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Biomedical subjects

P Linko

Publications and source records attributed to P Linko.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of the cellulose-binding ability of Geotrichum sp. M111 cells and its application to dehydration of the distilled waste of sweet potato shouchu.

The cellulose-binding ability of Geotrichum sp. M111 cells was investigated by the micro-tube method which gives an indication of the binding ability of M111 cells. The optimum pH value and temperature were 3-7 and below 50 degrees C, respectively, from measurement of the aggregation height for a mixture of cellulose powder and M111 cells. The binding constant of 0.3% for M111 cells to cellulose powder was obtained in a 20 mm citrate buffer of pH 5.0 at 30 degrees C. Aggregation was inhibited by such surfactants as sodium dodecylsulfate. The binding ability of M111 cells to cellulose fiber disappeared after a treatment with Driselase or Pronase E. This suggests that the binding ability might be related to the cell surface proteins. The dehydration rate of the distilled waste of sweet potato shouchu was accelerated by the addition of M111 cells. The analysis of dehydration by a linear viscoelastic model suggests that the acceleration effect might have been due to the space increase between cellulose fibers with the cell addition.

Cellulose↗
PubMed2000

Crystal transformation from anhydrous alpha-maltose to hydrous beta-maltose and from anhydrous trehalose to hydrous trehalose.

Anhydrous sugars such as maltose and trehalose are useful for making dry powder of foods and liquids. The crystal-transformation rate of maltose and trehalose were investigated under humid conditions and by kneading. The enthalpy for solubilization was 7.0 kJ/mol for the anhydrous maltose. The crystal-transformation rate of anhydrous alpha-maltose to hydrous beta-maltose depended on the temperature at 75% humidity. However, that of anhydrous trehalose did not depend on the temperature, and transformation was very rapid. An anomeric change to maltose and no such change to trehalose might have caused this. The activation energy of crystal transformation was 79 kJ/mol for maltose and zero for trehalose. The rate of crystal transformation of anhydrous maltose while kneading depended on the purity of the anhydrous alpha-maltose and the amount of water present. This crystal transformation rate fitted the Avrami equation.

Crystallization↗
PubMed2000

Refolding of denatured/reduced lysozyme at high concentration with diafiltration.

Refolding of reduced and denatured protein in vitro has been an important issue for both basic research and applied biotechnology. Refolding at low protein concentration requires large volumes of refolding buffer. Among various refolding methods, diafiltration is very useful to control the denaturant and red/ox reagents in a refolding solution. We constructed a refolding procedure of high lysozyme concentration (0.5-10 mg/ml) based on the linear reduction of the urea concentration during diafiltration under oxygen pressure. When the urea concentration in the refolding vessel was decreased from 4 M with a rate of 0.167 M/h, the refolding yields were 85% and 63% at protein concentrations, 5 mg/ml and 10 mg/ml, respectively, after 11 h. This method gave a high productivity of 40.1,microM/h of the refolding lysozyme. The change in refolding yields during the diafiltration could be simulated using the model of Hevehan and Clark.

Animals↗
PubMed2000

Cyclodextrin encapsulation to prevent the loss of l-menthol and its retention during drying.

The taste and flavor of spray-dried powdered products are the most important quality factors. In the present study, molecular encapsulation in cyclodextrin was applied to prevent the loss of a hydrophobic flavor compound (l-menthol) during the drying of a droplet. beta-Cyclodextrin appeared to be a better encapsulant for menthol than alpha- and gamma-cyclodextrin. The retention of menthol increased with increasing concentration of both cyclodextrin and maltodextrin. A simple mathematical model is proposed for estimating the flavor retention. The theoretical results by this model estimated well the final retention of menthol encapsulated in a blend of beta-cyclodextrin and maltodextrin.

Cyclodextrins↗
PubMed1999

Applying neural networks as software sensors for enzyme engineering.

The on-line control of enzyme-production processes is difficult, owing to the uncertainties typical of biological systems and to the lack of suitable on-line sensors for key process variables. For example, intelligent methods to predict the end point of fermentation could be of great economic value. Computer-assisted control based on artificial-neural-network models offers a novel solution in such situations. Well-trained feedforward-backpropagation neural networks can be used as software sensors in enzyme-process control; their performance can be affected by a number of factors.

Algorithms↗
PubMed1998

Neural networks as "software sensors" in enzyme engineering.

Industrial applications of enzyme technology are rapidly increasing. On-line control of enzyme production processes, however, is difficult owing to the uncertainties typical of biological systems and to the lack of suitable on-line sensors for key process variables and quality attributes. We demonstrate that well-trained feedforward backpropagation neural networks with one hidden layer can be employed to overcome such problems with no need for a priori knowledge of the relationships of the process variables involved. Neural network programs were written in Microsoft Visual C++ for Windows and implemented in a personal computer. The goodness of fit of the trained neural network to the reference data was determined by the coefficient of determination, R2. Case studies of beta-galactosidase, glucoamylase, lipase, and xylanase production processes will be used as examples.

Algorithms↗
PubMed1998

Biodegradable products by lipase biocatalysis.

The interest in the applications of biocatalysis in organic syntheses has rapidly increased. In this context, lipases have recently become one of the most studied groups of enzymes. We have demonstrated that lipases can be used as biocatalyst in the production of useful biodegradable compounds. A number of examples are given. 1-Butyl oleate was produced by direct esterification of butanol and oleic acid to decrease the viscosity of biodiesel in winter use. Enzymic alcoholysis of vegetable oils without additional organic solvent has been little investigated. We have shown that a mixture of 2-ethyl-1-hexyl esters can be obtained in a good yield by enzymic transesterification from rapeseed oil fatty acids for use as a solvent. Trimethylolpropane esters were also similarly synthesized as lubricants. Finally, the discovery that lipases can also catalyze ester syntheses and transesterification reactions in organic solvent systems has opened up the possibility of enzyme catalyzed production of biodegradable polyesters. In direct polyesterification of 1,4-butanediol and sebacic acid, polyesters with a mass average molar mass of the order of 56,000 g mol-1 or higher, and a maximum molar mass of about 130,000 g mol-1 were also obtained by using lipase as biocatalyst. Finally, we have demonstrated that also aromatic polyesters can be synthesized by lipase biocatalysis, a higher than 50,000 g mol-1 mass average molar mass of poly(1,6-hexanediyl isophthalate) as an example.

Biodegradation, Environmental↗
PubMed1998

Conformational change in the activation of lipase: an analysis in terms of low-frequency normal modes.

The interfacial activation of Rhizomucor miehei lipase (RmL) involves the motion of an alpha-helical region (residues 82-96) which acts as a "lid" over the active site of the enzyme, undergoing a displacement from a "closed" to an "open" conformation upon binding of substrate. Normal mode analyses performed in both low and high dielectric media reveal that low-frequency vibrational modes contribute significantly to the conformational transition between the closed and open conformations. In these modes, the lid displacement is coupled to local motions of active site loops as well as global breathing motions. Atomic fluctuations of the first hinge of the lid (residues 83-84) are substantially larger in the low dielectric medium than in the high dielectric medium. Our results also suggest that electrostatic interactions of Arg86 play an important role in terms of both the intrinsic stability of the lid and its displacement, through enhancement of hinge mobility in a high dielectric medium. Additional calculations demonstrate that the observed patterns of atomic fluctuations are an intrinsic feature of the protein structure and not dependent on the nature of specific energy minima.

Binding Sites↗
PubMed1996

Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity.

Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critical amino acids that determine the specificity of human amino acids that determine the specificity of human P450 2C19, we constructed chimeras of p450 2C9 replacing various proposed substrate binding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C19 and then replaced individual residues of P450 2C9 by site-directed mutagenesis. The 339 NH2-terminal amino acid residues (SRS-1-SRS-4) and amino acids 160-383 (SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P450 2C9. In contract, the COOH terminus of P450 2C19 (residues 340-490 including SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and residues 292-383 (part of SRS-4 and SRS-5) conferred only modest increases in activity. A single mutation Ile99 --> His increased omeprazole 5-hydroxylase to approximately 51% of that of P450 2C19. A chimera spanning residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase activity which was dramatically enhanced by the mutation Ile99 --> His. A combination of two mutations, Ile99 --> His and Ser200 --> Pro, converted P450 2C9 to an enzyme with a turnover number of omeprazole 5-hyrdroxylation, which resembled that of P450 /c19. Mutation of Pro221 --> Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids 220 and 221 are in the F-G loop and outside any known SRS. Mutation of these three amino acids did not confer significant S-mephenytoin 4'-hydroxylase activity to P450 2C9, although chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450 2C19 exhibited activity toward this substrate. Our results thus indicate that amino acids 99, 220, and 221 are key residues that determine the specificity of P450 2C19 for omeprazole.

Aryl Hydrocarbon Hydroxylases↗
PubMed1993

Expression of CYP1A1 and CYP1A2 genes in human liver.

Immunoblot analysis of human livers using a monospecific antibody to rat CYP1A2 section demonstrated that the expression of CYP1A2 protein is highly variable in human liver. Quantitative PCR analysis was then employed to examine the interindividual variability of both CYP1A1 and CYP1A2 mRNAs in human liver. Hepatic content of CYP1A2 mRNA correlated significantly with levels of CYP1A2 protein as analysed by immunoblot analysis (r = 0.58; p < 0.01). CYP1A2 mRNA content varied > 40-fold among individuals while CYP1A1 content varied > 20-fold. CYP1A2 mRNA was higher than CYP1A1 mRNA (approximately two to 30-fold) in livers of different individuals. The individual with the highest CYP1A1 and CYP1A2 mRNA amounts was a current smoker, but mRNA expression in two other smokers was within the range observed among nonsmokers. The expression of the two CYP1A mRNAs correlated highly (r = 0.72; p < 0.0005) when smokers were included, but the correlation was less significant (r = 0.62; p < 0.05) in nonsmokers. We amplified a full-length CYP1A2 cDNA clone by PCR from a liver which expressed extremely low amounts of CYP1A2 protein. Sequence analysis indicated that exon 4 was missing in this clone, but no other sequence changes were found. PCR analysis demonstrated that both the normally spliced mRNA and abnormally spliced mRNA could be detected in all human livers examined, but the normally spliced mRNA was more abundant than the splice variant. Therefore, sequence changes in the coding region of CYP1A2 did not account for the poor expression of CYP1A2 in this individual.

Adult↗
PubMed1992

A single amino acid mutation (Ser180----Cys) determines the polymorphism in cytochrome P450g (P4502C13) by altering protein stability.

Cytochrome P450g is polymorphic in the male rat. This polymorphism is characterized by a 20-40-fold difference in the hepatic content of P450g in the two phenotypes. Sequencing of cDNAs from high (+g) and low (-g) phenotype rats has shown that the low phenotype is due to a defective mRNA containing nine base mutations encoding 7 amino acid substitutions. To determine the role of these structural changes in the phenotypic expression of P450g, we altered each of these residues by site-directed mutagenesis in the present studies and expressed the normal and mutant cDNAs in Saccharomyces cerevisiae. P450+g protein was expressed at a level 4-6-fold higher than that of P450-g in yeast cells, despite the presence of identical mRNA levels. This difference in protein expression approaches the difference seen in the rat. A single amino acid change from Ser180 in P450+g to Cys in P450-g, in a highly conserved region in the P4502C subfamily, was found to be solely responsible for the phenotypic differences in expression of P450g. Protein half-life studies demonstrated that this mutation increases the degradation of P450g. This is the first example of a single amino acid substitution which alters the phenotypic expression of a P450 protein by affecting its stability.

Amino Acid Sequence↗
PubMed1992

Object-oriented fuzzy expert system for on-line diagnosing and control of bioprocesses.

An object-oriented fuzzy expert system to support on-line control of an automated fermentation plant is described. The major elements of the system consist of a fuzzy inference engine, a database, a knowledge base, and an expression evaluater. The expression evaluater calculates specific rates for growth, and substrate and product formation at different physiological states during the cultivation from the measured data. The specific rates are then compared with the standard target rates stored in the database. If differences outside the set tolerances were observed, the inference engine analyses the reasons for the faults on the basis of the knowledge represented in the form of a knowledge network and fuzzy membership functions of the process variables. The fuzzy expert system was developed on the basis of a shell constructed by using the object oriented Smalltalk/V Mac programming environment, with Lac-tobacillus casei lactic acid fermentation as the example of process application.

Biotechnology↗
PubMed1992

Fuzzy reasoning system for fault diagnosis of physiological activities in a cultivating process.

Aiming at development of a system which supports cultivating operations, a method to diagnose physiological activities in a cultivating process is presented, and a fuzzy expert system for diagnosing Lactobacillus casei cultivating process is implemented in this paper. This system can calculate specific rates of cell growth, substrate consumption, and product formation with measuring cell mass concentration, substrate concentration, and product concentration by using a turbidity sensor and HPLC. A database is implemented, where standard curves on specific rates representing characteristics of microorganisms are stored according to normalized substrate consumption. Comparing the calculated specific rates with standard values derived from the database, the system diagnoses physiological activities of the microorganisms. As a case study, a knowledge base for diagnosing lactic acid production process is implemented. The use of fault diagnosis on pH malfunctions by the expert system proves its reasonable performance.

Biotechnology↗
PubMed1991

Disposition of 2,3,7,8-tetrabromodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat: biliary excretion and induction of cytochromes CYP1A1 and CYP1A2.

The biologic activity and pharmacokinetic properties of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) are similar to those of the chlorinated congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Metabolism of both compounds appears to be rate-limiting for excretion, which is primarily via the feces. Therefore, the biliary elimination of TBDD and TCDD was examined as an indirect assessment of metabolism. Male F344 rats were anesthetized with pentobarbital, and 1 nmol/kg [3H]TBDD or [3H]TCDD was administered iv. Bile was collected for up to 8 hr while rats were maintained under anesthesia. The rate of biliary excretion of radioactivity was slightly greater for TCDD than TBDD (10% vs 7% in 5 hr). All biliary radioactivity was attributable to metabolites. High pressure liquid chromatographic (HPLC) profiles of biliary radioactivity were similar for [3H]TBDD and [3H]TCDD. To determine if pretreatment altered elimination kinetics, a single dose of 100 nmol/kg TBDD or TCDD was administered to rats by oral gavage 3 days prior to iv injection of 1 nmol/kg [3H]TBDD or [3H]TCDD, respectively. Biliary excretion of the radiolabeled dose was quantitatively and qualitatively unaffected by pretreatment despite a twofold increase in hepatic levels of radiolabel in the pretreated animals. Therefore, under these experimental conditions, autoinduction of TCDD and TBDD metabolism did not occur in the rat in vivo at doses which elicited enhanced hepatic uptake. In a second set of studies, the dose-response profiles for induction of cytochromes CYP1A1 and CYP1A2 by TBDD were characterized. The ED50 value for CYP1A1 induction (measured by ethoxyresorufin O-deethylase activity and radioimmunoassay (RIA) was estimated to be 0.8-1.0 nmol/kg, similar to what has been reported for TCDD. Induction of CYP1A2 (RIA) by TBDD appeared to be a more sensitive response over the dose range studied. Finally, comparison of hepatic CYP1A2 induction vs hepatic concentrations of TBDD 3 days following treatment with 10 vs 1 nmol/kg TBDD suggested that induction of CYP1A2 alone may not account for nonlinearities in dioxin disposition exemplified by dose-related increases in the ratio of dioxin concentrations in liver and adipose tissue.

Animals↗
PubMed1991

Neural network programming in bioprocess variable estimation and state prediction.

A neural network program with efficient learning ability for bioprocess variable estimation and state prediction was developed. A 3 layer, feed-forward neural network architecture was used, and the program was written in Quick C ver 2.5 for an IBM compatible computer with a 80486/33 MHz processor. A back propagation training algorithm was used based on learning by pattern and momentum in a combination as used to adjust the connection of weights of the neurons in adjacent layers. The delta rule was applied in a gradient descent search technique to minimize a cost function equal to the mean square difference between the target and the network output. A non-linear, sigmoidal logistic transfer function was used in squashing the weighted sum of the inputs of each neuron to a limited range output. A good neural network prediction model was obtained by training with a sequence of past time course data of a typical bioprocess. The well trained neural network estimated accurately and rapidly the state variables with or without noise even under varying process dynamics.

Algorithms↗