Hydrolysis of whey by whole cells of Kluyveromyces bulgaricus immobilized in calcium alginate gels and in hen egg white

Summary Whey hydrolysis was compared in column reactors containing whole yeast cells immobilized in Ca-alginate or in hen egg white in relation to cell -galactosidase activity, flow rates, temperature and time. With cells of 1.3 U/mg dry weight (ONPG method) immobilized in Ca-alginate, 80% hydrolysis was obtained at 4° and 20° C with, respectively 0.50 and 1.65 bed volume/H; the values were 0.2 and 0.74 with cells entrapped in hen egg white. When the flow rate was expressed as ml/H/g wet yeast, no significant difference was observed between both matrices and 80% hydrolysis was reached with a flow rate 1.7 and 5 according to the temperature. The best performance was achieved by the yeast egg white reactor. At 4°C, hydrolysis decreased by 10% after 13 days; by 20% after 17 days. The presence of lactose transport inhibitors in whey did not significantly influence lactose hydrolysis.M. Decleire et al.: Hydrolysis of whey by immobilized whole cells of Kluyveromyces bulgaricus     

Hydrolysis of whey lactose using CTAB-permeabilized yeast cells

Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by β-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to β-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 °C.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

Hydrolysis of lactose by β-galactosidase immobilized in polyvinylalcohol

Summary Fungal -galactosidase was immobilized in polyvinylalcohol gel formed in pores of contton material. Temperature and pH effects on the activity of free and immobilized enzymes were studied. The optimum temperatures of free and immobilized enzymes were 60° C and 55° C respectively. The pH optimum ranged from 4.5 to 5.0 for both enzymes. The thermal stability of the immobilized -galactosidase was slightly higher. The K_m values for soluble and immobilized enzymes were respectively 1.9 mM and 2.5 mM. The optimization of conditions for a highly effective hydrolysis of 4% lactose solution and reusability of the immobilized enzyme resulted in 75% hydrolysis after 5–6 h. The degree of conversion decreased to 50% after 30 repeated runs. The capacity of the immobilized enzyme to hydrolyze lactose in whey was also studied.     

Properties and potential advantages of β-galactosidase from Bacteroides polypragmatus

Summary A -galactosidase (EC 3.2.1.23) from the mesophilic obligate anaerobe, Bacteroides polypragmatus, was purified 172 fold by p-aminophenyl--D-thiogalactopyranoside agarose affinity chromatography followed by Bio-Gel P300 chromatography. The presence of Mg²⁺ and a reducing agent such as dithiothreitol (DTT) or mercaptoethanol was required for enzyme activity. The optimum pH and temperature, as determined from hydrolysis of the substrate analogue o-nitrophenyl--D-galactopyranoside (ONPG), for enzyme activity were 6.8 and 45°C, respectively. There was negligible activity loss during incubation at 35°C for up to 13 h. The K_m values obtained with ONPG and lactose as substrates were 0.43 mM and 9.09 mM respectively. The enzyme obtained by affinity chromatography was shown to hydrolyze the lactose component of cheese whey; the amount of lactose hydrolyzed was 32% of that expected with pure lactose as the substrate in buffer containing Mg²⁺ and DTT.NRCC Publication Number 24295     

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β‐galactosidase

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead‐Glu) or carboxyl groups through acid solution (Immobead‐Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β‐galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead‐Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10–500 mg g^?1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g^?1 support. Gal immobilized on Immobead‐Glu and Immobead‐Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half‐lifes than the soluble enzyme, where the half‐lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934–943, 2018     

Hydrolysis of lactose in acid whey using beta-galactosidase immobilized on porous glass particles: preparation and characterization of a reusable catalyst for the production of low-lactose dairy products

Partially purified lactoses (β-D -galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus niger, Ladobacillus helveticus, and Saccharomyces lactis were immobilized on diazotized porous glass particles (mean pore diameter, 86.5 nm: particle size diameters, 75–125 μm). In acid whey containing 4–4.5% lactose, A. niger lactase gave the highest activity (89 μmoles lactose hydrolyzed/g glass, min) at 55°C and pH 4.5. Glass-immobilized A. niger laclases (lactase-BG) retained much hydrolytic activity after storage and periodic use for 165 days at 55°C. For values of X greater than 30%, hydrolysis of 0.12M lactose in acid whey by a continuous flow column packed with 2 ml of lactase-BG particles could be correlated by X = 17.2(V/F) + 12.5 where X = lactose hydrolysis, percent of lactose originally present; V = volume of packed bed of lactase-BG, ml; F = flow rate of acid whey, ml/min.     

Growth of Euglena gracilis on whey

Summary To extend the use of industrial wastes, we have studied the growth of Euglena cells on demineralized whey powder, an industrial dairy waste from cheese making. The demineralized whey powder was solubilized (15 g/l) in 0.04 N HCl and autoclaved for two hours at 120°C. The solution was then brought to pH 3.5 with NH₄OH and tested for its ability to support Euglena growth. In the dark, cell densities of 4.5 to 5.5×10⁶ cells/ml were obtained when vitamin B₁₂, thiamine and minerals were added to the hydrolyzed whey solution. Although growth of Euglena is possible on whey, the industrial application may be limited due to the need to hydrolyze the whey and to the low utilization of carbon (20%) as the glucose, but not the galactose, released during hydrolysis is used.     

Characterization of Extracellular beta-d-Galactosidase from Fusarium moniliforme Grown in Whey

Extracellular lactase (beta-d-galactosidase, EC 3.2.1.23) was prepared as an ethanol precipitate from a culture of Fusarium moniliforme grown on whey. The enzyme functioned optimally at pH 3.8 to 5.0 and at 50 to 60 degrees C on both o-nitrophenyl-beta-d-galactopyranoside (ONPG) and lactose. The activation energy of the enzymic hydrolysis of ONPG and lactose in the range of 20 to 55 degrees C was 8,500 and 7,200 cal (ca. 3.57 x 10 and 3.02 x 10 J)/mol, respectively. The K(m) values were 4.4 and 12.4 mM for ONPG and lactose, respectively. At optimum pH, the enzyme lost half of its activity when it was heated at 50 degrees C for 6 h; at the same pH, the loss was only 5% when the enzyme was heated at 37 degrees C for 6 h. At optimum conditions, 50% of the lactose in whey was hydrolyzed by 10 U of this enzyme in 50 h.     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for β-galactosidase activity, were used for the synthesis of 1-hexyl-β- -galactoside and 1-octyl-β- -galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6–36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50 mM lactose for at least a week.     

Production of yeast lactase from sauerkraut brine

Kluyveromyces marxianus NRRL Y-1196 yielded the highest lactase activity when cultivated in shake flasks for 24 h in sauerkraut brine with 0.2% lactose as an inducer. The enzyme was purified 4-fold and had a specific activity of 28 units/mg protein. The K_m value was 3.94 mM. The pH and temperature optima of the enzyme were 7.0 and 50°C, respectively. It was stable between pH 6.0 to 7.6, but lost its activity at 60°C.     

Purification and biochemical properties of a galactooligosaccharide producing β-galactosidase from Bullera singularis

A beta-galactosidase, catalyzing lactose hydrolysis and galactooligosaccharide (GalOS) synthesis from lactose, was extracted from the yeast, Bullera singularis KCTC 7534. The crude enzyme had a high transgalactosylation activity resulting in the oligosaccharide conversion of over 34% using pure lactose and cheese whey permeate as substrates. The enzyme was purified by two chromatographic steps giving 96-fold purification with a yield of 16%. The molecular weight of the purified enzyme (specific activity of 56 U mg(-1)) was approx. 53 000 Da. The hydrolytic activity was the highest at pH 5 and 50 degrees C, and was stable to 45 degrees C for 2 h. Enzyme activity was inhibited by 10 mM Ag3+ and 10 mM SDS. The Km for lactose hydrolysis was 0.58 M and the maximum reaction velocity (V(max)) was 4 mM min(-1). GalOS, including tri- and tetra-saccharides were produced with a conversion yield of 50%, corresponding to 90 g GalOS l(-1) from 180 g lactose l(-1) by the purified enzyme.     

The characteristics of encapsulated whole cell β-galactosidase

We prepared encapsulated whole cell β-galactosidase using E. coli. The cell culture was divided into two steps for the cell accumulation inside the capsule and enzyme production in the cell. Growth and production media were used individually for this purpose. The dry cell weight of the free cell culture was increased 2.8 times by controlling the pH of the growth medium during cultivation. However, the weight of cells accumulated in the capsule reduced 40% with pH control. The dry cell weight increased with lactose concentration of the production medium for both cases of free and capsule cultures. The dry cell weights were 1.5?g/l for free culture and 100?g/l in the capsule when the lactose concentration of the production medium was 10?g/l. The dry cell weight increased about 60% for both cases as the lactose concentration increased from 10 to 50?g/l. The specific activity of whole cell enzyme decreased with lactose concentration from 5 to 1.4?unit/g dry cell for free culture and from 1.1 to 0.65?unit/g dry cell in the capsule. The value of Michaelis constant, K_m, of whole cell enzyme increased 3 times because of the resistance of mass transfer through the capsule membrane. The constants of Michaelis-Menten equation for the whole cell enzyme in the capsule were V_m: 0.0479?mM/min and K_m: 44.86?mM. These constants of the membrane-free cells were V_m: 0.0464?mM/min and K_m: 15.64?mM. To increase the whole cell enzyme activity, we treated encapsulated cells with organic solvents. The activity of encapsulated whole cell enzyme was increased 3.5 times with the treatment of chloroform and ethanol. The activity of the encapsulated whole cell enzymes was reserved after repeating the process 30 times.     

Pullulan production from deproteinized whey by Aureobasidium pullulans

Aureobasidium pullulans P56 was investigated using an adaptation technique and a mixed culture system. The adaptation of A. pullulans and the mixed cultures of A. pullulans and/or Lactobacillus brevisX20, Debaryomyces hansenii 194 and Aspergillus niger did not increase the production of polysaccharide. Enzymic hydrolysis of lactose in deproteinized whey gave a higher polysaccharide concentration and polysaccharide yield than acidic hydrolysed lactose. Maximum polysaccharide concentration (11.0 ± 0.5 g L⁻¹), biomass dry weight (10.5 ± 0.4 g L⁻¹), polysaccharide yield (47.2 ± 1.8%) and sugar utilization (93.2 ± 2.8%) were achieved using enzyme-hydrolysed whey (pH 6.5) containing 25 g L⁻¹ lactose and supplemented with K₂HPO₄ 0.5%, L-glutamic acid 1%, olive oil 2.5%, and Tween 80 0.5%. In this case the pullulan content of the crude polysaccharide was 40%. Received 16 December 1997/ Accepted in revised form 12 March 1999     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-d-galactoside and 1-octyl-beta-d-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50mM lactose for at least a week.     

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)⁻¹(min)⁻¹. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K _s ) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a K_s of 0.20 mM, but the K_s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport. Received: 22 January 1996 / Accepted: 18 March 1996     

Characterization of the Lactose Transport System in the Strain Bifidobacterium bifidum DSM 20082

Lactose was fermented but not assimilated by the strain Bifidobacterium bifidum DSM 20082. The sugar uptake was measured with lactose ¹⁴C. K _m and V _max values were respectively 2.6 mM and 12.11 nmol/min/mg of cell protein. The lactose transport system and the β-D-galactosidase were stimulated when the cells were grown with lactose, but isopropyl-β-D-thiogalactopyranoside had no effect. Lactose uptake was inhibited by compounds which interfered with proton and metal ionophore. Na⁺, Li⁺, or K⁺ did not affect incorporation of lactose. Furthermore, the lactose uptake decreased when an inhibitor of ATP synthesis was used. From the results of this study, the strain contained an active lactose transport system, probably a proton symport as described for Escherichia coli but with a different regulation system.     

Treatability of cheese whey for single-cell protein production in nonsterile systems: Part II. The application of aerobic sequencing batch reactor (aerobic SBR) to produce high biomass of Dioszegia sp. TISTR 5792

This study aimed to investigate the efficiency of an aerobic sequencing batch reactor (aerobic SBR) in a nonsterile system using the application of an experimental design via central composite design (CCD). The acidic whey obtained from lactic acid fermentation by immobilized Lactobacillus plantarum sp. TISTR 2265 was fed into the bioreactor of the aerobic SBR in an appropriate ratio between acidic whey and cheese whey to produce an acidic environment below 4.5 and then was used to support the growth of Dioszegia sp. TISTR 5792 by inhibiting bacterial contamination. At the optimal condition for a high yield of biomass production, the system was run with a hydraulic retention time (HRT) of 4 days, a solid retention time (SRT) of 8.22 days, and an acidic whey concentration of 80% feeding. The chemical oxygen demand (COD) decreased from 25,230 mg/L to 6,928 mg/L, which represented a COD removal of 72.15%. The yield of biomass production and lactose utilization by Dioszegia sp. TISTR 5792 were 13.14 g/L and 33.36%, respectively, with a long run of up to 180 cycles and the pH values of effluent were rose up to 8.32 without any pH adjustment.     

Cloning,purification, and characterization of β-galactosidase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> DSM 13

The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K _m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na⁺ and K⁺ in the concentration range of 1–100 mM as well as the divalent metal cations Mg²⁺, Mn²⁺, and Ca²⁺ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.     

Ester synthesis in an aqueous environment by<Emphasis Type="Italic"> Streptococcus thermophilus</Emphasis> and other dairy lactic acid bacteria

The ability of Streptococcus thermophilus ST1 and 19 other dairy lactic acid bacteria (LAB) to synthesize esters was investigated in an aqueous environment. These LAB were able to synthesize esters from alcohols and glycerides via a transferase reaction (alcoholysis) in which fatty acyl groups from glycerides were transferred to alcohols. S. thermophilus ST1 was active on tributyrin and on di- or monoglycerides of up to C10 with ethanol as the acyl acceptor. This strain was also active on a diglyceride of C6 and monoglyceride of C8 with 2-phenyl ethanol as the acyl acceptor. Alcoholysis occurred preferentially over hydrolysis. S. thermophilus ST1 had an apparent K_m value of 250 mM for ethanol and an apparent K_m value of 1.3 mM for tributyrin, measured against whole cells. Around 80% of both the transferase activity and the esterase activity were detected in the cell-free extract (CFE) of strain ST1. Both activities in the CFEs of five LAB tested were, to a similar degree, enhanced slightly by growth in the presence of ethanol and tributyrin. Using tributyrin and ethanol as substrates, the transferase activities ranged over 0.006–1.37 units/mg cell dry weight among the LAB tested and were both species- and strain-dependent.