Characterization of tributyrin hydrolysis by immobilized lipase on woolen cloth using conventional batch and novel spinning cloth disc reactors

Optimal loading and operating conditions for a new, superior immobilization of amano lipase from Pseudomonas fluorescens on woolen cloth were determined. The optimal enzyme loading was 46.8 mg g dry cloth⁻¹ with activity of 200 U. A batch reactor was used to characterize process conditions important to industrial application of the wool immobilized lipase. The optimal pH for immobilized lipase in tributyrin hydrolysis was 7, slightly lower than that of free lipase (pH 8). The optimal temperature for both free and immobilized lipase was 45 °C. The immobilized lipase was more stable to reuse than some other lipase immobilizations, maintaining 85% of its activity after 6 long term runs and 75.8% of the original activity after storage of 40 weeks at 4 °C. The thermal stability of lipase was improved by 2.4 times after immobilization. The thermal deactivation rate of immobilized lipase followed the Arrhenius law with E_d = 199 kJ mol⁻¹. The Michaelis–Menten constant (K_m) of the lipase increased from 1.63 mM to 4.48 mM after immobilization. The immobilized lipase was also successfully applied for tributyrin hydrolysis in a novel enzyme process intensification technology – the spinning cloth disc reactor (SCDR): conversion increased by around 13% under similar conditions compared to a conventional batch stirred tank reactor. The SCDR is therefore key to exploiting the advantages of the wool immobilized lipase developed in this work.     

Formulation and Evaluation of an Automatic Dishwashing Detergent Containing T1 Lipase

The use of enzymes in detergent formulations is becoming popular due to the concerns about the environment. T1 lipase (E.C. 3.1.1.3) was evaluated for its stability and performance in dishwashing along with other common components of an automatic dishwashing detergent. Therefore, the process of formulating the detergent would depend on the stability of T1 lipase, which may also reflect the performance during the washing. T1 lipase was mostly stable in nonionic surfactants, especially those that were made of polyhydric alcohols. T1 lipase was also stable in a mixture of sodium carbonate and glycine. However, sodium carbonate alone destabilized T1 lipase possibly due to the interaction between carbonates and Ca²⁺. These results indicated that polyhydric alcohols and glycine had stabilizing effects on T1 lipase. The dishwashing performance was evaluated in term of percent soil removed. The dishwashing performance of the formulated detergent was positively affected by the increase in temperature but negatively affected by the presence of hard water, specifically Ca²⁺ and Mg²⁺. However, T1 lipase was not negatively affected by the presence of hard water, and this enzyme was enhanced by the presence of polyacrylates. The presence of Ca²⁺ improved the structural integrity of T1 lipase. It is generally known that most enzymes that depend on Ca²⁺ for their structural integrity would be greatly destabilized in the presence of metal chelators; thus, stabilizing strategies such as adding glycine would be essential to maintain enzyme activity during the wash.     

Stereodivergent Biocatalytic Formal Reduction of α-Angelica Lactone to (R)- and (S)-γ-Valerolactone in a One-Pot Cascade

The formal asymmetric and stereodivergent enzymatic reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a one-pot cascade by uniting the promiscuous stereoselective isomerization activity of Old Yellow Enzymes with their native reductase activity. In addition to running the cascade with one enzyme for each catalytic step, a bifunctional isomerase-reductase biocatalyst was designed by fusing two Old Yellow Enzymes, thereby generating an unprecedented case of an artificial enzyme catalyzing the reduction of nonactivated C=C bonds to access (R)-valerolactone in overall 41 % conversion and up to 91 % ee. The enzyme BfOYE4 could be used as single biocatalyst for both steps and delivered (S)-valerolactone in up to 84 % ee and 41 % overall conversion. The reducing equivalents were provided by a nicotinamide recycling system based on formate and formate dehydrogenase, added in a second step. This enzymatic system provides an asymmetric route to valuable chiral building blocks from an abundant bio-based chemical.     

酶与中国洗涤剂工业的发展

论述中国加酶洗涤剂的发展历程。目前酶制剂正在成为市场竞争的有力武器 ,利用它推出新品、提高产品质量和优化产品性能 /价格比。酶制剂可成为中国洗涤剂组成的明日之星 ,为中国洗涤剂工业发展作出更大贡献。     

ON THE BEHAVIOR OF CONSECUTIVE ENZYMATIC REACTIONS IN CYCLICALLY OPERATED REACTORS

The dynamics of a process involving consecutive enzymatic reactions were investigated for a case in which the reactions are carried out in a cyclically operated reactor. Each cycle of the operation protocol involves three phases. During phase I the reactor operates in a semi-batch mode involving input of the reactants. During the second phase, the vessel operates in a batch mode. During phase III the reactor has an output only and a fraction of its contents are emptied before another identical cycle begins.

It was found that there are regimes in the operating parameter space where the system can reach more than one limit cycle (multistability). Using computer software based on the bifurcation theory for forced systems, as well as one- and two-parameter continuation algorithms, the impact of various parameters on the dynamics of the system was investigated. The results are presented in the form of diagrams. Conditions under which formation of the intermediate product in the reaction sequence is maximized were also investigated.

Production of the intermediate product in a limit cycle was compared to that obtained in batch and semi-batch operation. Implications of the proposed operation protocol for process optimization and pollution prevention are discussed.     

Experimental Validation of a Fundamental Model for PCR Efficiency

Recently a theoretical analysis of PCR efficiency has been published by Booth et al. (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii) the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii) the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. Experiments have been designed that are specifically controlled by each one of the efficiencies and the results are consistent with the mathematical model. The experimental data has also been used to quantify six key parameters of the theoretical model. An important application of the fully characterized model is to calculate initial template concentration from real-time PCR data. Given the PCR protocol, the midpoint cycle number (where the template concentration is half that of the final concentration) can be theoretically determined and graphed for a variety of initial DNA concentrations. Real-time results can be used to calculate the midpoint cycle number and consequently the initial DNA concentration, using this graph. The application becomes particularly simple if a conservative PCR protocol is followed where only the annealing efficiency is controlling.     

Ultrasonic enhancement of waste activated sludge hydrolysis and volatile fatty acids accumulation at pH 10.0

Volatile fatty acids (VFA), the preferred carbon source for biological nutrients removal, can be produced by waste activated sludge (WAS) anaerobic fermentation. However, because the rate of VFA accumulation is limited by that of WAS hydrolysis and VFA is always consumed by methanogens at acidic or neutral pHs, the ultrasonic pretreatment which can accelerate the rate of WAS hydrolysis, and alkaline adjustment which can inhibit the activities of methanogens, were, therefore, used to improve WAS hydrolysis and VFA accumulation in this study. Experiment results showed that the combination of ultrasonic pretreatment and alkaline adjustment caused significant enhancements of WAS hydrolysis and VFA accumulation. The study of ultrasonic energy density effect revealed that energy density influenced not only the total VFA accumulation but also the percentage of individual VFA. The maximal VFA accumulation (3109.8 mg COD/L) occurred at ultrasonic energy density of 1.0 kW/L and fermentation time of 72 h, which was more than two times that without ultrasonic treatment (1275.0 mg COD/L). The analysis of VFA composition showed that the percentage of acetic acid ranked the first (more than 40%) and those of iso-valeric and propionic acids located at the second and third places, respectively. Thus, the suitable ultrasonic conditions combined with alkaline adjustment for VFA accumulation from WAS were ultrasonic energy density of 1.0 kW/L and fermentation time of 72 h. Also, the key enzymes related to VFA formation exhibited the highest activities at ultrasonic energy density of 1.0 kW/L, which resulted in the greatest VFA production during WAS fermentation at pH 10.0.     

Incidence of adenoviruses in raw and treated water

Adenoviruses are of major public health importance and are associated with a variety of clinical manifestations, i.e. gastroenteritis, eye infections and respiratory infections. The importance of water in the epidemiology of adenoviruses and the potential health risks constituted by adenoviruses in water sources and supplies are widely recognised. This study was conducted to assess the incidence of human adenoviruses in raw and treated water systems. Various raw and treated water were routinely monitored for the presence of adenoviruses, over a 1-year period (July 2000-June 2001). The supplies were derived from acceptable quality surface water sources using treatment processes, which conform to international standards for the production of safe drinking water. Adenoviruses were detected by firstly amplifying the viruses in cell cultures and then amplifying the extracted nucleic acids of these viruses using molecular techniques (nested PCR). The results indicated human adenoviruses present in 13 (12.75%) of the raw and 9 (4.41%) of the treated water samples tested. The combination of cell culture and nested PCR has proved to be a quick and reliable method for the detection of adenoviruses in water environments.