Kluyveromyces Lactis Β-galactosidase Research Articles

Immobilization of Kluyveromyces lactis β-Galactosidase on Meso-macroporous Silica: Use of Infrared Spectroscopy to Rationalize the Catalytic Efficiency.

Enzyme immobilization on adequate carriers is a challenging strategy. Understanding the enzyme-carrier interactions and their effects on enzyme conformation and bioactivity is critical. In this study, a meso-macropores silica (MMS) was used to immobilize β-galactosidase from the yeast Kluyveromyces lactis (β-gal-KL) by physical adsorption. The bioactivity of the immobilized β-gal-KL was altered, evidenced by the increased Km , decreased Vmax and kcat , and increased activity at alkaline values. By performing infrared spectroscopy analysis and subsequent secondary structure assessment from the amide I band, the immobilized β-gal-KL suffered a β-sheet (∼31-35 %) to α-helix (∼15-19 %) transition with increased turns (∼21-22 %) with respect to the free β-gal-KL having ∼12 % α-helix, ∼42 % β-sheet, and ∼17 % turns. These findings led us to correlate the observed bioactivity performance to structural alterations to a non-native conformation. The presented line of thought can lead to a better understanding of the reasons causing bioactivity alterations upon enzyme immobilization.

Effect of Polyethylene Glycol-Induced Molecular Crowding on the Enzymatic Activity and Thermal Stability of β-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 β-galactosidase (β-Gal). The β-Gal-catalyzed hydrolysis of o-nitrophenyl-β-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, β-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 β-Gal from K. lactis.

STABILIZATION STUDY OF TETRAMERIC Kluyveromyces lactis β-GALACTOSIDASE BY IMMOBILIZATION ON IMMOBEAD: THERMAL, PHYSICO-CHEMICAL, TEXTURAL AND CATALYTIC PROPERTIES

Abstract We investigated the immobilization of a tetrameric Kluyveromyces lactis β-galactosidase (EC: 3.2.1.23) (KL-Gal) on Immobead 150 using different support modification strategies. Immobead support was modified using an acid solution of H2SO4:HNO3 (3:1) (Immobead-Ac) or 5 % (v/v) glutaraldehyde (Immobead-Glu). Its unmodified form (Immobead) was also tested. Immobilization yields and efficiencies were evaluated by testing protein loads from 10 to 200 mg.g-1 support. The thermal, physico-chemical, textural and catalytic properties of the supports (modified and unmodified) and their derivatives (Immobead-KL-Gal, Immobead-Ac-KL-Gal and Immobead-Glu-KL-Gal) were analyzed. The highest immobilization yields and efficiencies were achieved with a protein load of 100 mg.g-1 support. Surface and pore areas of the Immobead support were greatly decreased after modification. Michaelis constant of the immobilized β-galactosidase increased in the derivatives. Maximum velocity decreased approximately 2.8 times for Immobead-KL-Gal and Immobead-Glu-KL-Gal, and approximately 1.4 times for Immobead-Ac-KL-Gal. In batch processes, the three derivatives could be reused successfully at least 15 times, maintaining high residual enzymatic activity during the lactose hydrolysis (in both cheese whey and milk). The tetrameric K. lactis β-galactosidase immobilized on Immobead supports via the tested treatments was stabilized and is an alternative tool for lactose hydrolysis in the dairy industry.

Quillaja bark saponin effects on Kluyveromyces lactis β-galactosidase activity and structure

Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis β-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin–protein interaction. Circular dichroism shows that saponin changes the β-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of β-galactosidase with the artificial substrate o-nitrophenyl-β-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.

Kluyveromyces lactis β-galactosidase immobilized on collagen: catalytic stability on batch and packed-bed reactor hydrolysis

The aim of this study was to evaluate the catalytic characteristics and operational stability of the tetrameric Kluyveromyces lactis β-galactosidase (KLG) enzyme immobilized on collagen. The support was submitted to four different treatments: aluminum, glutaraldehyde, acetic acid, and a combination of the methods with aluminum and glutaraldehyde. The four modified supports and the enzyme, both in its soluble and immobilized forms, were studied using thermogravimetric, differential exploratory calorimetry, infrared spectroscopy, and textural analyses. Operational pH, temperature and kinetic parameters of the soluble enzyme and of derivatives were characterized. Immobilized enzyme was applied in milk and whey lactose hydrolysis, via batch and continuous processing. There was no significant reduction (p < 0.05) in immobilization yields of all derivatives with loads up to 100 mgprotein/gsupport, with yields of approximately 70%. All derivatives reached a percentage of approximately 50% of lactose hydrolysis after 17 reuse cycles in the hydrolysis of milk and whey, using batch processing. On the other hand, hydrolysis percentages higher than 50% were achieved with packed-bed reactor even after 48 h of operation. Immobilized KLG on collagen maintained its catalytic characteristics and showed good operational stability in hydrolysis processes, especially in the continuous processes with packed-bed reactor.

Effect of tannic acid on the structure and activity of Kluyveromyces lactis β-galactosidase

Tannins are compounds with antinutrient properties that hinder food digestibility, prejudicing human and animal nutrition. This work aimed to evaluate the negative effects of tannic acid on Kluyveromyces lactis β-galactosidase catalytic activity and correlate these changes with the protein structure. β-Galactosidase activity decreased in the presence of tannins, which caused changes to the structure of the enzyme, as demonstrated by circular dichroism. It was verified that tannin binds to the protein by a static mechanism. Additionally, isothermal titration calorimetry suggested that tannic acid modified the molecular interaction between β-galactosidase and o-nitrophenyl-β-d-galactoside, reducing their affinity and prejudicing the protein activity. This study helps to understand the effects of tannins on the β-galactosidase structure and how they are related to the enzyme catalytic activity. The alterations in the conformation and activity of the enzyme should be taken into consideration when dairy products are consumed with tannin-rich food.

Glucoheptonic acid derivative as a new transgalactosylation product

Transgalactosylation is increasingly used for modification of selected compounds because it may introduce new bioactive properties or improve existing ones. This paper presents the application of transgalactosylation activity of Kluyveromyces lactis β-galactosidase (EC 3.2.1.23) for a new derivative of glucoheptonic acid synthesis. The study concerned the impact of following factors on the course of reaction: content and ratio of substrates, enzyme dose, pH of the solution and the presence of salts. In the most favourable conditions (without salt), the final product concentration reached 54.5 g/L, which corresponded to 10.9% of dry matter. Research has shown that higher initial dry matter content results in higher product content (as % dm). The addition of 0.5–0.75 M MgCl2 or 1 M NaCl led to significantly increased yield. In contrast, the presence of MnCl2 or the lowest enzyme dose seemed to slow down the synthesis process. Increasing the pH over the optimal value for hydrolytic activity of β-galactosidase caused inhibition of transgalactosylation reaction. The molar ratio of 1.9:1 (sodium glucoheptonate:lactose) was the best among tested options. The described method allowed to successfully obtain a new compound with satisfactory yield in comparison to other transgalactosylation products.

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications

Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis.

Kluyveromyces lactis β-galactosidase immobilization in calcium alginate spheres and gelatin for hydrolysis of cheese whey lactose

ABSTRACT: One of the greatest challenges for dairy industries is the correct destination of all the whey generated during cheese making, considering its high impact, the large volume created, and its technological potential. Enzymatic hydrolysis of cheese whey lactose is a biotechnological alternative. However, one of the limiting factors of its use is the relatively high cost of the enzymes, which could be lowered with the immobilization of these biocatalysts. Considering this context, the objective of this research was to evaluate the commercial Kluyveromyces lactis β-galactosidase enzyme immobilized in calcium alginate spheres and gelatin, using glutaraldehyde and concanavalin A (ConA) as modifying agents in the hydrolysis of cheese whey lactose process. Results have shown that the enzyme encapsulation complexed with ConA in alginate-gelatin spheres, without glutaraldehyde in the immobilization support, has significantly increased the hydrolysis of lactose rate, achieving a maximum conversion of 72%.

Recovery and purification of a Kluyvermyces lactis β-galactosidase by Mixed Mode Chromatography.

Mixed Mode Chromatography (MMC) is a potential separation technique that allows simultaneous ionic and hydrophobic interactions between the adsorbent and the adsorbate. The aim of this work was to assess the recovery and purification of a Kluyveromyces lactis β-galactosidase employing MMC. Protein precipitation and dialysis were performed in order to concentrate the enzyme of interest and eliminate cell debris and other interferences inherent in the fermentation medium. The best conditions for both adsorption and desorption were attained by a non-factorial Central Composite Experimental Design and employed in the chromatographic runs with resin CAPTO MMC. Fermentation yielded mean values of total enzyme concentration of 0.44 mg/mL, enzymatic activity (employing lactose as a substrate) of 74 U/mL and specific activity of 168 U/mg. The Purification Factor (PF) obtained was of 1.17. After precipitation and dialysis, the subsequent chromatographic run resulted in recovery values ​​of 41.0 and 48.2% of total protein concentration and enzymatic activity, respectively. SDS-PAGE electrophoresis confirmed the purification evolution throughout the unit operations employed, attesting the feasibility of the technique to obtain enzymes with not only considerable degree of purity but also possessing high-added value.

Manipulation of nanofiber-based β-galactosidase nanoenvironment for enhancement of galacto-oligosaccharide production

The nanoenvironment of nanobiocatalysts, such as local hydrophobicity, pH and charge density, plays a significant role in optimizing the enzymatic selectivity and specificity. In this study, Kluyveromyces lactis β-galactosidase (Gal) was assembled onto polystyrene nanofibers (PSNFs) to form PSNF-Gal nanobiocatalysts. We proposed that local hydrophobicity on the nanofiber surface could expel water molecules so that the transgalactosylation would be preferable over hydrolysis during the bioconversion of lactose, thus improve the galacto-oligosaccharides (GOS) yield. PSNFs were fabricated by electro-spinning and the operational parameters were optimized to obtain the nanofibers with uniform size and ordered alignment. The resulting nanofibers were functionalized for enzyme immobilization through a chemical oxidation method. The functionalized PSNF improved the enzyme adsorption capacity up to 3100mg/g nanofiber as well as enhanced the enzyme stability with 80% of its original activity. Importantly, the functionalized PSNF-Gal significantly improved the GOS yield and the production rate was up to 110g/l/h in comparison with 37g/l/h by free β-galactosidase. Our research findings demonstrate that the localized nanoenvironment of the PSNF-Gal nanobiocatalysts favour transgalactosylation over hydrolysis in lactose bioconversion.

경쟁적 저해를 갖는 고정화 β-galactosidase 반응기의 해석

The present study deals with the immobilization of Kluyveromyces lactis β-galactosidase on a weak ionic exchange resin (Duolite A568) as polymer support. β-Galactosidase was immobilized using the adsorption method. A kinetic study of the immobilized enzyme was performed in a packed-bed reactor. The adsorption of the enzyme followed a typical Freundlich adsorption isotherm. The adsorption parameters of k and n were 14.6 and 1.74, respectively. The initial rates method was used to characterize the kinetic parameters of the free and immobilized enzymes. The Michaelis-Menten constant (Km) for the immobilized enzyme (120 mM) was higher than it was for the free enzyme (79 mM). The effect of competitive inhibition kinetics was studied by changing the concentration of galactose in a recycling packed-bed reactor. The kinetic model with competitive inhibition by galactose was best fitted to the experimental results with Vm, Km, and KI values of 46.3 mmolmin -1 mg -1 , 120 mM, and 24.4 mM, respectively. In a continuous packed-bed reactor, increasing the flow rate of the lactose solution decreased the conversion efficiency of lactose at different input lactose concentrations. Continuous operation of 11 days was conducted to investigate the stability of a long-term operation. The retained activity of the immobilized enzymes was 63% and the half-life of the immobilized enzyme was found to be 15 days.

Construction of an Expression Vector for Production and Purification of Human Somatostatin in Escherichia coli

Somatostatin/growth hormone-inhibiting hormone is the peptide that inhibits secretion of somatotropin/growth hormone. Solid-phase synthesis methods are being currently used to produce somatostatin. Recombinant peptide synthesis is widely described for the production of small proteins and peptides; however, the production at industrial scale of peptides for biopharmaceutical applications is limited for economic reasons. Here, we propose the use of a new pGB-SMT plasmid to produce Somatostatin, as a C-terminal fusion protein with a Kluyveromyces lactis β-galactosidase fragment. To facilitate removal of that fragment by CNBr cleavage, a methionine residue was introduced at the N-terminal of the hormone peptide. The use of this construction enables an IPTG-free expression system. The suitability of this procedure has been assessed in a 15 l scale-up experiment yielding almost 300 mg, with purity >99 % and it is being implemented for commercial scale. The plasmid pGB-SMT here described is an alternative option for a cheap and high expression of other short peptide hormones.

Improvement of lactulose synthesis through optimization of reaction conditions with immobilized β-galactosidase

Kluyveromyces lactis β-galactosidase was immobilized on silica gels using a covalent bonding method. To improve lactulose synthesis using immobilized β-galactosidase, the optimal reaction conditions, such as lactose and fructose concentrations, pH and ionic strength of the buffer, loading amount of the enzyme and temperature, were determined. Lactulose synthesis using the immobilized β-galactosidase was markedly improved after optimization of the reaction conditions. When the lactulose synthesis was carried out at 47 °C using 40% (w/v) lactose, 20% (w/v) fructose and immobilized β-galactosidase of 12 U/ml in 50 mM sodium phosphate buffer at pH 7.5, the lactulose concentration and specific productivity were 15.80 g/l and 1.32 mg/U·h, respectively. In addition, when the immobilized β-galactosidase was reused for lactulose synthesis, its catalytic activity retained 60.5% after 10 reuses.

Effect of Glutaraldehyde on the Conformational Properties of Kluyveromyces lactis β-Galactosidase and Its Application to Sucrose Biosensor Preparation

Effect of Glutaraldehyde on the Conformational Properties of Kluyveromyces lactis β-Galactosidase and Its Application to Sucrose Biosensor Preparation

Structural Insights into the Substrate Specificity of Streptococcus pneumoniae β(1,3)-Galactosidase BgaC

The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the β(1,3)-linked galactose and N-acetylglucosamine (Galβ(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Å and its complex with galactose at 1.95 Å. At pH 5.5-8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (β/α)(8) TIM barrel, followed by two all-β domains (ABDs) of unknown function. The side walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galβ(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.

Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols.

Performance of β-galactosidase pretreated with lactose to prevent activity loss during the enzyme immobilisation process

In this study, Kluyveromyces lactis β-galactosidase was pretreated with lactose to prevent loss of activity during the immobilisation process, and glutaraldehyde was used as a linker to immobilise β-galactosidase on the surface of a silica gel. The pretreatment of β-galactosidase strongly improved its activity after immobilisation. Specifically, the activity of pretreated immobilised β-galactosidase was 2.6 times greater than that of non-pretreated immobilised β-galactosidase. The optimal temperature, pH and ionic strength of buffer for pretreated immobilised β-galactosidase were 37 °C, pH 7.5 and 20 mM potassium phosphate buffer, respectively. These values were shifted by 5 °C and pH by 0.5 when compared to the soluble β-galactosidase. Moreover, the pretreated immobilised β-galactosidase showed a better reusability than did non-pretreated immobilised β-galactosidase, with 63.9% of its original activity being retained after 10 reuses.

Development of a continuous solid phase process for reduction and thiol-dependent immobilization of yeast β-galactosidase

Reduction and covalent immobilization of Kluyveromyces lactis β-galactosidase through disulfide bonds onto thiolsulfinate-agarose was performed using two fixed-bed mini-reactors connected in series, one packed with thiopropyl-agarose (a solid phase reducing agent) and the other with thiolsulfinate-agarose (a thiol-reactive support). With the aim of optimizing the whole process, two reactor systems were assessed. In System I, the percolate from thiopropyl-agarose containing the reduced enzyme was re-circulated through the thiolsulfinate-agarose reactor alone. In System II, re-circulation was performed through both the reactors, improving the immobilization yield from 17% (System I) to 42% and the expressed activity from 25% (System I) to 56%. When the bio-reactor achieved with System II was fed with skimmed milk at 22 °C at a flow rate of 48 ml/h, steady state lactose hydrolysis reached 80%. In addition, it could be reused four times without losing its lactose hydrolysis capacity.

Role of Lysine ε-Amino Groups of β-Lactoglobulin on Its Activating Effect of Kluyveromyces lactis β-Galactosidase

Native beta-lactoglobulin binds and increases the activity of Kluyveromyces lactis beta-galactosidase. Construction of a three-dimensional (3D) model of beta-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with beta-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys(138) is the most likely to react with both. Affinity chromatography demonstrated that succinylated beta-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated beta-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys epsilon-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of beta-lactoglobulin on beta-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys epsilon-amino groups of beta-lactoglobulin.