Controllable Display of Sequential Enzymes on Yeast Surface with Enhanced Biocatalytic Activity toward Efficient Enzymatic Biofuel Cells.

A precisely localized enzyme cascade was constructed by integrating two sequential enzymes, glucoamylase (GA) and glucose oxidase (GOx), on a yeast cell surface through an a-agglutinin receptor as the anchoring motif with cohesin-dockerin interaction. The overall catalytic activities of the combinant strains were significantly dependent on the assembly method, enzyme molecular size, enzyme order, and enzyme stoichiometry. The combinant strain with GA-DocC initially bound scaffoldin prior to GOx-DocT exhibited a higher overall reaction rate. The highest overall reaction rate (29.28 ± 1.15 nmol H2O2 min-1mL-1) was achieved when GA/GOx ratio was 2:1 with enzyme order: yeast-GA-GOx-GA, 4-fold enhancement compared to free enzyme mixture. Further, the first example of starch/O2 enzymatic biofuel cells (EBFCs) using codisplayed GA/GOx based bioanodes were assembled, demonstrating excellent direct biomass-to-electricity conversion. The optimized EBFC registered an open-circuit voltage of 0.78 V and maximum power density (Pmax) of 36.1 ± 2.5 µW cm-2, significantly higher than the Pmax for other starch/O2 EBFCs reported so far. Therefore, this work highlights rational organization of sequential enzymes for enhanced biocatalytic activity and stability, which would find applications in biocatalysis, enzymatic biofuel cells, biosensing, and bioelectro-synthesis.

Bacterial cell-surface displaying of thermo-tolerant glutamate dehydrogenase and its application in L-glutamate assay.

In this paper, glutamate dehydrogenase (Gldh) is reported to efficiently display on Escherichia coli cell surface by using N-terminal region of ice the nucleation protein as an anchoring motif. The presence of Gldh was confirmed by SDS-PAGE and enzyme activity assay. Gldh was detected mainly in the outer membrane fraction, suggesting that the Gldh was displayed on the bacterial cell surface. The optimal temperature and pH for the bacteria cell-surface displayed Gldh (bacteria-Gldh) were 70°C and 9.0, respectively. Additionally, the fusion protein retained almost 100% of its initial enzymatic activity after 1 month incubation at 4°C. Transition metal ions could inhibit the enzyme activity to different extents, while common anions had little adverse effect on enzyme activity. Importantly, the displayed Gldh is most specific to l-glutamate reported so far. The bacterial Gldh was enabled to catalyze oxidization of l-glutamate with NADP(+) as cofactor, and the resultant NADPH can be detected spectrometrically at 340nm. The bacterial-Gldh based l-glutamate assay was established, where the absorbance at 340nm increased linearly with the increasing l-glutamate concentration within the range of 10-400µM. Further, the proposed approach was successfully applied to measure l-glutamate in real samples.

Sensitive electrochemical microbial biosensor for p-nitrophenylorganophosphates based on electrode modified with cell surface-displayed organophosphorus hydrolase and ordered mesopore carbons.

A novel electrochemical microbial biosensor for the rapid monitoring of p-nitrophenyl-substituted organophosphates (OPs) compounds based on glass carbon electrode (GCE) modified with both ordered mesopore carbons (OMCs) and cell surface-expressed organophosphorus hydrolase (OPH) (OPH-bacteria/OMCs/GCE) was described in this paper. The genetically engineered Escherichia coli strain surface displayed mutant OPH (S5) with improved enzyme activity and favorable stability was constructed using a newly identified N-terminal of ice nucleation protein as an anchoring motif, which can be used directly without further time-consuming enzyme-extraction and purification, thereafter greatly improved the stability of the enzyme. Compared to OPH-bacteria modified GCE (OPH-bacteria/GCE), the OPH-bacteria/OMCs/GCE not only significantly enhanced the current response but also reduced the oxidation overpotential towards oxidizable p-nitrophenol (p-NP), which was the hydrolysate of p-nitrophenyl-substituted OPs. Under the optimized experimental conditions, at +0.84 V (vs. SCE), the current-time curve was performed with varying OPs concentration. The current response was linear with paraoxon concentration within 0.05-25 µM. Similarly, linear range of 0.05-25 µM was found for parathion, and 0.08-30 µM for methyl parathion. The low limits of detection were evaluated to be 9.0 nM for paraoxon, 10nM for parathion and 15 nM for methyl parathion (S/N=3). Thus, a highly specific, sensitive and rapid microbial biosensor was established, which holds great promise for on-site detection of trace p-nitrophenyl-substituted OPs.

Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates.

Organophosphates (OPs) widely exist in ecosystem as toxic substances, for which sensitive and rapid analytical methods are highly requested. In the present work, by using N-terminal of ice nucleation protein (INP) as anchoring motif, a genetically engineered Escherichia coli (E. coli) strain surface displayed mutant organophosphorus hydrolase (OPH) (S5) with improved enzyme activity was successfully constructed. The surface location of INP-OPH fusion was confirmed by SDS-PAGE analysis and enzyme activity assays. The OPH-displayed bacteria facilitate the hydrolysis of p-nitrophenol (PNP) substituted organophosphates to generate PNP, which can be detected spectrometrically at 410 nm. Over 90% of the recombinant protein present on the surface of microbes demonstrated enhanced enzyme activity and long-term stability. The OPH activity of whole cells was 2.16 U/OD600 using paraoxon as its substrate, which is the highest value reported so far. The optimal temperature for OPH activity was around 55 °C, and suspended cultures retained almost 100% of its activity over a period of one month at room temperature, exhibiting the better stability than free OPH. The recombinant E. coli strain could be employed as a whole-cell biocatalyst for detecting PNP substituted OPs at wider ranges and lower detection limits. Specifically, the linear ranges of the calibration curves were 0.5-150 µM paraoxon, 1-200 µM parathion and 2.5-200 µM methyl parathion, and limits of detection were 0.2 µM, 0.4 µM and 1 µM for paraoxon, parathion and methyl parathion, respectively (S/N=3). These results indicate that the engineered OPH strain is a promising multifunctional bacterium that could be used for further large-scale industrial and environmental applications.

Simultaneously improving stability and specificity of cell surface displayed glucose dehydrogenase mutants to construct whole-cell biocatalyst for glucose biosensor application.

The improved stability and substrate specificity of cell surface displayed glucose dehydrogenase (GDH) mutants by replacing four amino acids from Bacillus subtilis by using site-directed mutagenesis was systematically investigated. A series of mutated GDHs including E170R/Q252L, V149K/E170R/Q252L, E170R/Q252L/G259A and V149K/E170R/Q252L/G259A, were fused to the ice nucleation protein for displaying on cell surface of Eschericia coli. Q252L/E170R/V149K, Q252L/E170R/G259A and Q252L/E170R/V149K/G259A variants were found stable at a wide pH range and shown excellent thermostability. Especially, the Q252L/E170R/V149K/G259A mutant showed half-life of ~3.8days at 70 °C. Q252L/E170R/V149K/G259A variant exhibited the narrowest substrate specificity for d-glucose. The whole cell displayed GDH mutant could be cultured in a large scale with excellent enzyme activity and productivity. In addition, a sensitive and stable electrochemical glucose biosensor can be prepared using the GDH-mutant bacteria modified electrode. Thus, the whole cell biocatalysts are promising candidates for exploitation in a wide range of industrial applications.

Yeast surface displaying glucose oxidase as whole-cell biocatalyst: construction, characterization, and its electrochemical glucose sensing application.

The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.

Microbial surface display of glucose dehydrogenase for amperometric glucose biosensor.

A genetically engineered Escherichia coli (E. coli) strain displaying glucose dehydrogenase (GDH) with ice-nucleation protein (INP) as the anchoring motif was first constructed. The surface localization and functionality of the fusion protein containing GDH were verified by SDS-PAGE, Western blotting and enzymatic activity assay. The fusion of INP had no effects on the functionality of GDH cofactor binding domain. The activity assay showed that 74.6% of the cell lysate GDH activity was detected in the outer membrane fractions. Compared with the crude enzyme solution from E. coli expressing intracellular GDH, the GDH-displaying bacteria (GDH-bacteria) was stable within pH 6-10 below 40°C. Further, a novel electrochemical glucose biosensor was developed by construction of Nafion/GDH-bacteria/multiwalled-carbon-nanotube modified electrode. The as-prepared biosensor is linear with the concentration of d-glucose within the range of 50-800 µM and a low detection limit of 4 µM D-glucose (S/N=3). Excess saccharides including D-galactose, D-fructose, D-cellbiose, L-arabinose and D-sucrose, D-maltose, D-mannose and D-xylose as well as common interfering substances (acetaminophen, ascorbic acid and uric acid) did not affect the detection of D-glucose (0.1mM). The proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective, which can be used for detection of real samples. It is envisioned that this GDH-bacteria will be found promising applications in biofuel cell, glucose detection and cofactor reproduction system.

Direct energy conversion from xylose using xylose dehydrogenase surface displayed bacteria based enzymatic biofuel cell.

Xylose is an important and major monosaccharide that extensively exists in the cellulose fermentation industry. Here we present the first report on the direct energy conversion from xylose achieved by using novel xylose dehydrogenase (XDH) surface displayed bacteria (XDH-bacteria) based enzymatic biofuel cell. The maximum power density and the open-circuit potential of the cell are 63 µWcm(-2) and 0.58 V, respectively. The as-prepared BFC holds great potential to make use of biomass from lignocellulose degradation as source energy, which avoids the bottleneck in conversion of xylose to ethanol met by conventional fermentation method.

[Systematic evaluation on clinical literature related with treatment of Parkinson's disease with traditional Chinese medicine].

OBJECTIVE: To analyze the quality of scientific research design of clinical literature related with treatment of Parkinson's disease with traditional Chinese medicine, so as to objectively evaluate the therapeutic effect of TCM. METHODS: According to principles of evidence-based medicine, clinical epidemiology/design measurement evaluation (DME), the "Table of Systematic Evaluation of Quality and Information Collection for TCM Clinical Research Literature" were established and used to evaluate clinical control trial literature related with treatment of Parkinson's disease with TCM published during 1979 to 2000. RESULTS: The method of randomization was not described in 66.7% of the literature. Although randomized design was declared in 33.3 %, problems or mistakes of randomized allocation still existed in them. No record about the state of dropped out or absconded cases in follow-up study and without any record of samples screening presented in all literature. There were some problems of key links concerning samples' homogeneity, outcome indexes selection, conclusion deduction and so on, which could also influence the quality and reliability of randomized controlled trials. CONCLUSION: Methodological design of clinical research of TCM on Parkinson's disease should be strengthened.

A follow-up study of 69 discharged SARS patients.

Sixty-nine patients with severe acute respiratory syndrome (SARS) discharged from Guangdong Provincial TCM Hospital were followed up from January to April 2003 during which the patients were asked to fill the questionnaire form and at the same time received blood routine examination, hepatic, renal, pulmonary and immune function tests, and spiral computerized tomography (CT) of the chest, color B-ultrasonography of the heart with the collected data treated by descriptive analysis and deductive analysis. The results showed that in the 69 followed-up patients, impairment of the hepatic function was found in 5 cases, hypoimmune state in 18, impediment of ventilation in the distal air passages with normal major air passages in 15, increased residual volume in 40, mild disturbance of pulmonary diffusion function in 14, incomplete absorption of inflammatory exudates, focal or multiple interstitial lesions, pulmonary interstitial fibrosis and pleural adhesion in 24; increased resistance or mild systolic hypertension in the pulmonary circulation, and segmental ischemia of the left myocardium in 34; and decreased visual acuity in 2. According to TCM differentiation 24 cases belonged to the type of deficiency of both qi and yin, 8 deficiency of both the heart and spleen, 37 depression of the liver and deficiency of the spleen, 18 intermingling with damp-heat, and 7 intermingling with stagnant blood. Some patients still had psychological problems. The study indicates that though clinically cured and discharged from hospital, some SARS patients have functional impairment of the heart, lung and liver, hypoimmune state as well as psychological problems, and need to be treated accordingly for a complete recovery. A rationale for suggested TCM treatment is expounded.