适冷β-半乳糖苷酶及其在食品工业中的应用

β-半乳糖苷酶,又称乳糖酶,广泛存在于细菌、真菌、酵母菌等微生物中,主要功能是乳糖水解和合成低聚半乳糖。利用β-半乳糖苷酶生产低乳糖牛奶等乳制品成为解决乳糖不耐受问题最有效的途径,然而常用的商业化β-半乳糖苷酶的最适反应温度大多较高,对pH的要求比较严格,存在生产成本较高、消耗能量高等问题。适冷β-半乳糖苷酶在低温下也具有较高的酶活性,广泛应用于食品行业中,尤其在乳品工业。在低温下水解乳糖,生产低乳糖或无乳糖乳制品,供乳糖不耐受者食用,可降低成本、节约能源,具有重要意义。该文综述了β-半乳糖苷酶的微生物来源、特性、催化特性的研究现状,对适冷β-半乳糖苷酶的来源、特性、耐冷机制及工业化应用进行了系统阐述,并对其前景进行了展望。     

海藻酸钙固定化β-半乳糖苷酶催化合成低聚半乳糖

研究了强化海藻酸钙固定化β-半乳糖苷酶的方法以及固定化酶的性质,并用于制备低聚半乳糖。研究表明,用海藻酸钙包埋、戊二醛进行交联、对β-半乳糖苷酶进行固定,方法简便、酶的活力回收率高。所得固定化酶强度和活力高,对热、pH值耐受范围较游离酶宽,最佳反应温度和pH与游离酶相同,且贮存稳定性好。以乳糖为原料,用海藻酸钙固定化β-半乳糖苷酶催化合成低聚半乳糖,随着时间的延长,低聚半乳糖合成率呈抛物线变化。在温度55℃、pH6.0、乳糖浓度40%、反应时间为30h时,低聚半乳糖的合成率达最大值36.37%。     

高产β-半乳糖苷酶植物乳杆菌的筛选及其酶学性质研究

β-半乳糖苷酶不仅能通过分解乳制品中乳糖解决乳糖不耐症问题,同时能通过转糖苷作用合成具有益生功能的低聚半乳糖。从8株植物乳杆菌中筛选出高产β-半乳糖苷酶菌株K2,比活力高达6620 U/g,并对β-半乳糖苷酶酶学性质进行研究。结果表明:β-半乳糖苷酶最适和最稳定的pH值为6.5,最适温度为60℃,而在40℃稳定性最强。Mg2+对β-半乳糖苷酶活力有明显促进作用,而Cu2+有强烈抑制作用,通过推导求得米氏常数Km,oNPG=1.15 mmol/L,最大反应速率Vmax,oNP=6.34μmol/(min.mg)。研究结果为具有解决乳糖不耐受症的植物乳杆菌微生态制剂的开发奠定了基础。     

具有β-半乳糖苷酶转苷能力的菌株筛选及鉴定

以乳糖为惟一碳源,5-溴-4-氯-3-吲哚- β -D-半乳糖苷(X-gal)为显色剂,从土壤中筛选出10株产β- 半乳糖苷酶较高、生长较好的菌株.在30 ℃下发酵产酶,测定邻硝基苯- β- D-半乳糖苷(ONPG)水解能力.在50 mmol/L磷酸钾缓冲液(pH 7.0)中,加入400 g/L乳糖和200 g/L果糖,并分别添加10株菌所产β- 半乳糖苷酶, 至酶活为400 U/L,37 ℃下反应8 h,经高效液相色谱分析,编号为2-1样品中含有乳果糖.通过形态特征和16S rDNA 序列分析,鉴定菌株2-1为节杆菌属.     

β-半乳糖苷酶的研究现状与进展

β-半乳糖苷酶是一种重要的食用生物催化剂,具有催化β-1,4糖苷键水解和转糖苷作用,并被广泛应用于乳糖水解及低聚半乳糖合成。作为糖苷水解酶家族的重要成员,β-半乳糖苷酶在理论与应用方面均积累了丰富的研究成果。作者从酶的家族分布、催化机制形成、分子结构分析及特殊酶学性质发掘等方面对β-半乳糖苷酶的研究现状进行综述,以期为β-半乳糖苷酶催化调控的深入探索与应用开发提供参考。     

微泡菌ALW1重组β-半乳糖苷酶的异源表达和酶学性质

β-半乳糖苷酶催化乳糖水解为葡萄糖和半乳糖,是乳制品加工中重要的酶。该研究将微泡菌ALW1菌株的β-半乳糖苷酶在大肠杆菌BL21（DE3）中进行异源表达和纯化,研究其酶学性质。结果表明,微泡菌ALW1的β-半乳糖苷酶属于GH1家族,利用Ni-NTA Agarose亲和层析获得的重组β-半乳糖苷酶分子量约为64 ku。重组酶的最适反应温度为30 ℃,最适pH为4.5。温度低于25 ℃、pH 4.0~5.0条件下,β-半乳糖苷酶具有良好稳定性。重组β-半乳糖苷酶对DTT、吐温20和吐温80具有良好的耐受性;离子型去垢剂SDS和CTAB存在时,β-半乳糖苷酶几乎丧失活性。重组β-半乳糖苷酶的Km和Vmax分别为10.98 mmol/L和7.48 U/mg。结构模拟显示,微泡菌β-半乳糖苷酶的催化酸/碱残基和亲核残基分别为Glu186和Glu370。该研究为来自微泡菌ALW1的β-半乳糖苷酶在食品领域的应用奠定理论基础。     

芽孢杆菌35家族β-半乳糖苷酶的性质及其在合成低聚半乳糖中的应用

低聚半乳糖是一种重要的益生元,芽孢杆菌(Bacillales sp.)来源的β-半乳糖苷酶具有转半乳糖苷活性,能合成低聚半乳糖。研究了一个新的芽孢杆菌来源的β-半乳糖苷酶的表达、酶学性质及其在合成低聚半乳糖中的应用。从芽孢杆菌中克隆了一个糖苷水解酶35家族的β-半乳糖苷酶基因(BABgal35A),并在大肠杆菌BL21(DE3)中表达。多重序列比对结果表明:BABgal35A与环状芽孢杆菌(Bacillus circulans)来源的β-半乳糖苷酶同源性最高,为79.9%,是一个新型的β-半乳糖苷酶(命名为BABgal35A)。粗酶液经Ni-IDA亲和层析纯化得到电泳级纯酶,蛋白电泳分析表明其分子质量为60kDa左右,最适pH值和温度分别为5.0和55℃,并且该酶在pH 值为4.5~8.0,温度为20~45℃时稳定性好。BABgal35A对oNP-β-galactopyranoside具有较高的催化活性。该酶以乳糖为底物合成低聚半乳糖的产率达34%。研究结果表明,芽孢杆菌35家族β-半乳糖苷酶在低聚半乳糖的制备中具有潜在的应用价值。     

α-半乳糖苷酶的研究进展概况

论述了α-半乳糖苷酶性质、应用及国内外的研究概况,并简单介绍了本实验室利用生物技术将毛霉中克隆到的α半乳糖苷酶基因转化至酵母高效表达的研究情况.     

米曲霉β-半乳糖苷酶催化合成低聚半乳糖的工艺研究

研究了反应时间、乳糖浓度、温度、加酶量、pH对低聚半乳糖合成的影响,结果表明,加酶量、乳糖浓度、反应温度的影响较大。通过正交实验确定的米曲霉β-半乳糖苷酶催化合成低聚半乳糖的最佳工艺条件为:pH6.0、温度55℃、乳糖浓度40%、加酶量40U/g、反应时间32h。在最佳工艺条件下,低聚半乳糖的合成率为32.4%。     

米曲霉β-半乳糖苷酶催化合成低聚半乳糖的工艺研究

研究了反应时间、乳糖浓度、温度、加酶量、pH对低聚半乳糖合成的影响,结果表明,加酶量、乳糖浓度、反应温度的影响较大。通过正交实验确定的米曲霉β-半乳糖苷酶催化合成低聚半乳糖的最佳工艺条件为:pH6.0、温度55℃、乳糖浓度40%、加酶量40U/g、反应时间32h。在最佳工艺条件下,低聚半乳糖的合成率为32.4%。      

紫外线与氯化锂诱变选育高产乳糖酶米曲霉

以产乳糖酶米曲霉（ATCC20423）为出发菌株,通过紫外线和紫外复合氯化锂诱变处理选育高产乳糖酶菌株。利用紫外线辐照诱变得到突变株UV-15-20的乳糖酶活力为114.08U/mL,比出发菌株提高了49.22%;以紫外线复合氯化锂选育得到突变株UV-LiCl-38乳糖酶活力为121.42U/mL,比出发菌株提高了58.82%。传代实验表明两个突变株均具有稳定的遗传性。      

高效液相色谱法测定低聚半乳糖的含量

研究了高效液相色谱法测定乳制品及糖浆中低聚半乳糖含量的方法。以水和乙腈为流动相,用氨基柱将葡萄糖,半乳糖和乳糖与其他物质分离,用示差折光检测器检测,定量。通过计算低聚半乳糖酶解成的半乳糖含量,来对低聚半乳糖定量。葡萄糖,半乳糖,乳糖和低聚半乳糖的相对标准偏差分别为2.72%、4.16%、1.16%、4.26%。此法的线性相关系数为0.9990～0.9995,回收率达到95%～110%。此法能快速准确的测定低聚半乳糖的含量。     

Purification and Characterization of α-Galactosidase from Feijão bean Phaseolus vulgaris

α-Galactosidase is an important enzyme which degrades galactooligosaccharide in legumes. α-Galactosidase from feijão beans was purified and its characteristics established. The purified enzyme exhibits multiple forms –enzymes I and II with molecular weights of 140,000 and 49,000 daltons, respectively. Optimum pH was 5.5 for enzyme I and 6.0 for enzyme II. Optimum temperature for both was 55°C. Kinetically, enzyme I is more reactive. Heavy metal ions completely inhibited both enzymes I and II. Galactose is a potential inhibitor for both enzymes.     

β-Galactosidase-immobilised microreactor fabricated using a novel technique for enzyme immobilisation and its application for continuous synthesis of lactulose

In this study, several surface functionalisation techniques were used to immobilise β-galactosidase in a microreactor. β-Galactosidase was pretreated with lactose before immobilisation, and functionalised multi-walled carbon nanotubes (MWNTs), DNA-wrapped single-walled carbon nanotubes and glutaraldehyde as a linker were used to immobilise β-galactosidase on a microchannel surface. When functionalised MWNTs were used as a linker for immobilisation of pretreated β-galactosidase, the enzyme microreactor showed the highest performance. The K_m(app) and V_max were 2.84 mM and 0.52 mM/min, respectively, and the conversion of o-nitrophenyl-β-d-galactopyranoside (ONPG) reached 78.3% during the continuous flow reaction at a flow rate of 2.5 μL/min. In an enzyme microreactor, continuous synthesis of lactulose was performed, and the lactulose concentration was maintained at about 1.29 g/L for 48 h.     

Cell wall-modifying enzymes and firmness loss in ripening ‘Golden Reinders’ apples: A comparison between calcium dips and ULO storage

Calcium treatment and storage under ultra-low oxygen (ULO) conditions are common post-harvest practices aimed at delaying ripening-related softening of apple (Malus × domestica Borkh.) fruit, but the biochemical mechanisms underlying these effects have not been determined conclusively to date. In this study, commercially mature ‘Golden Reinders’ apples were dipped in 2% calcium chloride prior to storage at 1 °C and 92% RH under either regular air or ultra-low oxygen (ULO; 1kPa O₂:2kPa CO₂) for 19 or 31 weeks, and kept thereafter at 20 °C for 0, 7 or 14 days in order to simulate the usual marketing time. Cell wall composition and cell wall-modifying enzyme activities were determined in relation to fruit firmness. ULO-storage and calcium dips were effective for firmness preservation, seemingly due to decreased pectin solubilisation. β-Galactosidase, α-l-arabinofuranosidase and pectate lyase activities were correlated positively with firmness loss of ‘Golden Reinders’ fruit after storage.     

Substrate specificities and mechanism of action of multiple α-galactosidases from Streptomyces griseoloalbus

α-Galactosidase or melibiase is a versatile enzyme with many potential biotechnological and industrial applications. The substrate specificities of three α-galactosidases – α-Gal I, α-Gal II, and α-Gal III – from Streptomyces griseoloalbus were studied using different galactose-containing natural substrates like melibiose, raffinose and stachyose. The kinetic parameters K_m and V_max were determined from the Lineweaver–Burk plot. α-Gal I showed highest affinity towards melibiose where as α-Gal II and α-Gal III showed highest affinity towards stachyose. The ¹H NMR studies showed that all the three α-galactosidases had a retaining mechanism of hydrolysis.     

米曲霉β-半乳糖苷酶系的克隆表达与酶学特性分析

本研究通过对米曲霉的3个β-半乳糖苷酶基因O158、AO及O76进行了克隆与表达,成功构建了相应的重组菌GS115（p PIC-O158）、GS115（p PIC-AO）和GS115（p PIC-O76）,并获得相应的重组酶。进一步分析发现,重组酶O158、AO和O76的最适作用p H分别为4.0、5.5和7.0;最适作用温度均为50℃;在p H5.0⁷.5之间或30⁴0℃均较为稳定。Mn2+对重组酶O158、AO和O76有明显的激活作用,而Fe2+、Cu2+和Zn2+则强烈抑制它们的酶活。O158、AO和O76只对乳糖具有水解与转苷活性,而且AO对乳糖的亲和力和催化效率均高于O158和O76。此外,在所试米曲霉菌种中不存在参照基因组中的β-半乳糖苷酶基因O42。本文系统地对米曲霉的3个β-半乳糖苷酶进行了异源表达及酶学性质的研究,为其大规模生产及工业化应用奠定了基础。      

Immobilization of β-galactosidase from Cicer arietinum (gram chicken bean) and its catalytic actions

β-Galactosidase (β-d-galactosidase galactohydrolase EC 3.2.1.23) isolated and purified from Cicer arietinum (gram chicken bean) was immobilized on two kinds of modified resin D202 with glutaraldehyde. Both the immobilized enzymes had high protein-binding capacity and high yield of enzyme activity. Kinetics results showed that the enzyme activity attained its maximum at 57°C, pH 6 for the immobilized β-galactosidase I and 52°C, pH 6 for the immobilized β-galactosidase II, respectively. The operational pH range was increased. Kinetic constants (K_m, V_max and E_a) for the free and bound enzymes, with ONPG as substrate, were studied. Results showed that K_m and V_max of immobilized enzymes were decreased while E_a of them was increased. The effects of some compounds and organic solvents for the free and immobilized enzymes were discussed. Inhibitory constants for raffinose, lactose and d-galactose, which were all reversible inhibitors of the enzymes, were also obtained.     

Carbohydrate and protein sources influence the induction of α- and β-galactosidases in Lactobacillus reuteri

The induction of α- and β-galactosidases in six strains of Lactobacillus reuteri (L. reuteri) by six carbohydrate sources and four protein sources was studied. L. reuteri grown on raffinose had the highest α-galactosidase activity (10.55 Gal U/ml), while lactose exhibited the highest β-galactosidase activity (43.82 Gal U/ml) when compared to other carbohydrate sources. L. reuteri grown on yeast extract exhibited the highest α- and β-galactosidases activity (15.27 and 12.88 Gal U/ml, respectively) when compared to other protein sources. MF14C and SD2112 grown on raffinose had the highest α-galactosidase activity (14.75 and 14.18 Gal U/ml, respectively) followed by CF2-7F (13.38 Gal U/ml). CF2-7F grown on lactose had the highest β-galactosidase activity (82.01 Gal U/ml). SD2112, MM2-3 and CF2-7F grown on yeast extract (20.96, 19.67, 19.67 Gal U/ml, respectively) showed the highest α-galactosidase activity. MM2-3 and CF2-7F grown on yeast extract showed the highest β-galactosidase activity (18.1 and 17.59 Gal U/ml, respectively). Raffinose and lactose were the best carbohydrate sources to produce α- and β-galactosidases, respectively. Yeast extract was the best protein source to produce both enzymes and CF2-7F strain was the best producing strain on all tested conditions.     

β-Galactosidase Activity and Cell Wall Breakdown in Apricots

ABSTRACT: Apricots ( Prunus armeniaca L., cultivars Magyar and Bergeron ) were harvested 3 d apart (1st, 2nd and 3rd harvest). Fruits were stored at 4 to 6 °C, 90 % relative humidity, for 3 to 25 d. (β-Galactosidase activity, pectin degradation, and softening were studied as a function of harvest and storage time. (β-Galactosidase activity increased as a function of harvest; it increased continuously during storage in the case of cv. Bergeron. With Magyar enzyme activity reached a maximum value during storage in the case of 2nd and 3rd harvest fruits, then it declined. Total pectin and that solubilized neutral carbohydrate contents decreased as a function of storage. Solubilized pectin quantity did not depend either on harvest or on storage.