Sequence of the Kluyveromyces lactis β-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis

doi:10.1016/0378-1119(92)90248-N

Gene

Volume 118, Issue 1, 1 September 1992, Pages 55-63

This paper is dedicated to the memory of Dr. J.-P. Lecocq.

https://doi.org/10.1016/0378-1119(92)90248-N Get rights and content

Abstract

The LAC4 gene encoding the β-galactosidase (βGal) of the yeast, Kluyveromyces lactis, was cloned on a 7.2-kb fragment by complementation of a lacZ-deficient Escherichia coli strain. The nucleotide sequence of the structural gene, with 42 bp and 583 bp of the 5'- and 3'-flanking sequences, respectively, was determined. The deduced amino acid (aa) sequence of the K. lactis βGal predicts a 1025-aa polypeptide with a calculated M_r. of 117618 and reveals extended sequence homologies with all the published prokaryotic βGal sequences. This suggests that the eukaryotic βGal is closely related, evolutionarily and structurally, to the prokaryotic βGal's. In addition, sequence similarities were observed between the highly conserved N-terminal two-thirds of the βGal and the entire length of the β-glucuronidase (βGlu) polypeptides, which suggests that βGlu is clearly related, structurally and evolutionarily, to the N-terminal two-thirds of the βGal. The structural analysis of the βGal alignment, performed by mean secondary structure prediction, revealed that most of the invariant residues are located in turn or loop structures. The location of the invariant residues is discussed with respect to their accessibility and their possible involvement in the catalytic process.

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Citation Excerpt :
However, only one of these domains shows catalytic function, and its active site is formed mainly by residues from the third domain. The Lac4 Kl-β-gal gene was sequenced in 1992 by Poch et al. [25], who confirmed that this yeast could ferment lactose [25,26]. The enzyme is formed by oligomers consisting of individual monomers with a molecular weight of 119 kDa that can form active dimeric or tetrameric structures.
A review on the enzyme β-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 β-galactosidases were reported. This review gathers information to allow the development of biocatalysts based on the β-galactosidase enzyme from K. lactis, aiming to improve existing bioprocesses or develop new ones.
Intestinal enzyme delivery: Chitosan/tripolyphosphate nanoparticles providing a targeted release behind the mucus gel barrier
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Furthermore β-galactosidase in strains of Propionibacterium freudenreichii was inactivated by tryptic degradation [21]. Owing to extended sequence homologies with several published prokaryotic β-galactosidase sequences, among them E. coli [45], it can be presumed that β-galactosidase from Kluyveromyces lactis is a target of trypsin as well. Enzymatic activity of β-galactosidase encapsulated in nanoparticles was preserved to a 3-fold higher extent as in a control containing the same amount of free β-galactosidase (Fig. 7).
The aim of this study was to evaluate the potential of chitosan/tripolyphosphate (TPP) nanoparticles to provide a targeted release of β-galactosidase behind the intestinal mucus gel barrier.
Nanoparticles were prepared by ionic gelation of chitosan and TPP in the presence of β-galactosidase. Particles were characterized regarding size, polydispersity index and drug load. Target mediated hydrolysis of the TPP cross-linker followed by particle degradation and release of β-galactosidase was investigated during incubation with isolated as well as cell and tissue associated intestinal alkaline phosphatase (IAP). Phosphate content in the media was quantified via malachite assay, whereas particle disintegration was monitored in parallel by measuring the decrease in particle size as well as in optical density at 600 nm. The released amount of β-galactosidase was either determined utilizing bicinchoninic acid (BCA) protein detection or via an enzymatic activity assay with 2-nitrophenyl β-D-galactopyranoside (ONPG) as substrate. Protection towards tryptic degradation was verified by ONPG assay.
The size of nanoparticles was 573 ± 34 nm and a payload of 376 ± 18 µg β-galactosidase per mg particles was achieved. Degradation studies with isolated IAP revealed a maximum phosphate cleavage of 118 ± 1 µg/mg particles, a size decrease up to 38 ± 7 % and a release of 58 ± 0.5 % β-galactosidase. Release of 94 ± 6 % of the incorporated initial amount of β-galactosidase was proven after 3 h incubation on porcine mucosa. Furthermore a protection against tryptic degradation was attained resulting in a 3-fold higher residual enzymatic activity of encapsulated β-galactosidase compared to a control of free enzyme.
Chitosan/TPP nanoparticles seem to be qualified as a suitable carrier for a targeted delivery of active ingredients to mucosal tissues expressing alkaline phosphatase.
Recovery of ß-galactosidase from the yeast Kluyveromyces lactis by cell permeabilization with sarkosyl
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The S5 extract was filtered through a 1-μm glass-fiber microfilter and then diafiltered and concentrated by centrifuging in a 2-mL Centricon™ tube containing an Ultracell YM-100 membrane (nominal-molecular-weight cut-off, 100 kDa). This pore size was selected in order to retain ß-gal, of estimated MW 240–270 kDa [32,33]. Two washing-out steps were carried out for each 2.0-mL batch processed, consisting in a ∼10x concentration of the supernatant followed by a dilution with membrane-filtered EB buffer without methylparabene in order to restore the original volume.
We present here a novel method for recovering ß-galactosidase (ß-gal) from the yeast Kluyveromyces lactis (NRRL 1118) by means of sarkosyl permeabilization. The yeast was permeabilized with 0.2% (w/v) sarkosyl in 0.1 M potassium phosphate buffer for 30 min at 25 °C. After centrifuging and washing, the permeabilized cells were incubated in buffer at pH 7.2, 35 °C for 6–7 h resulting in a nearly soluble extract containing 80% of the ß-galactosidase and about 40% of the cell protein. Transmission electron microscopy showed that permeabilized cells underwent gross structural changes during enzyme release but did not exhibit significant physical breakage. An extract obtained from a 40 mg mL^–1 suspension of permeabilized cells was partially purified and concentrated 10-fold through diafiltration and ultrafiltration. The concentrated enzyme solution mixed with 50% (v/v) glycerol proved to be stable for at least 10 months at 5 °C. The kinetic properties of the ß-galactosidase preparation including the hydrolysis of milk, were similar to those exhibited by a commonly used commercial ß-galactosidase derived from the same yeast species. Sarkosyl is a biodegradable detergent with extremely low toxicity, thus making this compound an attractive permeabilizing agent for the downstream -processing of enzymes used in the food industry.
Interaction of residue tetracycline hydrochloride in milk with β-galactosidase protein by multi-spectrum methods and molecular docking
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In addition, Fig. 5A displayed a significant change in negative ellipticities of the CD spectrum in the presence of TCH, which was a characteristic of secondary structure conformation change [27]. Through calculation, a higher proportion of β-structure (54–55%, a β-type protein) with respect to α-helix (which was about 12–13%), similar to the structure predicted by Poch et al. [28]. Moreover, it could be found from Table 3 and Fig. 5B that the α-helix, β-sheet content for β-Gal was significantly decreased in the presence of TCH; whereas the content of β-turn and random coil did not exhibit obvious change.
The purpose of this study was to explore the effect of residue tetracycline hydrochloride (TCH) in milk on molecular structure and activity of β-Gal. Inhibition kinetics assay showed the TCH inhibited β-Gal activity reversibly in a competitive manner. In addition, differences in the activity of β-Gal in the absence and presence of TCH as a function of pH and temperature were found although the optimum pH and temperature of β-Gal remained similar. Fluorescence experiment results showed that TCH effectively quenched the intrinsic fluorescence of β-Gal via static quenching. Thermodynamic parameters delineated the major roles of electrostatic forces played between β-Gal and TCH. Additionally, synchronous fluorescence and circular dichroism spectra (CD spectra) results indicated the secondary structure of β-Gal was changed due to the formation of β-Gal−TCH complexes. The molecular docking further revealed that TCH interacted with some amino acid residues of β-Gal, affecting the active site of the enzyme and thus leading to change in enzyme activity. These alterations in conformation and activity of β-Gal should be taken into consideration while using β-Gal for producing oligosaccharide prebiotics on dairy industries.
Identification of a permease gene involved in lactose utilisation in Aspergillus nidulans
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Citation Excerpt :
Fig. 1 shows a phylogenetic study of the proteins deduced from the DNA sequences mined from the sequence databases using the BgaD protein as the TBLASTN bait. BgaD appears to be related to the structurally characterised β-galactosidase Lac4p from Kluyveromyces lactis (Poch et al., 1992) and more distally, to the two β-galactosidases from E. coli (Kalnins et al., 1983; Stokes et al., 1985). There are quite a few pezizomycotina that appear to harbour one or more genes for intracellular (pH neutral) β-galactosidase.
Lactose is intracellularly hydrolysed by Aspergillus nidulans. Classical mutation mapping data and the physical characteristics of the previously purified glycosyl hydrolase facilitated identification of the clustered, divergently transcribed intracellular β-galactosidase (bgaD) and lactose permease (lacpA) genes. At the transcript level, bgaD and lacpA were coordinately expressed in response to d-galactose, lactose or l-arabinose, while no transcription was detectable in the additional presence of glucose. In contrast, creA loss-of-function mutants derepressed for both genes to a considerable extent (even) under non-inducing or repressing growth conditions. Lactose- and d-galactose induction nevertheless occurred only in the absence of glucose, indicating a regulatory role for CreA-independent repression. Remarkably, bgaD deletion mutants grew normal on lactose. In contrast, lacpA deletants grew at a much slower rate in lactose liquid medium than wild-type while strains that carried more than one copy of lacpA grew faster, showing that transport is the limiting step in lactose catabolism. The effect of lacpA gene deletion on lactose uptake was exacerbated at lower substrate concentrations, evidence for the existence of a second transport system with a lower affinity for this disaccharide in A. nidulans.
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Galacto-oligosaccharides (GOS) have many biological functions. They are produced from lactose by a glycosyl transfer of one or more d-galactosyl units onto the d-galactose moiety of lactose catalysed by β-galactosidase. This reaction can proceed in batch or in continuous system. Membrane reactor with ultrafiltration ceramic membrane (150 kDa) was used and batch system as well. Lactose in phosphate buffer (197.9 g L⁻¹), recombined whey (197 g L⁻¹) and evaporated ultrafiltration permeate (200.7 g L⁻¹) were used as substrates. The used concentration of enzyme Maxilact LX 5000 was 6 U mL⁻¹. The highest concentration of GOS in buffer was reached after 30 min of batch reaction and it was 25.5 g L⁻¹, for recombined whey it was 31.1 g L⁻¹ and for ultrafiltration permeate it was 32.9 g L⁻¹. For continuous process was obtained 8.8 g L⁻¹ of GOS for buffer and 21.0 g L⁻¹ for recombined whey and 16.7 g L⁻¹ of GOS for ultrafiltration permeate.

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^∗: Present address: Transgène S.A., 11 rue de Molsheim, 67000 Strasbourg (France) Tel. (33-88)279100.

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Sequence of the Kluyveromyces lactis β-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis

Abstract

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

Gene

J. Biol. Chem.

Plasmid

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Genomics

J. Mol. Biol.

J. Biol. Chem.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

The SWISS-PROT protein sequence databank

Nucleic Acids Res.

Linker mutagenesis in the lacZ gene of Escherichia coli yields variants of active β-galactosidase

Eur. J. Biochem.

Analysis of a eukaryotic β-galactosidase gene: the N-terminal end of the yeast Kluyveromyces lactis protein shows homology to the Escherichia colilacZ gene product

Nucleic Acids Res.

Nucleotide sequence of Klebsiella pneumoniae lae genes

J. Bacteriol.

Prediction of protein conformation

Biochemistry

Effects of amino acid substitutions at the active site in Escherichia coli β-galactosidase

Genetics

A positive regulatory element is involved in the induction of the β-galactosidase gene from Kluyveromyces lactis

EMBO J.

A comprehensive set of sequence analysis programs for the VAX

Nucleic Acids Res.

Molecular cloning and expression in E. coli of a yeast gene coding for β-galaetosidase

Cell

Genetic evidence for similar negative regulatory domains in the yeast transcription activators GAL4 and LAC9

Nucleic Acids Res.

Characterization of the promoter, signal sequence, and amino terminus of a secreted β-galactosidase from ‘Streptomyces lividans’

J. Bacteriol.

The use of limited proteolysis to probe interdomain and active site regions of β-galactosidase (Eschericlina coli)

J. Biol. Chem.