Recent Advances in the use of Immobilized Lipases Directed Toward the Asymmetric Syntheses of Complex Molecules

Water-insoluble compounds can be substrates for enzymatic reactions when lipases are immobilized properly and suitable organic solvents are used. In this review, three type of lipase immobilization method and their application to the asymmetric syntheses of complex molecules are described. Lipases immobilized with Celite or synthetic prepolymers such as urethane prepolymer and photo-crosslinkable resin prepolymer have been applied for the kinetic resolution of many kinds of water-insoluble substrate.

Phospholipid-lipase aggregates with ether linkages are novel and have been found to function effectively as immobilized lipases in asymmetric hydrolysis or esterification reactions in water-saturated organic solvent. The phospholipid-lipase aggregates are considered to have a stacked bilayer based on X-ray diffraction analysis structure of the lipid in the crystalline phase.     

Carotenogenesis in Phycomyces

Time course studies of carotenoid production and of mycelial growth in liquid cultures of Phycomyces blakesleeanus wild type [NRRL 1555 (?)], red mutants C9, C10 and C13 and the heterokaryon C2 * C9 are reported. The ratios of the concentrations of lycopene, γ-carotene and β-carotene in the red mutant C13 and in the heterokaryon C2 * C9 during the growth periods were measured. In these strains the concentration of lycopene is close to its final value after 2 days of growth, at a time at which β-carotene is just beginning to be produced. It is suggested that the β-carotene produced late is possibly synthesized via β-zeacarotene.     

Sucrose phosphorylase as cross-linked enzyme aggregate: Improved thermal stability for industrial applications

Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60°C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity.     

The effect of soil aggregate size on early growth and shoot-root ratio of maize (Zea mays L.)

Shoot dry mass and leaf area of 16-d old maize plants decreased as soil aggregate size in greenhouse pots increased in diameter from 0.075–0.5 to 4–8 mm. Root length was also much greater on the finer aggregate beds, due primarily to increased growth of second-order laterals. In a subsequent experiment in which shoot dry matter again decreased with increasing aggregate size, it was found that a similar change in root morphology as noted in experiment I resulted in increased root dry mass as aggregate size increased. The associated change in shoot-root ratio was significant eight days after emergence. This change was due to a change in allocation of fixed carbon rather than allocation of seed reserves. Neither transpiration rate per unit leaf area, nor net assimilation rate were affected by aggregate size. Likewise nutrition could not account for the differences in shoot or root growth.     

Restoration scenario planning at a Spanish quarry can be informed by assessing ecosystem services

We carried out an ecosystem service (ES) assessment at Soto de Pajares, a 400 ha active gravel quarry site located close to protected areas in the southeast of the Community of Madrid, Spain. The currently approved restoration plan is for quarry‐made excavations to be restored back to agricultural land. However, the site has been identified as being important for nature, so different restoration strategies should be considered. To better understand how these compare in terms of ES provision, which had not previously been done at mineral extraction sites in Spain, we used an established toolkit: the Toolkit for Ecosystem Service Site‐based Assessments (TESSA). We compared the agriculture‐focused restoration plan and two nature‐focused alternative scenarios, one without public access (conservation scenario) and the other with public access (compromise scenario). Monetary estimations of the value of agricultural production, climate change mitigation, and recreation were calculated in three scenarios. Results indicated that the compromise scenario provided the greatest annual value (€91,409), mainly due to its potential visitors, surpassing both agricultural (€68,504) and conservation (€48,556) scenarios. Considering the higher costs associated with restoring sites to agricultural production, nature conservation may be an attractive option for extraction companies. The use of TESSA at this site has provided valuable information to quarry managers to help guide their decision‐making.     

Molecular phylogeny,pigment composition,toxicology and life history of Pseudochattonella cf. verruculosa (Class Dictyochophyceae) from Wellington Harbour,New Zealand

Pseudochattonella verruculosa is a heterokont flagellate and has frequently been found associated with multi-species harmful algal blooms in Wellington Harbour. In this study the partial sequences of the nuclear encoded LSU rDNA and the large subunit of ribulose bisphosphate carboxylase (rbcL) of Pseudochattonella isolated from Wellington Harbour indicate that it is similar to P. verruculosa, while sequences of mitochondrial encoded COI, are similar to those of Pseudochattonella farcimen. As with P. farcimen, the Wellington Pseudochattonella lacked violaxanthin, lutein and anteroxanthin, three pigments detected only in P. verruculosa. The Wellington isolate also contains zeaxanthin which is absent in P. farcimen. Among all Pseudochattonella, cells of the Wellington isolate are the most variable in terms of both size and shape. Mucocysts of the Wellington Pseudochattonella also have the greatest degree of variation – from small, ‘bullet’-shape to large oval, oblong or ‘sausage’-like. In the sexual reproduction phase two gametes of the Wellington isolate fuse to form a zygote which gives rise to a large multi-nucleate cell. At times two or more of these large multi-nucleate cells fuse further to form a ‘massive’, plasmodium-like aggregate (up to 200 μm long). Positive feeding and toxicity tests on rotifers confirmed that the Wellington Pseudochattonella is cytotoxic and probably also contributed to the May 2010 fish kills. As molecular phylogenies do not conclusively support the separation of the Wellington Harbour Pseudochattonella from P. verruculosa or P. farcimen, it is tentatively named as Pseudochattonella cf. verruculosa.     

Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates

We employed a cross-linked enzyme aggregate (CLEA) method to immobilize formate dehydrogenase (FDH) from Candida boidinii. The optimal conditions for the preparation of CLEAs were determined by examining effects of various parameters: the nature and amount of cross-linking reagent, additive concentration, cross-linking time, and pH during CLEA preparation. The recovered activities of CLEAs were significantly dependent on the concentration of glutaraldehyde; however, the recovered activity was not severely influenced by the content of dextran polyaldehyde as a mild cross-linker. Bovine serum albumin (BSA) was also used as a proteic feeder and enhanced the activity recovery by 130%. The highest recovered activity of CLEA was 18% for formate oxidation reaction and 25% for CO₂ reduction reaction. The residual activity of CLEA prepared with dextran polyaldehyde (Dex-CLEA) was over 95% after 10 cycles of reuse. The thermal stability of Dex-CLEA was increased by a factor of 3.6 more than that of the free enzyme. CLEAs of FDH could be utilized efficiently for both NADH regeneration and CO₂ reduction.     

Environmental Enhancement Through Use of Recycled Aggregate Concrete in a Two-Stage Mixing Approach

As a high priority of waste management and recycling by the Hong Kong government, Recycled Aggregate (RA) has been used in various construction applications, mainly as sub-grade, roadwork, and unbound materials. However, higher-grade applications are rare. The major barrier encountered is the variation of quality within RA, which causes lower strength, resulted from crystallization of Recycled Aggregate Concrete (RAC). Therefore, the objective of this study is to examine the crystallization of RAC in a Two-Stage Mixing Approach. Following are the five areas of interest: (i) investigate the waste problems in construction activities; (ii) examine the crystal development on the hydration of cement paste; (iii) develop a two-stage mixing approach (TSMA) for improving the performance of RAC; (iv) explore the crystallization of TSMA in comparison with the Normal Mixing Approach (NMA) through use of Differential Scanning Calorimetry (DSC); and (v) verify the results obtained from DSC analysis with those obtained from compressive strength testing. This study adopted 0, 20, and 100% RA substitution in virgin aggregate and measured by DSC and compressive strength on both TSMA and NMA. TSMA uses only half the water for mixing, forming a thin layer of cement slurry on the surface of RA that will permeate into the porous old cement mortar and fill old cracks and voids in the pre-mix process. The results from DSC analysis clearly demonstrated that TSMA can give a better crystallization of CaO·SiO₂·H₂O [CSH] and Ca(OH)₂[CH]. The optimal situation occurs on 20% RA substitution in virgin aggregate, balancing the advantages of each, a finding supported by the results from compressive strength testing. Therefore, TSMA is a superior methodology and opens a wider application for the use of RAC.     

Variables of Microbial Response in Natural Soil Aggregates for Soil Characterization in Different Fluvial Land Shapes

The main objective of this study was to determine changes in microbial response in natural soil aggregates for soil characterization in different fluvial land shapes. This study was carried out in fluvial lands formed on accumulated sediment depositions carried by K?z?l?rmak River. The majority soils of the study area were classified as Typic Ustifluvent and Typic Haplustept in Soil Taxonomy. It was found that macroaggregates (especially >6300 μm and 2000–4750 μm diameters) of all soil samples were higher than microaggregate of soils. In addition, it was determined that the C_org content varies between 0.41–0.91% in soil samples. C_mic content was also found higher level in aggregates involved <250 and 250–425 μm diameters as compared to other aggregate size classes. Moreover, we detected that C_org:C_mic ratio was much higher in macroaggregates than in microaggregate fractions. BR levels were also greater in macroaggregates of >6,300, 4,750–6,300 and 2,000–4,750 μm than in the other macroaggregates sizes and microaggregates. Consequently, macroaggregates have relatively more C_org level than the C_org level in microaggregates, even if the absolute values of C_mic were the lower. This study thus evidenced contrasting microbial habitats and their response in different soil aggregate size formed in various developed soils.