Several novel methods for immobilization of enzymes, microbial cells and organelles

Two novel methods--"photo-crosslinkable resin prepolymer method" and "urethane prepolymer method"--have been developed in our laboratory. These methods have the following advantages : 1) Prepolymers of desired properties, such as optional chain length, hydrophilicity or hydrophobicity, and ionic character etc., can be used for entrapment of biocatalysts : (2) preparation of gel-entrapped biocatalysts can be easily achieved under very mild conditions. Photo-crosslinked gels are conveniently obtained by several minutes illumination with near-UV light, of a mixture of liquid prepolymers having photo-sensitive functional groups, an appropriate sensitizer and the solution or suspension of biocatalyst. Formation of polyurethane gels is completed by only mixing water-miscible urethane prepolymers with the aqueous solution or suspension of biocatalyst. The biocatalysts entrapped by these methods are useful for a variety of purposes.     

Adzuki bean: A new resource of biocatalyst for asymmetric reduction of aromatic ketones with high stereoselectivity and substrate tolerance

A new resource of biocatalyst for asymmetric reduction of aromatic ketones has been discovered for the first time from a common plant seed, adzuki bean, i.e. Phaseolus angularis (Willd.) W.F. Wight. The study investigated the best methods to prepare the biocatalyst and its ability to reduce ketones. Our results indicated that the biocatalyst from adzuki bean could reduce various aromatic ketones at relatively high concentrations (e.g. 100 mM), exhibiting excellent stereoselectivity (>98% e.e.). In addition, it was found that NADPH acts as the reducing cofactor, which can be regenerated by the crude enzyme system itself using glucose as an auxiliary substrate.     

Degradation of carbazole,dibenzothiophene and polyaromatic hydrocarbons by recombinant <Emphasis Type="Italic">Rhodococcus</Emphasis> sp.

Objectives

With the view of designing a single biocatalyst for biorefining, carbazole dioxygenase was cloned from Pseudomonas sp. and expressed in Rhodococcus sp.

Results

The recombinant, IGTS8, degraded both carbazole and dibenzothiophene at 400 mg/l in 24 h. Maximum carbazole degradation was in 1:1 (v/v) hexadecane/aqueous phase. Anthracene, phenanthrene, pyrene, fluoranthene and fluorine were also degraded without affecting the aliphatic component.

Conclusions

Recombinant Rhodococcus sp. IGTS8 can function as a single biocatalyst for removing major contaminants of fossil fuels viz. dibenzothiophene, carbazole and polyaromatic compounds.

     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.     

Particle-bound phytochrome: Association with a ribonucleoprotein fraction from Cucurbita pepo L.

Summary In the absence of ethylenediaminetetraacetic acid (EDTA) and added Mg²⁺, the phytochrome, RNA, protein, cytochrome c oxidase and NADPH-cytochrome c reductase in 20000 x g pellets from hypocotyl hooks of red-irradiated Cucurbita seedlings are more or less coincident in a single, broad band on linear sucrose gradients. The inclusion of 3 mM EDTA in the extraction, resuspension and gradient media has three major effects: (a) The phytochrome profile splits into two main bands; (b) the main RNA population shifts to a sharp peak which co-sediments with the lighter phytochrome band at 31S; (c) the main NADPH-cytochrome c reductase peak shifts to a lower density. This indicates that the EDTA dissociates a rough-endoplasmic-reticulum fraction into separate membrane and ribonucleoprotein (RNP) components, and that part of the phytochrome is associated with the latter. The 31S RNP fraction is 35–40% RNA, has a 260/235 nm absorption ratio of 1.36 and the RNA dissociates into small fragments in sodium dodecyl sulfate. More than 90% of the phytochrome and RNA in the isolated 31S fraction becomes pelletable upon the addition of 10 mM Mg²⁺. Higher Mg²⁺ levels release the phytochrome and some of the other protein present from the RNA which remains pelletable. The data indicate that the 31S RNP fraction may be degraded ribosomal material with extraneously bound protein, including phytochrome. Several aspects of phytochrome-binding to particulate fractions which have been reported in the literature are consistent with an interaction of P_fr with ribosomal material—degraded or otherwise.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - RNase ribonuclease - RNP ribonucleoprotein - SDS sodium dodecyl sulfate     

Competing Risks Data Analysis with High-dimensional Covariates:An Application in Bladder Cancer

Analysis of microarray data is associated with the methodological problems of high dimension and small sample size. Various methods have been used for variable selection in highdimension and small sample size cases with a single survival endpoint. However, little effort has been directed toward addressing competing risks where there is more than one failure risks. This study compared three typical variable selection techniques including Lasso, elastic net, and likelihood-based boosting for high-dimensional time-to-event data with competing risks. The performance of these methods was evaluated via a simulation study by analyzing a real dataset related to bladder cancer patients using time-dependent receiver operator characteristic(ROC) curve and bootstrap.632+ prediction error curves. The elastic net penalization method was shown to outperform Lasso and boosting. Based on the elastic net, 33 genes out of 1381 genes related to bladder cancer were selected. By fitting to the Fine and Gray model, eight genes were highly significant(P 0.001). Among them, expression of RTN4, SON, IGF1 R, SNRPE, PTGR1, PLEK, and ETFDH was associated with a decrease in survival time, whereas SMARCAD1 expression was associated with an increase in survival time. This study indicates that the elastic net has a higher capacity than the Lasso and boosting for the prediction of survival time in bladder cancer patients.Moreover, genes selected by all methods improved the predictive power of the model based on only clinical variables, indicating the value of information contained in the microarray features.     

严重急性呼吸道综合征冠状病毒疫苗研究现状

冠状病毒被认为是新近爆发的严重急性呼吸道综合征的病原体.迄今公共数据库中已发表了不同国家和地区分离的12个SARS病毒株基因组完整序列.突起蛋白是冠状病毒的主要抗原,包含许多抗原决定簇.SARS冠状病毒突起蛋白的相对保守性为有效疫苗的开发奠定了很好的研究基础.灭活或减毒的冠状病毒疫苗存在一定的局限性和危险性.开展基因工程疫苗和核酸疫苗的制备研究以及相关候选疫苗的联合应用研究将是一个切实可行的途径.     

Biotechnological applications of Candida antarctica lipase A: State-of-the-art

The yeast Candida antarctica produces two different lipases, lipases A and B. While lipase B (CAL-B) is probably the mostly employed hydrolase in the biocatalysis field, the use of the lipase A (CAL-A) has been rather scarce and consequently its tridimensional structure has not been elucidated yet. However, CAL-A is a useful biocatalyst with many different applications that have been described especially in the last few years. Its attractiveness results from its unique features among hydrolases: the high thermostability, allowing operation at T > 90 °C; the ability to accept tertiary and sterically hindered alcohols, which has recently been attributed to the existence of a specific aminoacidic sequence in the active site; the sn-2 recognition in hydrolysis of triglycerides; the selectivity towards trans-fatty acids; the stability in the acidic pH range. Furthermore, it is considered to be an excellent biocatalyst for the asymmetric synthesis of amino acids/amino esters, due to its chemoselectivity towards amine groups. Considering all these aspects, in the present review, the origin, the properties and the applications of the CAL-A are briefly described and discussed, pointing out the unique characteristics of this biocatalyst.     

Intermembrane protein transfer. Band 3, the erythrocyte anion transporter, transfers in native orientation from human red blood cells into the bilayer of phospholipid vesicles

Band 3, the erythrocyte anion transporter, has been shown to transfer between human erythrocytes and sonicated vesicles (Newton, A. C., Cook, S. L., and Huestis, W. H. (1983) Biochemistry 22, 6110-6117). Functional band 3 becomes associated with dimyristoylphosphatidylcholine vesicles incubated with human red blood cells. Proteolytic degradation patterns reveal that the transporter is transferred to the vesicles in native orientation. In erythrocytes, native band 3 is degraded on the exoplasmic membrane face by chymotrypsin and on the cytoplasmic surface by trypsin (Cabantchik, Z. I., and Rothstein, A. (1974) J. Membr. Biol. 15, 227-248; Jennings, M. L., Anderson, M. P., and Monaghan, R. (1986) J. Biol. Chem. 261, 9002-9010). Band 3 in intact protein-vesicle complexes is degraded by exogenous chymotrypsin but not by trypsin. In contrast, trypsin entrapped in the lumen of the vesicles proteolyses the vesicle-bound band 3 quantitatively. Band 3 remaining in the membranes of vesicle-treated cells and in cell fragments is not degraded detectably by vesicle-entrapped trypsin. These observations indicate that band 3 is unlikely to transfer between cell and vesicle membranes via a water-soluble form or to adhere nonspecifically to the vesicle surface; the aqueous contents of vesicles and cells (or membrane fragments) are not pooled during cell-vesicle incubations, hence no cell-vesicle fusion occurs; and the band 3 associated with the sonicated vesicle fraction is inserted in the vesicle bilayer in native orientation, with its cytoplasmic segment contacting the aqueous contents of the vesicle lumen.     

Development of a magnetic biocatalyst useful for the synthesis of ethyloleate

Candida antarctica Lipase B was successfully immobilized on magnetite (Fe₃O₄) nanoparticles functionalized with chitosan and glutaraldehyde. The obtained magnetic catalyst was characterized and its performance was evaluated in solvent-free synthesis of ethyl oleate at room temperature. The performance of this biocatalyst was compared with the commercial Novozym 435, as a tool to estimate the efficiency of immobilization. It was found that using 33 mg of the biocatalyst it was possible to reach almost the same activity that was obtained using 12 mg of Novozym 435. Furthermore, this new biocatalyst presents the advantages of not being degraded by short alcohols, being easily recovered from the reaction media by magnetic decantation, and low fabrication cost. The possibility of reutilization was also studied, keeping a significant activity up to eight cycles. A special sampling protocol was also developed for the multiphasic reaction system, to assure accurate results. This novel biocatalyst is an interesting alternative for potential industrial applications, considering the above-mentioned advantages.     

Taking the sting out of darting: Risks,restraint drugs and procedures for the chemical restraint of Southern Hemisphere otariids

The need to manage otariid populations has necessitated the development of a wide range of capture methods. Chemical restraint by remote drug delivery (i.e., darting) is a highly selective method that can be used to facilitate otariid capture in a range of scenarios, when other methods may be impracticable. However, the risks associated with darting otariids are not widely known and guidelines necessary to promote and refine best practice do not exist. We review the risks associated with darting and in light of our findings, develop darting guidelines to help practitioners assess and minimize risks during capture, anesthesia and recovery. Published studies reveal that mortalities associated with darting predominantly result from complications during anesthetic maintenance (e.g., prolonged respiratory depression, apnea, or hyperthermia), rather than from complications during capture or recovery. In addition to monitoring vital signs and proper intervention, the risk of irreversible complications during anesthesia can be reduced by administering drug doses that are sufficient to enable the capture and masking of animals, after which anesthetic depth can be regulated using gas anesthesia.     

Demonstration of glycoprotein antigens associated with stomach tumors

After precipitation of the glycoproteins from gastric tumor tissues by caprylic acid, immunochemical methods were applied to the research of specific gastric tumor antigens; this precipitation process by caprylic acid was less aggressive than the use of urea or proteases. The purification of a fraction associated with tumor tissue has been carried out by affinity chromatography with a specific antiserum immobilized on Sepharose. The obtained fraction contains 4 proteic antigens. One of them is common with all the gastric extracts. Neither CEA, nor alpha-foetoprotein has been detected in this fraction. The corresponding antiserum seems to show a tumor specificity, whereas tissue specificity has to be demonstrated.     

Biocatalytic processes for the production of fatty acid esters

A comparative study of the oleyl oleate production using conventional and enzymatic catalysts has been carried out. The present paper describes the flow diagrams for these processes and compares operation conditions for batch reaction and the downstream proceedings. In addition, continuous and batch processes have been studied. In order to compare the different processes three major points are considered: product properties improvements, operation improvements and improvements on safer and environmental aspects. Experience has shown that biocatalyst is in favour only when most of these issues are far positive for biocatalyst. Enzymatic catalysts involve simpler processes carried out under milder reaction conditions.     

Orofacial clefts, parental cigarette smoking, and transforming growth factor-alpha gene variants.

Results of studies to determine whether women who smoke during early pregnancy are at increased risk of delivering infants with orofacial clefts have been mixed, and recently a gene-environment interaction between maternal smoking, transforming growth factor-alpha (TGFa), and clefting has been reported. Using a large population-based case-control study, we investigated whether parental periconceptional cigarette smoking was associated with an increased risk for having offspring with orofacial clefts. We also investigated the influence of genetic variation of the TGFa locus on the relation between smoking and clefting. Parental smoking information was obtained from telephone interviews with mothers of 731 (84.7% of eligible) orofacial cleft case infants and with mothers of 734 (78.2%) nonmalformed control infants. DNA was obtained from newborn screening blood spots and genotyped for the allelic variants of TGFa. We found that risks associated with maternal smoking were most elevated for isolated cleft lip with or without cleft palate, (odds ratio 2.1 [95% confidence interval 1.3-3.6]) and for isolated cleft palate (odds ratio 2.2 [1.1-4.5]) when mothers smoked > or =20 cigarettes/d. Analyses controlling for the potential influence of other variables did not reveal substantially different results. Clefting risks were even greater for infants with the TGFa allele previously associated with clefting whose mothers smoked > or =20 cigarettes/d. These risks for white infants ranged from 3-fold to 11-fold across phenotypic groups. Paternal smoking was not associated with clefting among the offspring of nonsmoking mothers, and passive smoke exposures were associated with at most slightly increased risks. This study offers evidence that the risk for orofacial clefting in infants may be influenced by maternal smoke exposures alone as well as in combination (gene-environment interaction) with the presence of the uncommon TGFa allele.     

Accepting space radiation risks

The human exploration of space inevitably involves exposure to radiation. Associated with this exposure are multiple risks, i.e., probabilities that certain aspects of an astronaut’s health or performance will be degraded. The management of these risks requires that such probabilities be accurately predicted, that the actual exposures be verified, and that comprehensive records be maintained. Implicit in these actions is the fact that, at some point, a decision has been made to accept a certain level of risk. This paper examines ethical and practical considerations involved in arriving at a determination that risks are acceptable, roles that the parties involved may play, and obligations arising out of reliance on the informed consent paradigm seen as the basis for ethical radiation risk acceptance in space.     

Highlights in Biocatalysis – Historical Landmarks and Current Trends

Biocatalysis has ancient roots, yet it is developing into a key tool for synthesis in a wide range of applications. Important events in the history of enzyme technology from the 19^th century onwards are highlighted. Considering the most relevant progress steps, the production of penicillanic acid and the impact of genetic engineering are traced in more detail. Applied biocatalysis has been defined as the application of a biocatalyst to achieve a desired conversion selectively, under controlled, mild conditions in a bioreactor. Biocatalysts are currently used to produce a wide range of products in the fields of food manufacture (such as bread, cheese, beer), fine chemicals (e.g., amino acids, vitamins), and pharmaceuticals (e.g., derivatives of antibiotics). They not only provide access to innovative products and processes, but also meet criteria of sustainability. In organic synthesis, recombinant technologies and biocatalysts have greatly widened the scope of application. Examples of current applications and processes are given. Recent developments and trends are presented as a survey, covering new methods for accessing biodiversity with new enzymes, directed evolution for improving enzymes, designed cells, and integrated downstream processing.     

Oxidation of ethanol by cumene hydroperoxide in the presence of cytochrome P-450 LM2 and hemoglobin

Ethanol oxidation by cumene hydroperoxide (CHP) with participation of cytochrome P-450 LM-2 (pH 7.4) and hemoglobin (pH 7.0) was studied at 37 degrees C in phosphate buffer. Both hemoproteins form complexes with CHP that are decomposed with the liberation of the RO2., RO. and HO. radicals, thus initiating the chain oxidation of ethanol. Ethanol oxidation catalyzed by cytochrome P-450 LM-2 and hemoglobin occurs only through a radical formation and is competitively inhibited by the radical scavenging agents, e.g., 1-naphthol, thiourea, mannitol and dimethylsulfoxide (DMSO). The values of effective inhibition constants were determined for all antioxidants whose activity decreases in the following order: 1-naphthol greater than thiourea greater than mannitol greater than DMSO. The non-inhibited oxidation of ethanol in "CHP-hemoproteins" systems is characterized by low ethanol conversion because of bimolecular termination of radicals and biocatalyst destruction.     

Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial- lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.     

Immobilization of thermophilic microbial cells in crude egg white

Summary Cells of thermophilic archaebacterium Caldariella acidophila have been trapped in chicken egg white by co-crosslinking with glutaraldehyde. The activity of cytosolic -galactosidase in trapped cells has been followed by varying temperature, pH, organic solvents. A biocatalyst with magnetic properties has been prepared by including magnetite in the resin.     

Optimizing lipases and related enzymes for efficient application

Although numerous reactions have been performed using lipases and related enzymes (e.g. esterases and phospholipases), it is still a challenge to identify the most suitable biocatalyst and best reaction conditions for an efficient application. Frequently used methods such as immobilization and optimization of the reaction medium cannot be transferred from one reaction system or substrate to another. However, in the past few years, rational protein design and directed evolution have emerged as efficient alternative methods to optimize biocatalytic reactions.