乳糖酶治疗小儿腹泻病的临床分析

目的 研究小儿腹泻病继发乳糖不耐受的年龄、病因及乳糖酶治疗继发乳糖不耐受的效果.方法 对382例腹泻继发乳糖不耐受患儿进行年龄、病因分析,同时将患儿分成治疗及对照两组,分析乳糖酶的疗效.结果 小儿腹泻继发乳糖不耐受以婴幼儿多见,轮状病毒感染是继发乳糖不耐受的主要病因,乳糖酶治疗继发乳糖不耐受疗效显著.结论 婴幼儿腹泻常规给予乳糖酶可以缩短病程,减少治疗费用,患儿及家长易于接受.     

乳糖酶缺乏的相关研究

乳及乳制品富含优质蛋白,多种矿物质和维生素,是改善人类营养、增强体质的理想食品。随着生活水平的改善,我国居民乳类的摄人量有所提高。但乳及乳制品中含有大量乳糖,必需经乳糖酶分解才能被吸收。由于遗传、饮食习惯等因素的影响,部分人因体内乳糖酶活性低或小肠黏膜乳糖酶缺乏（lactase deficiency,LD）,食用乳及乳制品后不能完全分解乳糖,     

新型乳糖酶的研制在中国农科院取得重要突破

乳糖酶又名β 半乳糖苷酶 ,广泛存在于植物、动物组织和微生物中 ,它的主要功能分解牛奶和乳制品中的乳糖。有些人群特别是儿童 ( 3～ 1 5岁 ) ,体内缺乏这种酶者占87% ,其中乳糖不适应症占 2 0 %左右 ,喝了鲜牛奶或某些乳制品后容易发生腹胀、腹泻或乳糖不适应症等不良后果。然后 ,乳糖酶在食品和乳制品工业中又未得到广泛应用 ,其因有 ( 1 )乳糖酶单位产量较低 ;( 2 )市场出售的乳糖酶系胞内酶 ,耐热性差 ;( 3 )源于微生物的胞内乳糖酶要经过细胞破碎、提取和纯化、工艺较为复杂 ,( 4 )酶售价过高 ,成本贵 ,适用范围窄 ,不能满足食品和乳…     

葛根芩连汤对泄泻肠道湿热证小鼠肠道双糖酶活性的影响

目的探究葛根芩连汤对泄泻肠道湿热证小鼠肠道双糖酶活性的影响。方法采用“高糖高脂+高温高湿+白酒复合灌胃冰水”的方法制备泄泻肠道湿热证小鼠模型,并运用葛根芩连汤进行干预治疗。分别在干预的第0、2、4、6天无菌采集各组小鼠肠黏膜,运用DNS法测定蔗糖酶活性,ONPG法测定乳糖酶活性,观察葛根芩连汤对泄泻肠道湿热证小鼠肠道双糖酶活性的影响。结果经肠道湿热泄泻造模后,小鼠肠道前段和后段黏膜的乳糖酶和蔗糖酶活性均显著下降,与正常组小鼠相比差异有统计学意义(均P<0.01)。随着治疗时间的延长,治疗组小鼠肠道乳糖酶和蔗糖酶活性与正常组相比有显著的提高(均P<0.05),治疗6 d后,肠道前段黏膜蔗糖酶和乳糖酶活性恢复至正常组水平(P>0.05)。肠道后段乳糖酶活性在第4天时最高,后段黏膜蔗糖酶活性在第6天时最高,与正常组和自愈组相比差异有统计学意义(均P<0.05)。结论泄泻肠道湿热证造模使小鼠肠道黏膜蔗糖酶、乳糖酶等双糖酶的活性显著下降,葛根芩连汤能调节泄泻肠道湿热证小鼠肠道黏膜乳糖酶和蔗糖酶的活性,从而发挥治疗泄泻肠道湿热证的疗效。     

两相体系中低聚半乳糖的合成

利用乳糖酶转移半乳糖苷活力,以环己烷和乙酸乙酯为有机相主体总蛋白在１５％水分手条件下,得到占总产物３５％以上的低聚半乳糖。在这两相体系中,研究了温度、缓冲液ＰＨ、乳糖浓度、半乳糖和葡萄糖,以及酶的固定化等因素对低地乳糖合成的影响：温度及起始乳糖浓度对低聚糖的影响不明显;加入半乳糖和葡萄糖对低聚糖的合成有一定的影响;以树脂Ｄ３４５固定化乳糖酶作生物催化剂,低聚糖的得率可达６４．７８％。     

警惕新生儿先天性乳糖酶缺乏

先天性乳糖酶缺乏(congenital lactase deficiency,CLD)是一种非常罕见终身性疾病属于常染色体隐性遗传病,始发于新生儿期,以第一次接触母乳后发生严重腹泻并伴有生长缓慢为主要特征,给婴幼儿生长发育带来严重的不良影响。经过国内外学者多年的研究发现其流行病率远比想象中的要高。因此,在临床工作中应警惕CLD的发生。CLD与原发性乳糖酶缺乏即成人型乳糖酶缺乏(adult-type hypolactasia,ATH)一样均属于常染色体隐性遗传,决定乳糖酶(lactase,LCT)的基因位于2q21染色体上,但它们的分子生物学机制却不同。CLD主要通过介导无意义的LCT基因的m RNA降解完成,ATH主要通过增强子多态性在LCT基因转录水平调节肠道细胞的乳糖酶合成。乳糖酶缺乏的实验室检测方法有很多,但目前对于新生儿CLD的检测方法较为实用的有基因检测及尿半乳糖酶试剂盒检测。目前较好的治疗方法是乳糖酶治疗。虽然目前为止我国未报道过一例CLD,但2012年日本已发现的2个CLD案例。因此我们应对该隐性遗传病提高应有的警惕性,并重视对其分子生物学机制进一步研究,以防患于未然,同时也有助于指导CLD患者的基因治疗。     

菌群失调腹泻造模及七味白术散治疗对小鼠乳糖酶基因13910多态性的影响

目的探讨抗生素及七味白术散对菌群失调腹泻小鼠乳糖酶基因13910位点多态性的影响。方法将36只SPF级小鼠随机分为正常组(12只)、模型组(12只)、七味白术散组(6只)和乳糖酶组(6只),除正常组外的其余小鼠采用混合抗生素造成菌群失调腹泻模型,然后分别灌胃七味白术散和乳糖酶治疗。造模成功和治疗后分别提取小鼠肠道内容物和肠道黏膜宏基因组,对包括小鼠乳糖酶基因13910位点在内的1 036bp长度的DNA片段进行单核苷酸多态性(SNP)测序分析。结果造模和治疗后,小鼠乳糖酶基因13910位点没有发生碱基的变异,均为A/A型,13910位点在内的1 036bp范围内也没有新的SNP位点出现。结论菌群失调腹泻造模与七味白术散治疗对小鼠乳糖酶活性的干预均与小鼠乳糖酶基因13910位点多态性没有相关性,可能存在其他多态性位点或其他方面的调控机制。     

乳糖酶研究进展

乳糖酶作为乳品添加剂,应用相当广泛,通过来源及其性质、基础研究和应用等方面对乳糖酶进行综述,重点介绍乳糖酶研究上的新进展和应用的新领域,由于近年来低温乳糖酶成为研究热点,特别对低温乳糖酶的研究进行介绍.     

微生物发酵法制备乳糖酶

乳糖酶是一种被广泛应用的酶。微生物发酵法生产乳糖酶具有周期短、产量高、造价低等优势。本文对近年来微生物（细菌、酵母菌、霉菌）发酵法生产乳糖酶的研究进展进行了综述。     

黑曲霉D2-26高温乳糖酶的酶学性质研究

为研究黑曲霉来源的高温乳糖酶的酶学性质,对黑曲霉D2-26发酵液进行分离纯化使酶蛋白达到电泳纯,并对其进行酶学性质研究.结果表明:乳糖酶的最适温度70℃,在30℃～60℃有较高的耐受性;最适pH为2.5,pH稳定范围在2.0～9.0;Mn2 对乳糖酶活性有显著的激活作用,Hg2 、Pb2 对酶活有较强的抑制作用;SDS严重抑制了酶活性;以ONPG为底物采用双倒数做图法测得Vmax为97 nmol/min,Km为8.77 mmol/L;单亚基蛋白的分子量为116.978 kD;糖基化程度为11.3%.     

Evolution of lactase persistence: an example of human niche construction

Niche construction is the process by which organisms construct important components of their local environment in ways that introduce novel selection pressures. Lactase persistence is one of the clearest examples of niche construction in humans. Lactase is the enzyme responsible for the digestion of the milk sugar lactose and its production decreases after the weaning phase in most mammals, including most humans. Some humans, however, continue to produce lactase throughout adulthood, a trait known as lactase persistence. In European populations, a single mutation (-13910*T) explains the distribution of the phenotype, whereas several mutations are associated with it in Africa and the Middle East. Current estimates for the age of lactase persistence-associated alleles bracket those for the origins of animal domestication and the culturally transmitted practice of dairying. We report new data on the distribution of -13910*T and summarize genetic studies on the diversity of lactase persistence worldwide. We review relevant archaeological data and describe three simulation studies that have shed light on the evolution of this trait in Europe. These studies illustrate how genetic and archaeological information can be integrated to bring new insights to the origins and spread of lactase persistence. Finally, we discuss possible improvements to these models.     

Human lactase and the molecular basis of lactase persistence

Human lactase purified from detergent extracts of the total membrane fraction of postmortem jejunum by means of monoclonal immunoadsorbent chromatography appears to be a dimer of subunits identical in M_r (160K). Trypsin or papain removes a small hydrophobic anchoring peptide from each subunit to give a hydrophilic enzyme which no longer interacts with detergent micelles. Lactase hydrolyzes, besides lactose, cellobiose and the synthetic substrates, 4-methylumbelliferyl--galactoside and -glucoside, as well as phlorizin; but it does not hydrolyze glucocerebroside. Phlorizin hydrolase is associated with lactase under all conditions investigated; coincident staining on immunodiffusion and immunoelectrophoresis, coincident elution on immunoadsorbent chromatography and on gel filtration in a dissociating buffer, and correlated reduction in activity in lactase-nonpersistent individuals. Adult and infant lactases are indistinguishable by titration or immunodiffusion against polyclonal rabbit antibodies. Adult individuals low in lactase activity also show a corresponding reduction in cross-reacting material. These observations suggest that lactase persistence is due to the continued synthesis of the infant enzyme.Financial support was provided by the Nuffield Foundation, the Medical Research Council, and the Open University Research Committee Fund.     

Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups.

Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

Selection of lactose-adapted cells in vinca minor and Dantura innoxia cultures; location and characterization of β-galactosidase and lactase activities

Lactose-adapted cells were obtained from Datura innoxia sucrose growing calli cultures and from Vinca minor glucose growing calli cultures. Lactose adaptation process points out the homogeneity of the cell population towards lactose uptake in V. minor cultures while it reveals the presence of heterogeneous population in D. innoxia cultures.In both species, lactose hydrolysis was only occurring in the cells; no lactase activity was detected in the culture medium. An intermittent lactase activity was determined in a cell-free extract during the culture period. Lactase activity was detected in Vinca glucose grown cells as well in Datura lactose-adapted cells cultured in absence of lactose; so lactase is a constitutive enzyme. Galactose liberated during lactose hydrolysis was not toxic for thecells; it was released into the culture medium and not metabolized in Vinca cultures while it was metabolized in Datura cultures at the end of the culture period.     

PCR-RFLP genotyping assay for a lactase persistence polymorphism upstream of the lactase-phlorizin hydrolase gene

The majority of the world's human population experiences a decline of lactase gene expression during maturation, so-called lactase nonpersistence. Thus, adults with lactase nonpersistence are susceptible to developing symptoms of lactose intolerance. By contrast, lactase persistence is an autosomal dominant heritable condition that results in a high level of lactase gene expression throughout adulthood and sustained lactose tolerance. Lactase persistence has recently been correlated with a single nucleotide genetic variant (a C --> T mutation) located 13,910 bases upstream from the lactase structural gene. We aimed to develop a restriction fragment length polymorphism (RFLP) method of detecting the C/T variants as a means of identifying individuals genetically inclined toward lactase persistence or nonpersistence. Genomic DNA in a 210-bp region surrounding the -13,910-bp variant site was PCR amplified with unique primers designed to avoid or mutate adjacent restriction sites. The amplified DNA was digested with a restriction enzyme, CviJI, that recognizes the base pair sequence generated by the lactase nonpersistence variant. Restriction digest gel analysis yielded DNA fragments of the expected diagnostic molecular weight sizes for individuals that were homozygote or heterozygote for the lactase persistence and nonpersistence variants. The genotypes predicted by the RFLP-based method were confirmed by DNA sequence analysis. The RFLP-based method provides a quick and noninvasive means of molecular detection of the presence or absence of the lactase persistence variant.     

Production of mold lactase

A process for production of mold lactase was developed. Tests were carried out in pilot and industrial scale with an Aspergillus niger strain selected after screening a number of molds.A computer coupled autoanalyzer system was used for monitoring enzyme formation in the pilot fermentor. Lactase production was investigated using different pH- and temperature-profiles. A. niger lactase has an acid pH optimum, a high temperature optimum and good stability. It does not require any metal ions. It is suitable for immobilization for hydrolysis of lactose in acid whey.Three-fold enhancement of lactase production was obtained by mutagenizing A. niger using NTG as mutagenic agent.The lactases produced by the mutants have the same pH and temperature optima and stability but the growth properties of the mutants were different from those of the original strain.Sufficient specific activity of the enzyme preparation for immobilization was obtained by purifying the enzyme by selective adsorption on Na-Ca-silicate.