Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis.

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

Revealing methyl-esterification patterns of pectins by enzymatic fingerprinting: Beyond the degree of blockiness.

Citrus pectins were studied by enzymatic fingerprinting using a simultaneous enzyme treatment with endo-polygalacturonase (endo-PG) from Kluyveromyces fragilis and pectin lyase (PL) from Aspergillus niger to reveal the methyl-ester distribution patterns over the pectin backbone. Using HILIC-MS combined with HPAEC enabled the separation and identification of the diagnostic oligomers released. Structural information on the pectins was provided by using novel descriptive parameters such as degree of blockiness of methyl-esterified oligomers by PG (DBPGme) and degree of blockiness of methyl-esterified oligomers by PL (DBPLme). This approach enabled us to clearly differentiate citrus pectins with various methyl-esterification patterns. The simultaneous use of PG and PL showed additional information, which is not revealed in digests using PG or PL alone. This approach can be valuable to differentiate pectins having the same DM and to get specific structural information on pectins and therefore to be able to better predict their physical and biochemical functionalities.

ß-Galactosidase from Kluyveromyces lactis: Characterization, production, immobilization and applications - A review.

A review on the enzyme ß-galactosidase from Kluyveromyces lactis is presented, from the perspective of its structure and mechanisms of action, the main catalyzed reactions, the key factors influencing its activity, and selectivity, as well as the main techniques used for improving the biocatalyst functionality. Particular attention was given to the discussion of hydrolysis, transglycosylation, and galactosylation reactions, which are commonly mediated by this enzyme. In addition, the products generated from these processes were highlighted. Finally, biocatalyst improvement techniques are also discussed, such as enzyme immobilization and protein engineering. On these topics, the most recent immobilization strategies are presented, emphasizing processes that not only allow the recovery of the biocatalyst but also deliver enzymes that show better resistance to high temperatures, chemicals, and inhibitors. In addition, genetic engineering techniques to improve the catalytic properties of the ß-galactosidases were reported. This review gathers information to allow the development of biocatalysts based on the ß-galactosidase enzyme from K. lactis, aiming to improve existing bioprocesses or develop new ones.

Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase.

Enzyme engineering toward catalytic-tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (kcat /KM = 47.3 s-1 ·mM-1 ), improved stereoselectivity (from moderate selectivity (deP = 66.7%) to strict (S)-selectivity (deP > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering.

The safety of a Kluyveromyces lactis strain lineage for enzyme production.

Kluyveromyces lactis is broadly considered as a safe yeast in food and a suitable organism for the production of food enzymes. The K. lactis enzyme production strains of DSM are used to produce a variety of enzymes, for example beta-galactosidase (lactase), chymosin and esterase. All of these production strains are derived from the same lineage, meaning they all originate from the same ancestor strain after classical mutagenesis and/or genetic engineering. Four different enzyme preparations produced with strains within this lineage were toxicologically tested. These enzyme preparations were nontoxic in repeated-dose oral toxicity studies performed in rats and were non-genotoxic in vitro. These studies confirm the safety of the DSM K. lactis strains as a production platform for food enzymes, as well as the safety of the genetic modifications made to these strains through genetic engineering or classical mutagenesis. The outcome of the toxicity studies can be extended to other enzyme preparations produced by any strain from this lineage through read across. Therefore, no new toxicity studies are required for the safety evaluation, as long as the modifications made do not raise safety concerns. Consequently, this approach is in line with the public ambition to reduce animal toxicity studies.

High performance biocatalyst based on ß-d-galactosidase immobilized on mesoporous silica/titania/chitosan material.

A new support for the immobilization of ß-d-galactosidase from Kluyveromyces lactis was developed, consisting of mesoporous silica/titania with a chitosan coating. This support presents a high available surface area and adequate pore size for optimizing the immobilization efficiency of the enzyme and, furthermore, maintaining its activity. The obtained supported biocatalyst was applied in enzyme hydrolytic activity tests with o-NPG, showing high activity 1223 Ug-1, excellent efficiency (74%), and activity recovery (54%). Tests of lactose hydrolysis in a continuous flow reactor showed that during 14 days operation, the biocatalyst maintained full enzymatic activity. In a batch system, after 15 cycles, it retained approximately 90% of its initial catalytic activity and attained full conversion of the lactose 100% (±12%). Additionally, with the use of the mesoporous silica/titania support, the biocatalyst presented no deformation and fragmentation, in both systems, demonstrating high operational stability and appropriate properties for applications in food manufacturing.

Structural basis for substrate recognition of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis.

Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of reducing power for nicotinamide adenine dinucleotide phosphate (NADPH) and is a precursor of 5-carbon sugar used by cells. To uncover the structural features of the enzyme, we determined the crystal structures of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis (KlG6PD) in both the apo form and a binary complex with its substrate glucose-6-phosphate. KlG6PD contains a Rossman-like domain for cofactor NADPH binding; it also presents a typical antiparallel ß sheet at the C-terminal domain with relatively the same pattern as those of other homologous structures. Moreover, our structural and biochemical analyses revealed that Lys153 contributes significantly to substrate G6P recognition. This study may provide insights into the structural variation and catalytic features of the G6PD enzyme.

Stabilization of ß-Galactosidase on Modified Gold Nanoparticles: A Preliminary Biochemical Study to Obtain Lactose-Free Dairy Products for Lactose-Intolerant Individuals.

The unique chemical, optical, and electrical characteristics of nanoparticles make their utilization highly successful in every field of biological sciences as compared to their bulk counterpart. These properties arise as a result of their miniature size, which provides them an excellent surface area-to-volume ratio, inner structure, and shape, and hence increases their surface characteristics. Therefore, this study was undertaken to engineer gold nanoparticles (AuNPs) for improving their catalytic activity and stability in biotechnological processes. The characterization of AuNPs was performed by XRD, UV spectra, and TEM. The synthesized AuNPs were surface-modified by polyvinyl alcohol (PVA) for binding the enzyme in excellent yield. The developed immobilized enzyme system (PVA-AuNPs-ß-galactosidase) displayed pH optima at pH 7.0 and temperature optima at 40 °C. Moreover, the stability of PVA-AuNPs-ß-galactosidase was significantly enhanced at wider pH and temperature ranges and at higher galactose concentrations, in contrast to the free enzyme. ß-galactosidase bound to PVA-modified AuNPs exhibited greater operational activity, even after its sixth reuse. The developed nanosystem may prove useful in producing lactose-free dairy products for lactose-intolerant patients.

Tailoring an aldo-keto reductase KmAKR for robust thermostability and catalytic efficiency by stepwise evolution and structure-guided consensus engineering.

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an advanced chiral diol intermediate of the cholesterol-lowering drug atorvastatin. KmAKRM5 (W297H/Y296W/K29H/Y28A/T63M) constructed in our previous work, displayed good biocatalytic performance on (3R,5R)-2. In the present work, stepwise evolution was applied to further enhance the thermostability and activity of KmAKRM5. For thermostability enhancement, N109 and S196 located far from the active site were picked out by structure-guided consensus engineering, and mutated by site-directed mutagenesis (SDM). For catalytic efficiency improvement, the residues A30 and T302 adjacent to the substrate-binding pocket were subjected to site-saturation mutagenesis (SSM). As a result, the "best" mutant KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C) was developed, of which T5015 and Tm were 5.0 °C and 8.2 °C higher than those of KmAKRM5. Moreover, compared to KmAKRM5, KmAKRM9 displayed a 1.9-fold (846 vs 2436 min) and 6.7-fold (126 vs 972 min) longer half-lives at 40 and 50 °C, respectively. Structural analysis suggested that beneficial mutations introduced additional hydrophobic interactions and hydrogen bonds, contributing rigidification of the flexible loops and the increase of internal forces, hence increasing the thermostability and activity. 5 g DCW (dry cell weight) L-1KmAKRM9 completely reduced 350 g L-1t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate ((5R)-1), within 3.7 h at 40 °C, yielding optically pure (3R,5R)-2 (d.e.p > 99.5%) with a space-time yield (STY) of 1.82 kg L-1 d-1. Hence, KmAKRM9 is a robust biocatalyst for the synthesis of (3R,5R)-2.

Construction of engineered RuBisCO Kluyveromyces marxianus for a dual microbial bioethanol production system.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO2 fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C5 and C6 sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks.

The ß-galactosidase immobilization protocol determines its performance as catalysts in the kinetically controlled synthesis of lactulose.

In this paper, 3 different biocatalysts of ß-galactosidase from Kluyveromyces lactis have been prepared by immobilization in chitosan activated with glutaraldehyde (Chi_Glu_Gal), glyoxyl agarose (Aga_Gly_Gal) and agarose coated with polyethylenimine (Aga_PEI_Gal). These biocatalysts have been used to catalyze the synthesis of lactulose from lactose and fructose. Aga-PEI-Gal only produces lactulose at 50 °C, and not at 25 or 37 °C, Aga_Gly_Gal was unable to produce lactulose at any of the assayed temperatures while Chi_Glu_Gal produced lactulose at all assayed temperatures, although a lower yield was obtained at 25 or 37 °C. The pre-incubation of this biocatalyst at 50 °C permitted to obtain similar yields at 25 or 37 °C than at 50 °C. The use of milk whey instead of pure lactose and fructose produced an improvement in the yields using Aga_PEI_Gal and a decrease using Chi_Glu_Gal. The operational stability also depends on the reaction medium and of biocatalyst. This study reveals how enzyme immobilization may greatly alter the performance of ß-galactosidase in a kinetically controlled manner, and how medium composition influences this performance due to the kinetic properties of ß-galactosidase.

ß-galactosidase from Kluyveromyces lactis in genipin-activated chitosan: An investigation on immobilization, stability, and application in diluted UHT milk.

The objective of this research was to evaluate the immobilization of the enzyme ß-galactosidase in a genipin-activated chitosan support. The influence of the number of spheres and substrate concentration on immobilization yield (IY) and enzyme activity (EA) was analyzed using experimental design. Thermal, operational and storage stabilities were assessed, and the enzymatic derivatives were characterized by thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The TGA showed that the enzymatic derivatives kept their thermal behavior, and the SEM images revealed smooth surfaces in all the spheres. The optimized conditions for the immobilization process were 4.57 mg·mL-1 of spheres and a substrate concentration of 10 mM (IY = 84.13%; EA = 24.97 U·g-1). Thermal stability was enhanced at 10 and 37 °C, enabling four successive cycles of lactose hydrolysis in diluted UHT milk. Therefore, the immobilized enzyme in genipin-activated chitosan has potential for lactose hydrolysis and applications in the food industry.

Metabolic Engineering of Saccharomyces cerevisiae for Ethyl Acetate Biosynthesis.

Ethyl acetate can be synthesized from acetyl-CoA and ethanol via a reaction by alcohol acetyltransferases (AATase) in yeast. In order to increase the yield of acetyl-CoA, different terminators were used to optimize the expressions of acetyl-CoA synthetase (ACS1/2) and aldehyde dehydrogenase (ALD6) to increase the contents of acetyl-CoA in Saccharomyces cerevisiae. ATF1 coding AATase was coexpressed in expression cassettes of ACS1/ACS2 and ALD6 to promote the carbon flux toward ethyl acetate from acetyl-CoA. Further to improve ethyl acetate production, four heterologous AATase including HuvEAT1 (Hanseniaspora uvarum), KamEAT1 (Kluyveromyces marxianus), VAAT (wild strawberry), and AeAT9 (kiwifruit) were introduced. Subsequently mitochondrial transport and utilization of pyruvate and acetyl-CoA were impeded to increase the ethyl acetate accumulation in cytoplasm. Under the optimal fermentation conditions, the engineered strain of PGAeΔPOR2 produced 1.69 g/L ethyl acetate, which was the highest value reported to date by metabolic engineering methods.

Effect of by-products from the dairy industry as alternative inducers of recombinant ß-galactosidase expression.

OBJECTIVE: The aim of the present study was to evaluate the efficiency of lactose derived from cheese whey and cheese whey permeate as inducer of recombinant Kluyveromyces sp. ß-galactosidase enzyme produced in Escherichia coli. Two E. coli strains, BL21(DE3) and Rosetta (DE3), were used in order to produce the recombinant enzyme. Samples were evaluated for enzyme activity, total protein content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after induction with isopropyl-ß-D-1-thiogalactoside (IPTG) (0.05 and 1 mM) and lactose, cheese whey, and cheese whey permeate solutions (1, 10, and 20 g/L lactose) at shake-flask cultivation, and whey permeate solution (10 g/L lactose) at bioreactor scale. RESULTS: The highest specific activities obtained with IPTG as inducer (0.05 mM) after 9 h of induction, were 23 and 33 U/mgprotein with BL21(DE3) and Rosetta(DE3) strains, respectively. Inductions performed with lactose and cheese whey permeate (10 and 20 g/L lactose) showed the highest specific activities at the evaluated hours, exhibiting better results than those obtained with IPTG. Specific activity of recombinant ß-galactosidase using whey permeate (10 g/L lactose) showed values of approximately 46 U/mgprotein after 24-h induction at shake-flask study, and approximately 26 U/mgprotein after 16-h induction at bench bioreactor. CONCLUSIONS: The induction with cheese whey permeate was more efficient for recombinant ß-galactosidase expression than the other inducers tested, and thus, represents an alternative form to reduce costs in recombinant protein production.

Identification and characterization of proteinase B as an unstable factor for neutral lactase in the enzyme preparation from Kluyveromyces lactis.

The stability of the commercial lactase enzyme is important for the dairy industry. A destabilizing factor for neutral lactase in the enzyme preparation from Kluyveromyces lactis was investigated. We found that lactase had lower thermal stability when fragmented bands of lactase were confirmed on SDS-PAGE. After the destabilizing factor of lactase was purified, that was identified by BLAST search as a hypothetical protein in K. lactis similar to proteinase B (PRB) of Saccharomyces cerevisiae. The molecular mass of protease was estimated to be approximately 30 kDa with SDS-PAGE. The purified protease exhibited activity toward lactase and FITC-casein but not toward bovine serum albumin or milk casein. The optimal pH and temperature of the protease were 8.0 and 40 °C, respectively. The protease activity was strongly inhibited by Fe2+, Cu2+, and a serine protease inhibitor, but activated by Ca2+. Based on these properties, the protease was identified as PRB. Lactase fragmentation was accelerated by the addition of purified PRB to the lactase preparation and was suppressed by protease inhibitors. Thus, this is the first report to identify and characterize PRB as the unstable factor of neutral lactase in the K. lactis preparation.

Engineering better catalytic activity and acidic adaptation into Kluyveromyces marxianus exoinulinase using site-directed mutagenesis.

BACKGROUND: Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis. RESULTS: Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s-1 and 568.93 L mmol-1 s-1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s-1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol-1 s-1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference. CONCLUSIONS: The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry.

Lactose hydrolysis using ß-galactosidase from Kluyveromyces lactis immobilized with sodium alginate for potential industrial applications.

The present study aimed to evaluate the lactose hydrolysis conditions from "coalho" cheese whey using ß-galactosidase (ß-gal) produced by Kluyveromyces lactis immobilized with sodium alginate. Three sodium alginate-based immobilization systems were evaluated (0.5, 0.7, and 1% w/v) for maximizing the immobilization yield (Y), efficiency (EM), and recovered activity (ar). The lactose hydrolysis capacity of the immobilized form of ß-gal was determined, and simulated environments were used to assess the preservation of the immobilized enzyme in the gastrointestinal tract. The results showed that ß-gal immobilization with 1% (w/v) sodium alginate presented the best results (EM of 66%, Y of 41%, and ar of 65%). The immobilization system maintained the highest pH stability in the range between 5.0 and 7.0, with the highest relative activity obtained under pH 5 conditions. The temperature stability was also favored by immobilization at 50 °C for 30 min was obtained a relative activity of 180.0 ± 1.37%. In 6 h, the immobilized ß-gal was able to hydrolyze 46% of the initial lactose content. For the gastrointestinal simulations, around 40% of the activity was preserved after 2 h. Overall, the results described here are promising for the industrial applications of ß-galactosidase from K. lactis.

Co-evolution of activity and thermostability of an aldo-keto reductase KmAKR for asymmetric synthesis of statin precursor dichiral diols.

Aldo-keto reductase KmAKR-catalyzed asymmetric reduction offers a green approach to produce dichiral diol tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoates, which are important building blocks of statins. In our previous work, we cloned a novel gene of NADPH-specific aldo-keto reductase KmAKR (WT) from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 and a mutant KmAKR-W297H/Y296W/K29H (Variant III) has been constructed and displayed strict diastereoselectivity towards tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) but moderate activity and stability. Herein, to further co-evolve its activity and thermostability, we performed semi-rational engineering of Variant III by using a combinational screening strategy, consisting of tertiary structure analysis, loop engineering, and alanine scanning. As results, the "best" variant KmAKR-W297H/Y296W/K29H/Y28A/T63M (Variant VI) was acquired, whose Km, kcat/Km towards (5R)-1 was 0.66 mM and 210.77 s-1 mM-1, respectively, with improved thermostability (half-life of 14.13 h at 40 °C). Combined with 1.5 g dry cell weight (DCW) L-1Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for NADPH regeneration, 4.5 g DCW L-1Variant VI completely reduced (5R)-1 of up to 450 g L-1 within 7.0 h at 40 °C, yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-3, >99.5% d.e.p) with a space-time yield (STY) of 1.24 kg L-1 day-1, and this was the highest level documented in literatures so far on substrate loading and STY of producing (3R,5R)-3. Besides (5R)-1, Variant VI displayed strong activity on tert-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-2). 4.5 g DCW L-1Variant VI completely reduced 400 g L-1 (5S)-2, within 5.0 h at 40 °C, yielding optically pure tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-4, >99.5% d.e.p) with a STY of 1.34 kg L-1 day-1. In summary, Variant VI displayed industrial application potential in statins biomanufacturing.

Purification and characterization of an inulinase produced by a Kluyveromyces marxianus strain isolated from blue agave bagasse.

Exo-inulinases are versatile enzymes that have gained attention in recent years due to their ability to hydrolyze linear and branched polyfructose chains found in inulines. Agavin, a branched inulin, is found in Agave plant, the raw matter to produce tequila. Our group has isolated several microbial strains from agave bagasse, an agro-industrial residue from tequila production that increases yearly. Strain ISO3, identified as Kluyveromyces marxianus, showed a remarkable activity towards agavin, and from its fermentation liquor an inulinolytic enzyme (Inu-ISO3) was purified. The isolated enzyme is a glycosylated dimeric protein with a molecular mass of ~256 kDa, as determined by DLS and SEC. The enzyme has an isoelectric pH of 4.6 and has both inulinase and invertase activities with an I/S ratio (ratio of activity with agavin to activity with sucrose) of 1.39. The enzyme has temperature and pH optima of 50 °C and 5.5, respectively, and follows hyperbolic kinetics with agavin (kcat of 339 ± 27 s-1 and KM of 11.8 ± 1.5 mM). The remarkable activity of Inu-ISO3 on linear and branched inulin spotlights this enzyme as a potential player in the treatment of agricultural residua for the generation of added-value products.

Effect of Polyethylene Glycol-Induced Molecular Crowding on the Enzymatic Activity and Thermal Stability of ß-Galactosidase from Kluyveromyces lactis.

Here, we report the effect of polyethylene glycol (PEG6000)-induced molecular crowding (MC) on the catalytic activity and thermal stability of Kluyveromyces lactis ß-galactosidase (ß-Gal). The ß-Gal-catalyzed hydrolysis of o-nitrophenyl-ß-d-galactopyranoside followed a Michaelian kinetics at [PEG6000] ≤ 25% w/v and positive cooperativity at higher concentrations (35% w/v PEG6000). Compared with dilute solutions, in the MC media, ß-Gal exhibited stronger thermal stability, as shown by the increase in the residual activity recovered after preincubation at high temperatures (e.g., 45 °C) and by the slower inactivation kinetics. Considering the effects of water thermodynamic activity on the reaction kinetics and protein structure and the effect of the exclusion volume on protein conformation, we suggest that changes in the protein oligomerization state and hydration could be the responsible for the behavior observed at the highest MC levels assayed. These results could be relevant and should be taken into account in industrial food processes applying ß-Gal from K. lactis.