Statistical Modeling of β-galactosidase Inhibition During Lactose Hydrolysis

A statistical model approach called response surface methodology was used to describe the product and substrate inhibition effect on β-galactosidase enzyme during lactose hydrolysis. The effect of independent variables, namely the initial concentrations of lactose (73 – 146 mM), galactose (44 – 122 mM) and glucose (83 – 167 mM) on the reaction rate of β-galactosidase was evaluated. The enzymatic reaction rate was influenced by both combined and individual effects of all the substrate and products. Although, glucose acted as an activator at low lactose and low galactose concentrations, glucose caused the inhibition of β-galactosidase at higher concentrations of lactose and galactose. The effect of galactose concentration on β-galactosidase enzyme was in the direction of inhibition. At low lactose concentrations and high glucose concentrations, galactose concentration became more effective on the reaction rate.     

Multi-enzyme biosensors with amperometric detection for determination of lactose in milk and dairy products

 In home-made sensors coimmobilizing enzymes in thin-layer plexi-cells on natural protein membranes, three enzyme cells: β-galactosidase and galactose oxidase (A), β-galactosidase and glucose oxidase (B) and β-galactosidase, galactose oxidase and glucose oxidase (C) were built into a flow-injection-analyzer system. The lactose was decomposed and oxidized by the immobilized enzymes and the hydrogen peroxide generated during the enzymatic reactions was determined by amperometric detection. The parameters for biochemical and electrochemical reactions (concentration of buffer, temperature, flow rate) were optimized in each enzyme cell. The pH optima of the lactose measurement was determined in the three enzyme cells mentioned above. The pH optimum of the cells A, B and C were 6.4, 4.5 and 4.8, respectively. The measuring ranges were 1–5 mM, 2–10 mM and 1–5 mM, while the detection limits were 0.5, 1.0 and 0.5 mM, respectively. More than 600, 1000 and 800 samples could be measured with these cells, respectively. Commercial milk and instant dessert powder products were analysed also. Our results showed that the cells B and C were more suitable for the determination of the lactose content of milk. For samples of dairy products containing added glucose, starch and other carbohydrates, enzyme cell A could be used for the efficient determination of lactose in one step. Received: 24 August 1998 / Revised version: 9 November 1998     

Batch and continuous synthesis of lactulose from whey lactose by immobilized β-galactosidase

In this study, lactulose synthesis from whey lactose was investigated in batch and continuous systems using immobilized β-galactosidase. In the batch system, the optimal concentration of fructose for lactulose synthesis was 20%, and the effect of galactose, glucose and fructose on β-galactosidase activity was determined for hydrolysis of whey lactose and the transgalactosylation reaction for lactulose synthesis. Galactose and fructose were competitive inhibitors, and glucose acted as a noncompetitive inhibitor. The inhibitory effects of galactose and glucose were stronger in the transgalactosylation reaction than they were in the hydrolysis reaction. In addition, when immobilized β-galactosidase was reused for lactulose synthesis, its catalytic activity was retained to the extent of 52.9% after 10 reuses. Lactulose was synthesized continuously in a packed-bed reactor. We synthesized 19.1 g/l lactulose during the continuous flow reaction at a flow rate of 0.5 ml/min.     

Detection of glucose,galactose, and lactose in milk with a microdialysis-coupled flow injection amperometric sensor

A microdialysis-coupled flow injection amperometric Sensor (microFIAS) was used to determine glucose, galactose, and lactose in milk. The sensor is based on enzyme-catalyzed reaction in combination with the three well-established analytical techniques, namely; microdialysis sampling, flow injection analysis (FIA), and amperometric detection. With the multianalyte sensor it was possible to detect glucose and galactose by sequential injection of their corresponding oxidase enzymes: glucose oxidase and galactose oxidase, while lactose was determined by injection of a mixture of beta-galactosidase and glucose oxidase enzymes. The sensor showed a linear response between 0.05 and 10 mM for glucose, between 0.1 and 20 mM for galactose and between 0.2 and 20 mM for lactose, respectively. The relative standard deviation values of the sensor measurements for glucose, galactose, and lactose were 3-4% (n = 3). The sensor measurements for lactose content in milk were compared with a standard method with an infrared spectrophotometer.     

TRICHODERMA REESEIα-GALACTOSIDASE ACTIVITY ON LOCUST BEAN AND GUAR GALACTOMANNANS

The effect of side-chain density on the kinetic parameters of a side-chain-cleaving hemicellulase was determined. Kinetic parameters were based on the rate of Trichoderma reesei α- galactosidase-catalyzed liberation of galactose from galactomannan (guar and locust bean) substrates. The focus enzyme was the predominant α-galactosidase obtained from the fungus'galactomannan-supplemented cell-free culture medium. Substrate concentrations were based on the number of galactosyl moieties per volume reaction mixture. The K_m values for the galactomannan substrates differed approximately 4.3-fold (28.36 and 121.16 μM), the more branched substrate having the higher K_m. In contrast, the corresponding V_max values were found to be essentially the same. The results indicate the enzyme preferentially acts at sites of low side-chain density .     

Lactobacillus plantarum 70810 from Chinese paocai as a potential source of β-galactosidase for prebiotic galactooligosaccharides synthesis

A novel food-grade strain Lactobacillus plantarum 70810 producing β-galactosidase with high transgalactosylation activity was isolated from Chinese paocai. The galactooligosaccharides (GOS) were synthesized by using this enzyme with a maximum yield of 44.3 % (w/w) from 400 g/L lactose at 45 °C for 10 h. The β-galactosidase from this strain was purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. It was a heterodimer arrangement of approximately 105 kDa composed of two subunits of 35 and 72 kDa. The optimal pH of the purified β-galactosidase was 8.0 for both o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose hydrolysis, and optimal temperature was 60 °C and 55 °C, respectively. Its K _m and V _max values for oNPG and lactose were 0.89 ± 0.05 mM, 194 ± 3.0 μmoL/min/mg protein, and 9.88 ± 0.16 mM, 15.88 ± 0.21 μmoL/min/mg protein, respectively. This enzyme was slightly inhibited by the hydrolysis products, that is, glucose and galactose. Since the β-galactosidase from L. plantarum 70810 exhibited higher transgalactosylation activity, strong affinity for lactose and low end-product inhibition, it was suggested to be a potential candidate for the synthesis of prebiotic GOS.     

OPTIMIZATION OF GALACTOOLIGO-SACCHARIDE PRODUCTION FROM LACTOSE USING β-GLYCOSIDASES FROM HYPERTHERMOPHILES

The maximal production of galactooligosaccharides (GOS) from lactose by (β-glycosidases from the hyperthermophilic archaea, Sulfolobus solfataricus (LacS) (derived from lacS gene) and Pyrococcus furiosus (CelB) (derived from celB gene) was optimized. The performance of these enzymes under extreme reaction conditions, temperatures up to 95°C and lactose concentrations up to 90% (w/v), were studied. The highly thermostable enzymes were shown to be very well suited for oligosaccharide synthesis. For both LacS and CelB the maximum yield of GOS increased with increasing lactose concentration, up to 70% (w/v). The maximum yield of GOS also increased with increasing temperature, and the optimal pH for synthesis was different at different temperatures. The sum of tri- and tetrasaccharides yielded 37% (w/w) in an optimally designed reaction for LacS, and a maximum yield of 40% (w/w) was attained for CelB. Compared to aqueous solution, an increase of the tetrasaccharide/trisaccharide ratio was obtained in two-phase systems with heptane and nonane. These two enzymes from hyperthermophilic organisms were shown to give higher GOS yields at high substrate concentrations than a β-galactosidase from a thermophilic/mesophilic organism (Aspergillus oryzae).     

The Effect of the Electric Field on Lag Phase, β-Galactosidase Production and Plasmid Stability of a Recombinant Saccharomyces cerevisiae Strain Growing on Lactose

Ethanol and ??-galactosidase production from cheese whey may significantly contribute to minimise environmental problems while producing value from low-cost raw materials. In this work, the recombinant Saccharomyces cerevisiae NCYC869-A3/pVK1.1 flocculent strain expressing the lacA gene (coding for ??-galactosidase) of Aspergillus niger under ADHI promoter and terminator was used. This strain shows high ethanol and ??-galactosidase productivities when grown on lactose. Batch cultures were performed using SSlactose medium with 50?g?L^?1 lactose in a 2-L bioreactor under aerobic and microaerophilic conditions. Temperature was maintained at 30?°C and pH?4.0. In order to determine the effect of an electric field in the fermentation profile, titanium electrodes were placed inside the bioreactor and different electric field values (from 0.5 to 2?V?cm^?1) were applied. For all experiments, ??-galactosidase activity, biomass, protein, lactose, glucose, galactose and ethanol concentrations were measured. Finally, lag phase duration and specific growth rate were calculated. Significant changes in lag phase duration and biomass yield were found when using 2?V?cm^?1. Results show that the electric field enhances the early stages of fermentation kinetics, thus indicating that its application may improve industrial fermentations?? productivity. The increase in electric field intensity led to plasmid instability thus decreasing ??-galactosidase production.     

以乳清为原料酶法水解乳糖条件的研究

乳糖溶解性差、甜度低,加之部分人群具有乳糖不耐症等原因,乳清中乳糖的利用率相对较低。利用β-半乳糖苷酶对乳清中的乳糖进行水解,既可以改善乳糖的溶解性能和甜度,又有利于提高乳清中乳糖的利用率。本实验通过研究乳清中乳糖的浓度、β-半乳糖苷酶的使用量、水解液温度、水解液pH对乳糖水解度的影响,确定了以乳清为原料酶法水解乳糖的适宜条件,即乳糖浓度15%、β-半乳糖苷酶的使用量为90U/g乳糖、水解液温度40℃、水解时间3h、水解液pH值6.5,在此条件下水解度为68%。     

Immobilization of β-galactosidase by bioaffinity adsorption on concanavalin A layered calcium alginate–starch hybrid beads for the hydrolysis of lactose from whey/milk

Aspergillus oryzae β-galactosidase was immobilized on the surface of a novel bioaffinity support: concanavalin A layered calcium alginate–starch beads. The maximum activity of the immobilized β-galactosidase was obtained at 60 °C, approximately 10 degrees higher than that of the free enzyme. The immobilized β-galactosidase exhibited significantly higher stability to heat, urea, MgCl₂, and CaCl₂ than the free enzyme. An enhancement of the activity of immobilized β-galactosidase by up to 5.0% MgCl₂ was seen, whereas the activity of the free enzyme decreased above 3.0% MgCl₂. Immobilized β-galactosidase retained 61%, 50% and 43% activity in the presence of 5% CaCl₂, 5% galactose and 4 m urea, respectively, when incubated for 1 h at 37 °C. The immobilized β-galactosidase had a much higher K_iapp value than the free enzyme, which indicated less susceptibility to product inhibition by galactose. The immobilized β-galactosidase preparation was superior to the free enzyme in hydrolysing lactose in whey or milk in a batch process: it hydrolyzed 89% of the lactose in whey in 3 h and 79% of the lactose in milk in 4 h. The immobilized β-galactosidase retained 61% of its original activity after 2 months storage at 4 °C, while the soluble enzyme showed only 37% of the initial activity under identical conditions.     

Engineering of a β-galactosidase from Bacillus coagulans to relieve product inhibition and improve hydrolysis performance

Most β-galactosidases reported are sensitive to the end product (galactose), making it the rate-limiting component for the efficient degradation of lactose through the enzymatic route. Therefore, there is ongoing interest in searching for galactose-tolerant β-galactosidases. In the present study, the predicted galactose-binding residues of β-galactosidase from Bacillus coagulans, which were determined by molecular docking, were selected for alanine substitution. The asparagine residue at position 148 (N148) is correlated with the reduction of galactose inhibition. Saturation mutations revealed that the N148C, N148D, N148S, and N148G mutants exhibited weaker galactose inhibition effects. The N148D mutant was used for lactose hydrolysis and exhibited a higher hydrolytic rate. Molecular dynamics revealed that the root mean square deviation and gyration radius of the N148D-galactose complex were higher than those of wild-type enzyme-galactose complex. In addition, the N148D mutant had a higher absolute binding free-energy value. All these factors may lead to a lower affinity between galactose and the mutant enzyme. The use of mutant enzyme may have potential value in lactose hydrolysis.     

Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol

The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae β-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40 °C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.     

高效离子交换色谱法测定半乳糖、葡萄糖、乳糖及低聚半乳糖含量

建立离子色谱法定量测定以乳糖为原料、使用β-半乳糖苷酶生产的低聚半乳糖产品的方法。样品前处理过程中用体积稀释法代替质量稀释法,采用高效CarboPac PA20阴离子交换色谱柱和积分安培法检测,并对淋洗液组成进行优化。优化后的方法加标回收率90.58%～99.45%,相对标准偏差为3.54%。半乳糖、葡萄糖、乳糖的方法检出限分别为0.24、0.59、0.75 μg/g。半乳糖和葡萄糖在0.2～7 μg/mL、乳糖在0.6～10 μg/mL的范围内,其质量浓度与峰面积线性关系良好,相关系数在0.999 2～0.999 9之间。用此方法测定6 种原料低聚半乳糖,目标组分总含量为95.84%～101.19%。     

驼乳中产转糖基活性β-半乳糖苷酶乳酸菌的筛选鉴定及其酶学特性分析

分离筛选高产转糖基活性β-半乳糖苷酶的乳源微生物,为高效合成低聚半乳糖（galacto-oligosaccharides,GOS）提供新酶源。以添加5-溴-4-氯-3-吲哚-β-D-半乳糖苷（X-Gal）的乳糖为碳源的乳酸细菌培养基（MRS）进行初级分离筛选,以产酶菌株粗酶液催化乳糖转糖基反应产物的薄层层析进行复筛,单因素优化最佳产酶条件和转糖基反应条件,硫酸铵分级沉淀纯化β-半乳糖苷酶并对其酶学特性进行初步分析。筛选获得产转糖基活性β-半乳糖苷酶乳酸菌20?株,选择产酶水平较高、转糖基活性最强的产β-半乳糖苷酶菌株L6进行进一步研究。生理生化和分子生物学鉴定确定L6菌株为Lactobacillus kefiri。该菌株在2?g/100?mL乳糖、1?g/100?mL氮源（蛋白胨、牛肉膏和酵母浸粉）及初始pH?5.5的条件下,37?℃培养20?h,产酶水平最高可达（3.81±0.02）U/mL。L6菌株所产β-半乳糖苷酶催化反应的温度范围较宽,45～70?℃均能保持50%以上相对酶活力。以45?g/100?mL乳糖为底物,该酶在65?℃、pH?7.0条件下,反应4?h生成转移二糖的得率为13.51%（m/m,下同）,转移三糖为13.85%,转移三糖以上的GOS为4.15%。     

响应面法优化低乳糖奶水解工艺

牛奶及乳制品营养丰富,容易消化吸收,人称"白色血液",是最理想的天然食品。近年来我国乳业发展迅速,但与世界平均水平仍存在巨大的差距,制约我国乳业发展的一个重要原因就是乳糖不耐症。利用β-半乳糖苷酶对牛奶进行水解可生成易被人体吸收的葡萄糖、半乳糖及"双歧因子"低聚半乳糖,不仅能解决乳糖不耐症问题,还能增加牛奶的营养价值。本文在单因素初步试验的基础上,以低聚半乳糖合成率为响应指标,通过响应分析法对低乳糖水解工艺进行优化。结果表明,当反应温度为45.3℃、加酶量为2.5mL及反应时间为69.7min时,低聚半乳糖得率达到最大值6.15%。通过乳果糖试剂盒测定水解后低乳糖奶中乳果糖含量,平均含量为158.17mg/L。     

PURIFICATION AND CHARACTERIZATION OF β-GALACTOSIDASE FROM MUCOR PUSILLUS

The β-galactosidase from Mucor pusillus was purified by acetone precipitation, gel filtration and ion-exchange chromatography. Its molecular weight was 129 kDa on SDS PAGE, and its pI was 4.55. Optimum activity was observed at pH 4 and at 65C. Thermal denaturation at temperatures above 60C was essentially first order with an activation energy of 26.4 KJ/mole. Activity was not affected by metal ions or EDTA but was inhibited by galactose and galactono 1–4 lactone. The K_m for lactose at 37C was 22 mM. The enzyme was devoid of cysteine/cystine and stained positive for carbohydrate. Overall the enzyme is similar in structural and kinetic properties to the β-galactosidase from Aspergillus niger.     

Changes in the concentrations of glucose and galactose in the peripheral blood of sucking piglets

Changes in the concentrations of glucose and galactose were measured in the peripheral blood of ten piglets after they had ingested milk during a natural sucking. In addition, the mild stress associated with the experimental procedure was determined by sampling nine fasted piglets over a period of 9 to 12 min. During this period there was a significant increase in the concentration of glucose in the blood of the piglets but no change in the concentration of galactose. After milk ingestion during a natural sucking the concentrations of both glucose and galactose increased from 5.7 mM and 19 microM to reach peak values of 7.7 mM and 122 microM, respectively, by 30 to 35 min. The concentrations of glucose and galactose returned to initial values in 60-80 min and 80-100 min, respectively, after sucking. Since the change in the concentration of galactose in the peripheral blood was much lower than the change in the concentration of glucose, we conclude that galactose was rapidly removed by the livers of sucking piglets. However, after the ingestion of milk the percentage increase (from initial to peak values) in the concentration of galactose in the blood was much larger (650%) than the increase in the concentration of glucose (43%). Thus, we propose that the determination of galactose in the peripheral blood may provide a qualitative method for monitoring the digestion and absorption of milk lactose in sucking piglets.     

The indirect determination of lactose using a glucose analyser with particular reference to solutions containing low levels of lactose in high levels of glucose and galactose

This investigation has used a Yellow Springs Instrument Co., Inc. (YSI) Model 23A glucose analyser to measure the glucose produced by the hydrolysis of the lactose solution with β-galactosidase. The analyser was also used to measure initial and final glucose values before and after hydrolysis of a low lactose high glucose/galactose product such as a lactose hydrolysed whey syrup. The accuracy and reproducibility of the glucose analyser is excellent provided that (i) operation and calibration techniques are consistent, (ii) the analyser is injected with samples which have ben suitably diluted to the optimum glucose range of 100–300 mg dl^-1, and (iii) the instrument and its 'syringepet' are both maintained. The error in measuring the small increase in glucose from the small quantity of lactose present is thereby greatly reduced.     

Production, health aspects and potential food uses of dairy prebiotic galactooligosaccharides

Galactooligosaccharides are sugars composed of 3-10 molecules of galactose and glucose via a transgalactosylation reaction mediated by the enzyme β-galactosidase. Prebiotics are non-digestible food ingredients that pass through the upper digestive system relatively intact and ferment in the lower colon, producing short-chain fatty acids that support the growth of supplemented or indigenous colonic microbiota. Galactooligosaccharides and other prebiotic ingredients are increasingly being recognized as useful dietary tools for the modulation of the colonic microflora toward a healthy balance. Galactooligosaccharides compare well to other oligosaccharides in terms of their prebiotic, immunomodulation, and functional properties in foods. This review elucidates the galactooligosaccharide production process from refined lactose and/or cheese whey permeates, galactooligosaccharide market share and economic value, their health properties, and potential food applications.     

Specificity, inhibitory studies, and oligosaccharide formation by beta-galactosidase from psychrotrophic Bacillus subtilis KL88

beta-Galactosidase from psychotrophic Bacillus subtilis KL88 was specific to the beta-D-glycosidic linkage normally present in lactose. The enzyme was completely inhibited by transition metal ions (Cu2+, Fe3+, Fe2+, Zn2+) and partially inhibited by high concentrations of glucose and galactose as well as Ca2+. It was activated by most of the alkaline earth metal ions (NA+, K+, Li+). Oligosaccharides were formed at the different levels of lactose concentrations reaching more than 20% for high lactose concentration (20%). Three types of oligosaccharides were formed in significant concentrations detected by HPLC analysis.