Influence of alternansucrase-derived oligosaccharides and other carbohydrates on alpha-galactosidase and alpha-glucosidase activity in Bifidobacterium adolescentis.

AIMS: To determine the influence of alternansucrase-derived oligosaccharides (AOS) and other carbohydrates on alpha-galactosidase and alpha-glucosidase activity in Bifidobacterium adolescentis. METHODS AND RESULTS: Activities for alpha-galactosidase and alpha-glucosidase were determined from cell extracts of B. adolescentis grown on 18 test carbohydrates including AOS. alpha-galactosidase activity was enhanced on a variety of alpha-linked or beta-linked carbohydrates regardless of a galactoside or glucoside. alpha-glucosidase, however, was enhanced only on alpha-linked carbohydrates. AOS significantly enhanced enzyme activity compared with most of the carbohydrates tested. Most of the AOS showed significant increases in activity for both enzymes over that displayed by their corresponding acceptor carbohydrates. CONCLUSIONS: alpha-galactosidase may serve as a biomarker for microbial metabolic activity within the large intestine for potential prebiotics composed of alpha-linked or beta-linked oligosaccharides whereas alpha-glucosidase activity may be restricted to assessing the influence of only alpha-linked carbohydrates. The AOS synthesis process provided a value-added component to carbohydrates by increasing metabolic activity (via alpha-galactosidase and alpha-glucosidase) over certain acceptor carbohydrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Fundamental knowledge of enzyme activity in Bifidobacterium may aid in the design of more effective prebiotics and may also help identify enzyme indicators of metabolic activity when assessing influence within the intestine.     

Growth of various intestinal bacteria on alternansucrase-derived oligosaccharides

AIMS: To determine whether alternansucrase (ASR)-derived oligosaccharides can support the in vitro growth of various intestinal bacteria. METHODS AND RESULTS: Growth was assessed from each culture after incubation in a medium containing ASR-derived oligosaccharide as sole carbohydrate source. Most of the Bifidobacterium spp. tested showed growth on all five of the oligosaccharides tested while the Lactobacillus spp., Bacteroides thetaiotaomicron, coliforms and pathogenic bacteria displayed no or little growth. CONCLUSIONS: The ASR-derived oligosaccharides were selectively utilized by many of the Bifidobacterium spp. tested but did not support significant growth of the Lactobacillus spp., Bact. thetaiotaomicron, coliforms and pathogenic bacteria tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Alternansucrase-derived oligosaccharides are a potential source of new prebiotics.     

Cloning and characterization of a novel α-galactosidase from Bifidobacterium breve 203 capable of synthesizing Gal-α-1,4 linkage

A novel α-galactosidase gene ( aga2 ) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p -nitrophenyl-α- d -galactopyranoside ( p NPG). The K _m value for p NPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-α-1, 4-Gal-α-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-α-1,4 linkage, a novel galactosidic link formed by microbial α-galactosidase. In the presence of p NPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.     

Substrate preference of Bifidobacterium adolescentis MB 239: compared growth on single and mixed carbohydrates

The utilization of mono-, di-, and oligosaccharides by Bifidobacterium adolescentis MB 239 was investigated. Raffinose, fructooligosaccharides (FOS), lactose, and the monomeric moieties glucose and fructose were used. To establish a hierarchy of sugars preference, the kinetics of growth and sugar consumption were determined on individual and mixed carbohydrates. On single carbon sources, higher specific growth rates and cell yields were attained on di- and oligosaccharides compared to monosaccharides. Analysis of the carbohydrates in steady-state chemostat cultures, growing at the same dilution rate on FOS, lactose, or raffinose, showed that monomeric units and hydrolysis products were present. In chemostat cultures on individual carbohydrates, B. adolescentis MB 239 simultaneously displayed α-galactosidase, β-galactosidase, and β-fructofuranosidase activities on all the sugars, including monosaccharides. Glycosyl hydrolytic activities were found in cytosol, cell surface, and growth medium. Batch experiments on mixtures of carbohydrates showed that they were co-metabolized by B. adolescentis MB 239, even if different disappearance kinetics were registered. When mono-, di-, and oligosaccharides were simultaneously present in the medium, no precedence for monosaccharides utilization was observed, and di- and oligosaccharides were consumed before their constitutive moieties.     

Production and properties of ααα-glucosidase from the thermotolerant bacteriumThermomonospora curvata

Thermomonospora curvata contains α-1,4-glucosidase that is induced duringgrowth on maltose and starch. Maltose acts as an inducer of α-glucosidase even in thepresence of glucose. An intracellular thermostable α-glucosidase from T. curvata wasdetected in the crude extract on SDS-PAGE by means of modified colour reaction afterrenaturation of the enzyme. The enzyme was purified 59-fold to homogeneity with a yield of17·7% by a combination of ion-exchange and hydrophobic interaction chromatography andgel filtration. The enzyme has an apparent molecular mass of 60±1 kDa and isoelectric point4·1. The α-glucosidase exhibits optimum activity at pH 7·0–7·5 and54°C. The activity is inhibited by heavy metals and is positively affected by Ca²+ andMg²+. The enzyme hydrolyses maltose, sucrose, p-nitrophenyl-α- d -glucopyranoside and maltodextrins from maltotriose up to maltoheptaose with a decreasingefficiency. The K_m for maltose and p-NPG are 12 and 2·3 mmol l⁻¹,respectively.     

青春双歧杆菌对2型糖尿病模型大鼠肠道菌群和脂质代谢的影响

目的通过观察青春双歧杆菌对2型糖尿病模型大鼠肠道菌群的变化,和血清中总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白（HDL-C）、超氧化物歧化酶（SOD）和丙二醛（MDA）的水平,探讨青春双歧杆菌对2型糖尿病模型大鼠肠道功能和脂质代谢的影响。方法采用青春双歧杆菌灌胃2型糖尿病模型大鼠,取粪便检查正常菌群,取血和脏器检测TC、TG、HDL-C、SOD和MDA含量。结果青春双歧杆菌导致肠道内双歧杆菌、乳杆菌的数量增加,而肠杆菌、肠球菌数量下降;TC、TG和MDA水平下降,而HDL-C和SOD水平升高。结论青春双歧杆菌具有改善2型糖尿病模型大鼠肠道功能和降血脂作用,与二甲双胍联合应用效果更佳。     

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.     

青春双歧杆菌对2型糖尿病模型大鼠免疫功能和肾脏的影响

目的通过观察青春双歧杆菌对2型糖尿病模型大鼠血清中细胞因子IL-2、IL-6和IFN-γ活性的影响,以及血清及尿中的NO与ET-1的变化,探讨青春双歧杆菌对2型糖尿病模型免疫功能和肾脏的影响。方法采用青春双歧杆菌灌胃2型糖尿病模型大鼠,取血液和尿液,ELISA法检测细胞因子IL-2、IL-4、IL-6、IFN-γ和ET-1活性,硝酸酶还原法测定NO水平。结果青春双歧杆菌提高IL-2、IL-4水平,降低IL-6、IFN-γ和ET-1活性,NO水平在病程中动态变化。结论青春双歧杆菌具有平衡2型糖尿病模型大鼠免疫功能,抑制ET-1,调节NO水平的作用,从而预防肾小球硬化的发生。     

α-glucosidase in Mycoplasma mycoides subspecies capri

Abstract Mycoplasma mycoides subsp. capri utilisede maltose in medium lacking serum and hence serum saccharolytic enzymes. The presence of α-glucosidase activity was demonstrated by p-nitrophenyl-α- d -glucoside hydrolysis in toluene-treated cells. Specific activities were approx. 4-fold higher in cells grown in the presence of maltose than in cells grown with other sugars or with glucose plus maltose. Extracellular activity was < 2% of cellular activity in growing cultures. α-Glucosidase activity was also demonstrated in cells grown in medium containing serum. It is suggested that the presence of α-glucosidase might be of value in mycoplasma chatacterisation; in a previous study, α-glucosidase activity was not detected in Mycoplasma mycoides subsp. mycoides .     

Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.     

Purification and characterization of α-glucosidase in Apis cerana indica

Apis cerana indica foragers were used for the isolation of a full‐length α‐glucosidase cDNA, and for purification of the active nascent protein by low salt extraction of bee homogenates, ammonium sulphate precipitation and diethylaminoethyl‐cellulose and Superdex 200 chromatographies. The molecular mass of the purified protein was estimated by polyacrylamide gel electrophoresis resolution, and the pH, temperature, incubation, and substrate optima for enzymic activity were determined. Conformation of the purified enzyme as α‐glucosidase was performed by BLAST software homology comparisons between matrix assisted laser desorption ionization time of flight mass spectroscopy analysed partial tryptic peptide digests of the purified protein with the predicted amino acid sequences deduced from the α‐glucosidase cDNA sequence.     

Synthesis and localization of α-amylase and α-glucosidase in Bacillus licheniformis grown in batch and continuous culture

In batch and continuous cultures of Bacillus licheniformis NC1B 6346 α-amylase was invariably extracellular and could not be detected in the cytoplasm or cell surface. α-Glucosidase however, was largely intracellular but at the end of exponential growth and during slow growth under Mg²⁺ limitation it was detected in the culture fluid. Both enzymes were susceptible to catabolite repression and glucose totally inhibited their synthesis in batch culture. In maltose-limited chemostat culture, synthesis of both enzymes was maximal at D = 0.2/h and declined at higher growth rates. α-Amylase synthesis was constitutive but α-glucosidase synthesis was induced by maltose and maltotriose but not by methyl-α-D-glucoside or phenyl-α-D-glucoside. α-Amylase was synthesized at pH 6.5 and above in maltose-limited chemostat culture but not below this pH. Intracellular α-glucosidase synthesis varied little with pH. Increasing temperature decreased the synthesis of both enzymes in chemostat culture to the extent that α-glucosidase was undetectable at 50° C. Polar lipid composition varied with pH and temperature but there was no correlation between this and enzyme secretion. Moreover cerulenin, an antibiotic that inhibits protein secretion in some bacteria by interacting with the membrane had no effect on α-amylase secretion but decreased the release of α-glucosidase upon protoplast formation.     

Synthesis of two distinct α-glucosidase activities in Bacillus licheniformis

When grown with maltose or starch as carbon source at 37°C, Bacillus licheniformis synthesized two α-glucosidases which could be resolved by polyacrylamide gel electrophoresis. One of these enzymes preferentially hydrolysed p -nitrophenyl-α-D-glucoside ( p NPG) and the other was inactive on p NPG but hydrolysed maltose (maltase). Only the maltase could be detected in induced cells grown at 50°C.     

青春双歧杆菌辅助治疗牙周炎的疗效及对常见致病菌和炎性因子水平的影响

目的探讨青春双歧杆菌辅助治疗牙周炎的疗效及对常见致病菌和炎性因子水平的影响。方法选择2017年6月-2018年6月于我院确诊并治疗的慢性牙周炎患者84例,按照随机数字表法将所有患者分为双歧杆菌组(n=43)和对照组(n=41),所有患者均予以一次牙周清洁治疗。双歧杆菌组在此基础上使用双歧杆菌活菌散含服3周,治疗结束后进行为期3个月的随访。观察比较两组患者治疗前后牙周炎相关指标、龈沟液(GCF)内双歧杆菌及常见致病菌水平及炎性因子水平之间的差异,并分析GCF内双歧杆菌与炎性因子水平的相关性。结果双歧杆菌组出血指数(BI)、菌斑指数(PLI)、牙龈指数(GI)及探诊深度(PD)均显著低于对照组(均P<0.05)。双歧杆菌组患者末次随访时GCF内牙龈卟啉单胞菌、福赛斯坦纳菌、中间普雷沃菌及齿垢密螺旋体水平显著低于对照组,双歧杆菌水平显著高于对照组(均P<0.05)。双歧杆菌组患者末次随访时GCF内IL-6、TNF-α、MMP-8及TIMP-2水平显著低于对照组(均P<0.05)。GCF内IL-6、TNF-α、MMP-8及TIMP-2含量均与双歧杆菌水平呈负相关(β=-0.311,-0.309,-0.306,-0.142,均P<0.05)。结论双歧杆菌能有效改善牙周炎患者口腔内微生物环境,降低患处GCF内炎性因子水平,对牙周炎的辅助治疗起到较好的效果。     

分叉双歧杆菌对小鼠腹水瘤的抑制作用

本文观察了分叉双歧杆菌对小鼠腹腔移植的淋巴细胞腹水瘤(SRS)的抑制作用。结果发现,分叉双歧杆菌在SRS瘤细胞移植前或移植后应用,均能明显抑制瘤细胞的生长,特别在移植后应用,抗瘤效果更显著。主要表现为荷瘤小鼠存活时间延长、存活率提高。将该菌预先进行处理或不处理后加入体外培养的SRS瘤细胞中,发现该菌对离体的瘤细胞生长也有直接抑制作用。     

Utilization of starch and synthesis of a combined amylase/αα-glucosidase by the human colonic anaerobe Bacteroides ovatus

Bacteroides ovatus preferentially utilized starch and pectin when grown on a mixture of polysaccharides in batch culture, indicating that these carbohydrates are important substrates for the bacterium in the human large intestine. Further studies on starch breakdown showed that continuous cultures grew on the polysaccharide when it provided the sole carbohydrate source, to yield a single hydrolytic product at low dilution rates ( D = 0·04 h⁻¹), with an estimated molecular mass of 13 kDa. In contrast, two major types of oligomeric products were formed at higher dilution rates ( D = 0·44 h⁻¹), with approximate molecular weights of 11 and 140 kDa. Analysis of cell-associated starch-degrading enzymes produced by Bact. ovatus using ion exchange chromatography and HPLC gel-filtration showed that amylase and α-glucosidase activities eluted in the same fractions. The single peak containing amylase and α-glucosidase activities obtained by HPLC gel-filtration chromatography corresponded to a molecular mass of approximately 140 kDa, and activity staining of gels for α-glucosidase activity after polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulphate, gave an estimated molecular mass of 70 kDa, indicating this enzyme to be a dimer. After renaturation, the 70 kDa band was cut from the gels and solubilized. The extract hydrolysed gelatinized starch and p -nitrophenyl-α- D -glucopyranoside.     

双歧杆菌DNA对巨噬细胞PKC的影响

目的探索青春型双歧杆菌的DNA对巨噬细胞PKC家族的影响.方法以激光共聚焦显微镜定量测定小鼠腹腔巨噬细胞PKCα、PKCβⅠ、PKCβⅡ、PKCγ、PKCε和PKCζ的含量.结果双歧杆菌DNA注射组小鼠腹腔巨噬细胞PKCα和PKCβⅡ的平均荧光强度明显高于对照组（P＜0.01）,而PKCβⅠ、PKCγ、PKCε和PKCζ的平均荧光强度在2组间则差异无显著性（P＞0.05）.结论青春型双歧杆菌的DNA能活化巨噬细胞的PKCα和PKCβⅡ.     

Effect of seawater on Escherichia coli β-galactosidase activity

An investigation of β-galactosidase activity of Escherichia coli strain H10407, under different physiological and environmental conditions, e.g. induced and uninduced osmotic stress, light, etc., was undertaken. In this study E. coli was employed as a model for faecal coliforms in waste water. β-Galactosidase activity was induced by isopropyl-β-D-thiogalactoside (IPTG). Enzyme activity (U cell^-1)/cell for sewage bacteria and for induced E. coli was similar, i.e. log U cell^-1= -8.5 whereas uninduced E. coli yielded log U cell^-1= -12.1. Initial enzyme activity was not dependent on phase of growth of the cell (exponential vs stationary phase) or whether marine or fresh water at the time of initial dilution. However, osmotic change resulted in a decrease in culturable cells, even though enzyme activity remained constant. A significant decrease in the number of culturable bacteria, followed by a decrease in β-galactosidase activity, was observed after exposure of cells to visible light radiation. It is concluded that β-galactosidase enzyme is retained in viable but non-culturable E. coli. Furthermore, β-galactosidase appears to offer a useful and rapid (25 min) measure of the viability of faecal coliforms, and therefore, of the water quality of bathing and shellfishing areas.     

Effect of tunicamycin on α-galactosidase secretion by Aspergillus nidulans and the importance of N-glycosylation

Abstract Aspergillus nidulans released α-galactosidase into the culture medium during the exponential growth on either lactose or galactose as the only carbon source. This enzyme is a glycoprotein. Its treatment with endoglycosidases produces a reduction in its molecular mass but the resulting enzyme conserved some of their carbohydrate components in addition to its enzymatic activity. Mycelia of A. nidulans growing in the presence of tunicamycin synthesized an underglycosylated α-galactosidase which was not released into the culture media but remained bound to the cell-wall. Tunicamycin did not prevent the synthesis and secretion of α-galactosidase by protoplasts. N-linked oligosaccharide chains seem not to be essential for the synthesis and secretion of α-galactosidase of A. nidulans , but they could be necessary for proper targeting at the extracellular level.