Properties and applications of β‐glycosidase from Bacteroides thetaiotaomicron that specifically hydrolyses isoflavone glycosides

To modify the glycan part of glycosides, the gene encoding β‐glycosidase was cloned from Bacteroides thetaiotaomicron VPI‐5482. The cloned gene, bt_1780, was expressed in Escherichia coli MC1061 and the expressed enzyme was purified using Ni‐NTA affinity chromatography. The purified enzyme, BTBG, showed optimal activity at 50 °C and pH 5.5. Interestingly, this enzyme did not have any hydrolysing activity on ordinary β‐linkage–containing substrates such as xylobiose, lactose and cello‐oligosaccharide, but specifically hydrolysed isoflavone glycosides such as daidzin, genistin and glycitin. Compared to a commercial beta glucosidase, BTBG selectively hydrolysed isoflavone glycosides in soybean extract mixture solution. These results suggest that BTBG may be a specialized enzyme for the hydrolysis of glycosides and that the substrate specificity of BTBG is applicable for the bioconversion of isoflavone glycosides in the food industry.     

Fimbriae of Bacteroides nodosus: protein engineering of the structural subunit for the production of an exogenous peptide

The pattern of sequence variation between Bacteroides nodosusfimbrial subuits of different serotypes suggests a degree offlexibility, which might be exploited for protein engineeringapproaches for the expression of other peptides. We have testedthis using the well-characterized peptide epitope from VP1 offoot-and-mouth disease virus (FMDV), residues 144–159:LRGDLQVLAQKVARTL (strain 01-BFS). Using bacterial codon usage,several oligonucleotides were designed for the substitutionof this sequence internally at hypervariable regions of thefimbrial subunit (aligned for maximum homology), and for itsaddition at the carboxy-terminus with a diglycine spacer asa flexible hinge. Following site-directed mutagenesis in phageM13, the modified genes were placed under P_L promoter controland placed in a broad host range vector. Analysis of the variantproteins expressed in E.coli showed that these substitutionsaffected, to varying extents, recognition by both anti-fimbrialand anti-FMDV antibodies, indicating that hypervariable region2 is a major antigenic determinant of the fimbrial subunit andthat local stereochemical effects can influence antibody bindingto the FMDV peptide antigenic determinant. In Pseudomonas aeruginosa,viable transformants could only be obtained with the mutantgene encoding the carboxy-terminal graft. These cells providedfimbrial preparations comprised of the modified subunit. Thisthen constitutes the prototype for the development of a generalexpression system for the production of vaccine epitopes andother bioactive peptides. Furthermore, there is considerablescope for further modification of the system, for example byengineering specific chemical or protease cleavage sites forrelease of the grafted peptide. Alternatively, the fimbriaethemselves may serve as a useful supramoleuclar carrier or adjuvantfor immune provocation.     

Sourcing faecal pollution: a combination of library-dependent and library-independent methods to identify human faecal pollution in non-sewered catchments

Library-dependent (LD) (biochemical fingerprinting of Escherichia coli and enterococci) and library-independent (LI) (PCR detection of human-specific biomarkers) methods were used to detect human faecal pollution in three non-sewered catchments. In all, 550 E. coli isolates and 700 enterococci isolates were biochemically fingerprinted from 18 water samples and compared with metabolic fingerprint libraries of 4508 E. coli and 4833 enterococci isolates. E. coli fingerprints identified human unique biochemical phenotypes (BPTs) in nine out of 18 water samples; similarly, enterococci fingerprints identified human faecal pollution in 10 water samples. Seven samples were tested by PCR for the detection of biomarkers. Human-specific HF134 Bacteroides and enterococci surface protein (esp) biomarkers were detected in five samples. Four samples were also positive for HF183 Bacteroides biomarker. The combination of biomarkers detected human faecal pollution in six out of seven water samples. Of the seven samples analysed for both the indicators/markers, at least one indicator/marker was detected in every sample. Four of the seven PCR-positive samples were also positive for one of the human-specific E. coli or enterococci BPTs. The results indicated human faecal pollution in the studied sub-catchments after storm events. LD and LI methods used in this study complimented each other and provided additional information regarding the polluting sources when one method failed to detect human faecal pollution. Therefore, it is recommended that a combination of methods should be used to identify the source(s) of faecal pollution where possible.     

Escherichia coli, enterococci, and Bacteroides thetaiotaomicron qPCR signals through wastewater and septage treatment

Fecal indicators such as Escherichia coli and enterococci are used as regulatory tools to monitor water with 24 h cultivation techniques for possible input of sewage or feces and presence of potential enteric pathogens yet their source (human or animal) cannot be determined with routine methods. This critical uncertainty has furthered water pollution science toward new molecular approaches. Members of Bacteroides genus, such as Bacteroides thetaiotaomicron are found to have features that allow their use as alternative fecal indicators and for Microbial Source Tracking (MST). The overall aim of this study was to evaluate the concentration and fate of B. thetaiotaomicron, throughout a wastewater treatment facility and septage treatment facility. A large number of samples were collected and tested for E. coli and enterococci by both cultivation and qPCR assays. B. thetaiotaomicron qPCR equivalent cells (mean: 1.8 × 10⁷/100 mL) were present in significantly higher concentrations than E. coli or enterococci in raw sewage and at the same levels in raw septage. The removal of B. thetaiotaomicron target qPCR signals was similar to E. coli and enterococci DNA during the treatment of these wastes and ranged from 3 to 5 log₁₀ for wastewater and was 7 log₁₀ for the septage. A significant correlation was found between B. thetaiotaomicron marker and each of the conventional indicators throughout the waste treatment process for both raw sewage and septage. A greater variability was found with enterococci when compared to E. coli, and CFU and equivalent cells could be contrasted by various treatment processes to examine removal and inactivation via septage and wastewater treatment. These results are compared and contrasted with other qPCR studies and other targets in wastewater samples providing a view of DNA targets in such environments.     

Evaluation of multiple sewage-associated Bacteroides PCR markers for sewage pollution tracking

The host specificity of the five published sewage-associated Bacteroides markers (i.e., HF183, BacHum, HuBac, BacH and Human-Bac) was evaluated in Southeast Queensland, Australia by testing fecal DNA samples (n = 186) from 11 animal species including human fecal samples collected via influent to a sewage treatment plant (STP). All human fecal samples (n = 50) were positive for all five markers indicating 100% sensitivity of these markers. The overall specificity of the HF183 markers to differentiate between humans and animals was 99%. The specificities of the BacHum and BacH markers were > 94%, suggesting that these markers are suitable for the detection of sewage pollution in environmental waters in Australia. The HuBac (i.e., 63%) and Human-Bac (i.e., 79% specificity) markers performed poorly in distinguishing between the sources of human and animal fecal samples. It is recommended that the specificity of the sewage-associated markers must be rigorously tested prior to its application to identify the sources of fecal pollution in environmental waters.     

Maximum volumetric production of β‐galactosidase by Kluyveromyces fragilis in fed‐batch culture with automatic control

The production of β‐galactosidase by Kluyveromyces fragilis was studied in different culture systems, with dissolved oxygen concentration control and using defined media. An operating strategy of fed‐batch culture with automatic control of substrate addition regulated by dissolved oxygen concentration, consisting of the replacement of variable volumes of broth by fresh medium (once the fed‐batch culture has finished), was designed. The volumetric enzyme productivity (Q_p, 13 600 UI dm^?3 h^?1) obtained was 38% higher than that reached in continuous culture of K fragilis with dissolved oxygen concentration control and far higher than that obtained by batch culture of K fragilis under the same aeration conditions. © 2002 Society of Chemical Industry     

不同拟杆菌的定殖对伪无菌小鼠血清生化指标和短链脂肪酸的影响

为探究不同拟杆菌的定殖对伪无菌小鼠肠道代谢及健康的影响,通过灌胃小鼠混合抗生素剔除大部分肠道微生物建立伪无菌模型.成模后,随机分为模型组和4组拟杆菌定殖组,进行人源耐酸性较强的4组拟杆活菌的移植.通过测定脏器指数、血清生化、盲肠内容物短链脂肪酸含量等指标比较评价拟杆菌的定殖对小鼠的影响.研究表明Bacteroides ...     

Research note: Effect of dissolved oxygen level on lactase production by Kluyveromyces fragilis

The influence of dissolved oxygen (% DO) on lactase production by Kluyveromyces fragilis (NRRL-Y-1109) in chemostat culture using a defined medium was studied. The aim was to determine conditions for both high specific enzyme activity and high volumetric enzyme productivity. Significant differences in the specific enzyme activity and specific and volumetric enzyme productivity were found at the corresponding steady states when the DO was varied between 0 and 90%. Maximum lactase production was attained at 10% DO. Under this condition the best results were an enzyme activity of 5910 IU g⁻¹, specific production rate of 1810 IU g⁻¹ h⁻¹ and volumetric production rate of 1530 IU dm⁻³ h⁻¹. This seem to be due to the fact that at low aeration conditions the yeast metabolism is more reductive and as a consequence it verifies both higher specific lactose consumption rate and higher enzyme expression than in full aeration conditions. The results of this investigation are also compared with those of other studies of lactase production by Kluyveromyces sp. © 1998 Society of Chemical Industry     

糖尿病患者肠道中4 种重要菌群的定量分析

目的：了解正常人群与Ⅱ型糖尿病人,以及Ⅱ型糖尿病人在是否有家族遗传、是否用药治疗和是否伴随肥胖等情况下肠道中拟杆菌、双歧杆菌、柔嫩梭菌和乳杆菌的含量与变化,为预防和治疗Ⅱ型糖尿病开辟新途径。方法：收集上述各类人群粪便样品（每组10 人）,分别提取样品中微生物的总DNA,运用荧光定量聚合酶链式反应（polymerase chain reaction,PCR）扩增4 种重要菌群的特异性片段,计算含量,分析上述菌群的变化差异。结果：正常人群肠道中的拟杆菌和双歧杆菌含量均显著低于Ⅱ型糖尿病人群;柔嫩梭菌和乳杆菌含量均显著高于后者。在Ⅱ型糖尿病人群中,用药患者组和肥胖＋高血糖患者组的肠道中拟杆菌和双歧杆菌含量均显著低于未用药患者组和单纯高血糖患者组,而柔嫩梭菌和乳杆菌含量均显著高于后者。有无Ⅱ型糖尿病遗传史对患者肠道内4 种菌群的含量均无显著影响。结论：4 种重要菌群与Ⅱ型糖尿病的发生和发展有密切关系;用药治疗后的患者肠道中4 种重要菌群含量比其他各组更接近正常人群;肥胖易加重Ⅱ型糖尿病病情;而有无Ⅱ型糖尿病家族遗传对这4 种菌群的含量几乎没有影响。