Improvement of intracellular β-galactosidase production in fed-batch culture of Kluyveromyces fragilis

Four fed-batch control strategies were evaluated to improve the specific lactase activity of Kluyveromyces fragilis. Control strategies tested included DO-stat control, exponential feeding, exponential feeding with manual feedback control and corrected feed-forward control. Each was implemented with standard sensors (i.e., temperature, dissolved oxygen and pH sensors) commonly installed in fermenters. The highest specific activity was obtained using the corrected feed-forward control strategy, a strategy incorporating a novel method for on-line estimation of specific growth rate. The control strategy was able to operate effectively to a final cell density of 69 g dry wt l^–1 with a specific lactase activity of 2 U mg^–1 cell dry wt.     

Inducible synthesis of β-galactosidase inKluyveromyces fragilis

Lactose,d-galactose, andl-arabinose induce the synthesis of β-galactosidase inKluyveromyces fragilis. Lactose is the best inducer with a maximum effect at 1.4 mm. The induced synthesis of the enzyme in glycerol grown stationary phase cells is triggered within 30 min of inducer addition, the full induction being achieved within subsequent 30–40 min.     

Covalent immobilization of Kluyveromyces fragilis β-galactosidase on magnetic nanosized epoxy support for synthesis of galacto-oligosaccharide

The novel magnetic nanobeads with epoxy groups on the surface were prepared from glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EGDMA) and hydroxyethyl methacrylate (HEMA) via emulsifier-free emulsion polymerisation, and they were characterized by scanning electron microscopy and vibrating sample magnetometer. The magnetic poly(GMA-EDGMA-HEMA) nanobeads were used as support for covalent immobilization of Kluyveromyces fragilis β-galactosidase, the maximum amount enzyme attached onto the support was 145.6?mg/g with activity recovery of 72.6%. The loading capacity of this novel support for K. fragilis β-galactosidase was improved 2.6-folds compared with Eupergit(?) C (commercial epoxy support). The immobilized K. fragilis β-galactosidase exhibited high catalytic activity for the reaction of galacto-oligosaccharide (GOS) synthesis, and a total of 2,240?g GOS were produced per gram of immobilized enzyme during consecutive batch reaction of 10 times. The immobilized biocatalyst retained 81.5% of its original activity after 10 reaction cycles.     

Molecular modelling of a psychrophilic β-galactosidase

An Antarctic strain of bacteria was isolated from the digestive tract of the crustacean Thysanoessa macrura and classified as Pseudoalteromonas sp. 22b based on 16SrRNA gene sequence and physiological as well as biochemical properties. This bacterium turned out to be a good producer of a cold-adapted β-galactosidase. The enzyme displays high catalytic and molecular adaptation to low temperatures. Here we present a homology model of the psychrophilic β-galactosidase based on the structural template of the mesophilic β-galactosidase from Escherichia coli (PDB code: 1JZ7, resolution 1.5 Å). Our aim was to identify and characterize potential cold-adaptational features of the target psychrophilic β-galactosidase at the level of the three-dimensional structure rather than solely from the analysis of the amino acid sequence. We report the results of comparisons between the psychrophilic and mesophilic β-galactosidases and point out similarities and differences in the catalytic site and in other parts of the structure. The model allowed us to pinpoint a number of characteristics that are frequently observed in psychrophilic enzymes and allowed interpretation of the results of immunochemical and biochemical analyses.     

Transferase reactions of the β-galactosidase from Streptococcus thermophilus

Summary The -galactosidase from Streptococcus thermophilus formed transferase products (including up to six disaccharides and two trisaccharides) during the hydrolysis of lactose to glucose and galactose. The extent of transferase products formed was dependent on the initial lactose concentration, reaching up to 40% of the total carbohydrate at 70% w/v lactose. At high lactose concentrations (40% w/v) trisaccharide transferase products were formed initially, followed by the appearance of disaccharide transferase products. In contrast, at low lactose concentrations (7.5 w/v), only traces of the trisaccharides were detected with disaccharides being the predominant transferase products. The disaccharide products accumulated to relatively high concentrations late in the overall hydrolysis of lactose, at both high and low initial lactose concentrations, while the trisaccharides peaked much earlier and were themselves subsequently hydrolysed prior to the complete disappearance of lactose. It was possible to study the hydrolysis of galactosyl lactose by the S. thermophilus -galactosidase using a semi-pure galactosyl lactose preparation containing 5% lactose. The hydrolysis of this trisaccharide occurred via at least four disaccharide intermidiates, which appeared chromatographically identical to the disaccharide transferase products formed during lactose hydrolysis. This suggests that the enzymic formation and subsequent hydrolysis of galactosyl lactose occurs via coincident reaction pathways. The initial rate of galactose over glucose formation during galactosyl lactose hydrolysis changed from a ratio of 3:1 at low (2–3% w/v) substrate concentrations to 1.5:1 at high (>20% w/v) concentrations. This indicates a shift in the preferred initial cleavage site from the galactose-galactose bond to the galactose-glucose bond.     

Structural basis of specificity in tetrameric Kluyveromyces lactis β-galactosidase

β-Galactosidase or lactase is a very important enzyme in the food industry, being that from the yeast Kluyveromyces lactis the most widely used. Here we report its three-dimensional structure both in the free state and complexed with the product galactose. The monomer folds into five domains in a pattern conserved with the prokaryote enzymes of the GH2 family, although two long insertions in domains 2 and 3 are unique and related to oligomerization and specificity. The tetrameric enzyme is a dimer of dimers, with higher dissociation energy for the dimers than for its assembly. Two active centers are located at the interface within each dimer in a narrow channel. The insertion at domain 3 protrudes into this channel and makes putative links with the aglycone moiety of docked lactose. In spite of common structural features related to function, the determinants of the reaction mechanism proposed for Escherichia coli β-galactosidase are not found in the active site of the K. lactis enzyme. This is the first X-ray crystal structure for a β-galactosidase used in food processing.     

Purification and characterisation of an inducible β-galactosidase from Corynebacterium murisepticum

The -galactosidase (EC 3.2.1.32) of Corynebacterium murisepticum (inducible by lactose and galactose) was purified by successive column chromatography on Sephadex G-200, DEAE-Sephadex A-50 and DEAE-cellulose (DE52). The enzyme was found to be a dimer of identical subunits of molecular mass 100,000 daltons. The K _m values of the enzyme for the substrates lactose and o-nitrophenyl--d-galactopyranoside (ONPG) are 16.7 mM and 4.4 mM, respectively, indicating, its low affinity for the substrates. The Ouchterlony immunodiffusion method exhibited immunological homogeneity of the enzyme preparation. The catalytic site of the enzyme does not take part in antigen-antibody reaction.     

Mechanism for reversible inactivation of immobilized β-galactosidase from Bacillus circulans during continuous production of galacto-oligosaccharides

Summary The specific activity-dependent stability of the immobilized -galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans during the continuous production of galactooligosaccharides from lactose was studied. This was done by measuring the elution pattern of saccharides from the various immobilized Merckogel (controlled pore silica gel) columns and the amount of saccharides remaining in the gel. It was suggested that oligosaccharides produced were trapped inside the three dimensional enzyme aggregate with the immobilized enzyme having a specific activity of 240 units/g of wet gel, causing gradual inactivation, while the immobilized enzyme with 15 units/g of wet gel was stable since the oligosaccharides were not accumulated.Free -galactosidase-2 was stable during continuous reaction in a membrane reactor.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Appearance of cell-bound β-galactosidase activity in Kluyveromyces bulgaricus resting cells

Summary The conditions involved in the appearance of cell-bound -galactosidase activity in Kluyveromyces bulgaricus resting cells were determined in relation to phospholipid content. The cells displayed an optimum -galactosidase activity when they were anaerobically growing in whey medium and kept at 25°C for more than 14 days. The activity was stimulated by a preliminary 3–5 hours shaking period and an adequate protein concentration of whey medium. The expression of the cell-bound -galactosidase was correlated to a 60% decrease in yeast phospholipid content.     

Purification and characterization of a novel thermophilic β-galactosidase from Picrophilus torridus of potential industrial application

Extremophiles - Intracellular β-galactosidase (E.C 3.2.1.23) produced by the thermoacidophilic archeon Picrophilus torridus DSM 9790 was purified to homogeneity using a combination of DEAE...     

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

Purification and analysis of an extremely halophilic β-galactosidase from Haloferax alicantei

As a first step in the development of a reporter system for gene expression in halophilic archaea, a β-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180±20 kDa in size, consisting of two monomers (each 78±3 kDa). It cleaves several different β-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has β-d-fucosidase activity. No β-glucosidase, β-arabinosidase or β-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

Expression of an α-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus

For expression of the -galactosidase gene from Cyamopsistetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identify of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high -galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low -galactosidase production levels (2 mg/l). Correspondence to: R. J. Planta     

Kinetics of improved productivity of β-galactosidase by a cycloheximide-resistant mutant of Kluyveromyces marxianus

The maximum volumetric productivity of beta-galactosidase by a Kluyveromyces marxianus mutant, grown on lactose/corn steep liquor medium for 3 d, was 150 IU l(-1) h(-1) which is twice that of the parent organism. During product formation, mutated cells provided more resistance against thermal inactivation.     

Growth and β-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10–12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4–8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in ‘substrate-accelerated death’. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO₂. Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol. Received 16 October 1997/ Accepted in revised form 17 April 1998     

Kinetic analysis of the active site of an intracellular β-glucosidase fromCellulomonas biazotea

Purified β-glucosidase fromCellulomonas biazotea had an apparentK _m andV for 2-nitrophenyl β-d-glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of β-glucosidase was 65 kJ/mol. The inhibition by Mn²⁺ vs. oNPG of parental β-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 μmol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK _a’s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of β-glucosidase decreased in the presence of 10 % ethanol. The half-life of β-glucosidase in 1.75 mol/L urea at 35 °C was 145 min, as determined by 0–9 mol/L transverse urea gradient-PAGE. This work was financed in part by a grant made by theUS Agency for International Development under PSTC proposal 6-163,USAID grant no. 9365542-G-00-89-42-00, and PAEC.