and stability under long-term storage (more than 2 years).