Relative Residual Activity Research Articles

Numerical analysis of polyphenol oxidase inactivation in non-Newtonian characteristic liquid food with high viscosity during radio frequency treatment

Enzymatic browning is one of the main problems in the food industry, especially in juice and jam processing. An efficient approach to reduce enzymatic browning is to inactivate key polyphenol oxidases (PPO) that cause color reactions. Radio frequency (RF) treatment has advantages of volumetric heating and no chemical residue, making it one of the most potential treatments to replace traditional heating for pasteurization. In this study, a finite element model was established using the commercial software, COMSOL, to investigate the feasibility and effectiveness of PPO inactivation in non-Newtonian characteristic liquid food with high viscosity during pilot-scale RF treatments. The PPO was represented by the tyrosinase, and four concentrations of carboxymethylcellulose (CMC) solutions were considered to have high viscosity and non-Newtonian characteristics for liquid food. The results showed that the relative residual enzyme activity rapidly decreased to 1.2% when the target temperature increased to 70 °C during a batch RF enzyme inactivation. Meanwhile, the simulated and experimental trends were consistent with an acceptable RMSE value. The low-concentration CMC solutions (0.5% and 1.0%) with shorter heating times may be more suitable for developing the batch RF enzyme inactivation technology. During continuous-flow RF enzyme inactivation, the F-value increased with an increase in the solution concentration but sharply decreased with an increase in the volumetric flow rate. The exponential function could be used to describe the relationship between the relative residual enzyme activity and the volumetric flow rate of the CMC solution after RF treatment. The optimal volumetric flow rates for reducing the activity of PPO in 0.5%, 1.0%, 1.5%, and 2.0% CMC solutions to 20% were 426.13, 467.87, 552.05, and 566.07 mL/min, respectively. This study can provide valuable information for inactivating enzymes in non-Newtonian characteristic liquid food with high viscosity.

A Recombinant Thermophilic and Glucose-Tolerant GH1 β-Glucosidase Derived from Hehua Hot Spring.

As a crucial enzyme for cellulose degradation, β-glucosidase finds extensive applications in food, feed, and bioethanol production; however, its potential is often limited by inadequate thermal stability and glucose tolerance. In this study, a functional gene (lq-bg5) for a GH1 family β-glucosidase was obtained from the metagenomic DNA of a hot spring sediment sample and heterologously expressed in E. coli and the recombinant enzyme was purified and characterized. The optimal temperature and pH of LQ-BG5 were 55 °C and 4.6, respectively. The relative residual activity of LQ-BG5 exceeded 90% at 55 °C for 9 h and 60 °C for 6 h and remained above 100% after incubation at pH 5.0-10.0 for 12 h. More importantly, LQ-BG5 demonstrated exceptional glucose tolerance with more than 40% activity remaining even at high glucose concentrations of 3000 mM. Thus, LQ-BG5 represents a thermophilic β-glucosidase exhibiting excellent thermal stability and remarkable glucose tolerance, making it highly promising for lignocellulose development and utilization.

Mining and Characterization of Thermophilic Glucose Isomerase Based on Virtual Probe Technology.

Fructose, which is produced by the isomerization of glucose isomerase, is a crucial precursor for the biosynthesis of rare sugars. In this study, thermophilic glucose isomerases (GI) from Caldicellulosiruptor acetigenus (CAGI), Thermoanaerobacter thermocopriae (TTGI), and Thermotoga petrophila (TPGI) were screened from GenBank database by a virtual probe and were successfully expressed in Escherichia coli BL21(DE3). The results of characterization demonstrated that the optimal pH for CAGI and TTGI were 8.0 and were maintained at 80% in a slightly acidic environment. The relative residual activities of CAGI and TTGI were found to be 40.6% and 52.6%, respectively, following an incubation period of 24h at 90 ℃. Furthermore, CAGI and TTGI exhibited superior catalytic performance that their reaction equilibrium both reached only after an hour at 85 ℃ with 200g/L glucose, and the highest conversion rates were 54.2% and 54.1%, respectively. This study identifies competitive enzyme candidates for fructose production in the industry with appreciable cost reduction.

Investigating diverse barley (Hordeum vulgareL.) germplasm for thermostability of β‐amylase: A key player of diastatic power

AbstractBackground and Objectivesβ‐amylase plays a vital role in starch hydrolysis during mashing to obtain fermentable wort for beer production. Since starch gelatinization requires a high temperature, the catalytic activity, and thermostability of β‐amylase are crucial for obtaining high‐quality malt owing to its heat‐labile character. This study aimed to determine the variation in thermostability of β‐amylase among a diverse set of barley genotypes (hulled, hulless, mutants, and wild).FindingsStatistical analysis indicated the significant effect of genotype and temperature on β‐amylase activity. The mutant genotypes had a wider variation for β‐amylase activity followed by hulled, hulless, and wild genotypes. The % relative residual activity was found to be the highest at 55°C for all the genotypes. Among 54 barley genotypes, PL891 (hulless) was reported to have the highest activity at all temperatures followed by BL1714 (hulled) and BL2105 (mutant) which shows the thermostable nature of β‐amylase in these genotypes. The β‐amylase activity was positively correlated with the amount of reducing sugars formed at a 1% level of significance (r = .852), further corroborating the results.ConclusionsThe β‐amylase activity declined after 55°C in the majority of the genotypes depicting its optimum temperature. Few genotypes, namely PL891, BL1714, and BL2105 had been identified that possessed high β‐amylase activity at all temperatures.Significance and Noveltyβ‐amylase with high thermostability may prove more helpful than selecting the genotype with higher β‐amylase levels if one attempts to increase the amount of fermentable sugars in the wort. Therefore, in the current study genotypes identified with high thermostability of β‐amylase may be exploited in the genetic improvement programs for incorporating beneficial traits into low malting efficiency cultivars but possessing high yield and resistance to various stresses.

Transarterial Radioembolization with Yttrium-90 Glass Microspheres: Distribution of Residual Activity and Flow Dynamics during Administration

PurposeTo evaluate the microsphere flow dynamics and residual yttrium-90 (90Y) activity during and after transarterial radioembolization with glass microspheres and to assess the distribution and predilection sites of residual activity in the administration devices. Materials and MethodsIn this laboratory investigation, after 18 consecutive clinical transarterial radioembolization and 4 ex vivo experimental procedures with 90Y glass microspheres, the distribution of residual activity in the administration devices was assessed by activimeter and positron emission tomography (PET)/CT measurements. During ex vivo procedures, microsphere outflow from the administration device was assessed by dynamic scintigraphic measurements. ResultsMean residual activity was 3.4% ± 1.7 (range, 0.9%–8.8%). Calculations showed a negative correlation between relative residual activity and prescribed activity (r = −0.4258, P = .0486) and a positive correlation between absolute residual activity and prescribed activity (r = 0.5345, P = .0104). The main predilection site was the Luer-Lok microcatheter connector. Lower activities were detected in the dose vial. Flow measurements showed that more than 98% of the final injected activity was transferred to the patient with the first 20 mL of saline solution. ConclusionsResidual activity in the standard administration device for glass microsphere radioembolization is considered to be low compared with similar procedures, but is variable. The microsphere flow profile shows an initial peak, resulting in a rapid activity transfer at the beginning of the injection process. The findings may have implications for safe handling of the administration device and for dose calculation of 90Y glass microspheres.

Effects of radio-frequency energy on peroxidase inactivation and physiochemical properties of stem lettuce and the underlying cell-morphology mechanism

This paper investigated the effects of radio-frequency (RF) energy and conventional hot-water blanching (95 °C for 2 min) on the peroxidase (POD) activity, physiochemical properties, and changes in the cellular morphology of stem lettuce. The relative residual POD activity significantly decreased (P < 0.05) from 66.03% to 6.46% with increased RF heating temperature (65 °C–85 °C). The weight loss (3.06%–7.64%), color, texture, relative electrolyte leakage (23.45%–67.90%), and residual vitamin C content (72.22%–16.67%) significantly changed (P < 0.05) with increased RF heating temperature (65 °C–85 °C). Micrographs indicated that the changes in physiochemical property can be attributed to the destruction of cell membranes, loss of cell turgor, reduced rigidity of cell walls, and loose adhesion between adjacent cells. Samples treated by RF heating at 75 °C showed lesser cell damage and better nutrient retention than those treated by hot-water blanching at a similar level of POD inactivation.

Effect of Applied Voltage on the Aggregation and Conformational Changes in Peroxidase Under Electrospray

The application of electrospray to treat enzyme solution can produce electrically charged micro- or nanoparticles. Peroxidase is usually regarded as a typical enzyme indicating quality changes. In this study, we evaluated the effect of voltage on peroxidase inactivation under electrospray, and the structural changes caused by increasing voltages were analyzed. The results showed that peroxidase activity decreased with the increasing applied voltages. The relative residual activity of peroxidase reduced to 7.7% by electrospray applied with the voltage of 25 kV. The alteration of particle size and zeta potential indicated that the applied voltage caused aggregation or dissociation of peroxidase, affecting the activity of the peroxidase. From the analysis of spectra for electrospray treatment and control (no voltage), the former had a significant effect (p < 0.05) on the damage of secondary structure (α-helix) stability and has changed the spatial position of tertiary structure. This study provides a sound basis for the application of electrospray in enzyme inactivation and advanced food processing.

Study on Thermostability of Bacillus Subtilis Lipase by Site-Directed Mutagenesis

BSLA is an extracellular hydrolase secreted by Bacillus subtilis, which is an enzyme at room temperature and its optimum temperature is only 35 °C. Thermal stability is an important indicator for the evaluation of industrial biocatalysts. The industrial increase of reaction temperature can improve the conversion rate, the solubility of the substrate and the possibility of reducing the microbial contamination. Therefore, by improving the heat resistance of BSLA through molecular modification, Adapt to the requirements of industrial production process, has important practical value. The mutant BSLAN174E was constructed by whole plasmid PCR and the thermal stability of the mutant strain was verified by experiments. After the purified BSLA and BSLAN174E enzyme solutions were stored at 55 °C for 5, 20 and 60 min respectively, the relative residual activity of BSLAN174E Respectively 1.6, 2.0, 2.3 times of the original strain BSLA.

Optimization of encapsulation of a microbial laccase enzymatic extract using selected matrices

Abstract The encapsulation of the laccase extract, obtained from Coriolus hirsutus, was investigated using selected matrices, including alginate, alginate-silica and silica sol-gel. Two methods, M1 and M2, were carried out for the hybridization of alginate with silica. The experimental findings indicated that the incorporation of silica into alginate, using M2, resulted by a higher (70%) encapsulation efficiency (EE) for the laccase extract as compared to that for the alginate (59%) alone. Furthermore, the encapsulation of the laccase extract in sol-gel resulted in an enhancement of its catalytic activity, with a concomitant increase (90%) in the EE. The alginate and sol-gel matrices also enhanced the laccase catalytic efficiency as compared to that for the free one, with a kcat of 89.9, 63.7 and 56.9 min−1, respectively. The protein release profiles indicated that the laccase was more effectively confined in the sol-gel and alginate-silica M2 as compared to that for the alginate and alginate-silica M1. In addition, the laccase extract in sol-gel exhibited an enhanced re-usability, with a relative residual enzyme activity of 82.7%, after 5 continuous reaction cycles. The alginate, alginate-silica M2 and sol-gel matrices demonstrated complete storage stability at 4 °C, without any loss in laccase activity after 6 weeks of storage.

Bioconversion of cellulose and simultaneous production of thermoactive exo- and endoglucanases by Fusarium oxysporum

Saccharification of cellulose is a promising method for production of biofuels. However, low bioconversion efficiency of cellulose to soluble sugars is a major challenge. In this study, a cellulolytic strain of Fusarium oxysporum was cultivated on pure cellulosic substrates (avicel, α-cellulose, carboxymethylcellulose and methylcellulose) and conversion efficiency into glucose was investigated. Production of exo- and endoglucanases during the bioconversion process was evaluated. Influence of pH on saccharification of cellulose and enzyme production by F. oxysporum were determined. Highest yield of glucose (1.76 μmol/ml) was obtained from F. oxysporum on methyl cellulose at 192 h under basal conditions. Liberated glucose under optimized condition of pH 6.0 at 96 h of fermentation was 2.12 μmol/ml with maximum production of exo- and endoglucanases (23.70 and 34.72 U/mg protein, respectively). The crude exo- and endoglucanases had optimum activities at pH 8.0, 70 °C and pH 7.0, 50 °C, respectively. The enzymes were stable over pH of 4.0–7.0 with relative residual activity above 60% after 1 h incubation. Exoglucanase activity was enhanced by Ca2+ and Cu2+ at 5 mM and Mg2+ at 10 mM. Endoglucanase activity was greatly enhanced in the presence of Mn2+, Ca2+, Mg2+, Cu2+ and Fe3+ at 5 and 10 mM. Activities of both enzymes were inhibited in the presence of Hg2+ at 5 and 10 mM. Results show that F. oxysporum possessed good cellulolytic enzyme system for efficient conversion of cellulose. Exhibited thermotolerance of exoglucanase with the striking tolerance of endoglucanase to metal ions demonstrate potentials of enzymes for biofuel industry.

Action of a GH115 α-glucuronidase from Amphibacillus xylanus at alkaline condition promotes release of 4-O-methylglucopyranosyluronic acid from glucuronoxylan and arabinoglucuronoxylan.

Glucuronic acid and/or 4-O-methyl-glucuronic acid (GlcA/MeGlcA) are substituents of the main xylans present in hardwoods, conifers, and many cereal grains. α-Glucuronidases from glycoside hydrolase family GH115 can target GlcA/MeGlcA from both internally and terminally substituted regions of xylans. The current study describes the first GH115 α-glucuronidase, AxyAgu115A, from the alkaliphilic organism Amphilbacillus xylanus. AxyAgu115A was active in a wide pH range, and demonstrated better performance in alkaline condition compared to other characterized GH115 α-glucuronidases, which generally show optimal activity in acidic conditions. Specifically, its relative activity between pH 5.0 and pH 8.5 was above 80%, and was 35% of maximum at pH 10.5; although the enzyme lost 30% and 80% relative residual activity after 24-h pre-incubation at pH 9 and pH 10, respectively. AxyAgu115A was also similarly active towards glucuronoxylan as well as comparatively complex xylans such as spruce arabinoglucurunoxylan. Accommodation of complex xylans was supported by docking analyses that predicted accessibility of AxyAgu115A to branched xylo-oligosaccharides. MeGlcA release by AxyAgu115A from each xylan sample was increased by up to 30% by performing the reaction at pH 11.0 rather than pH 4.0, revealing applied benefits of AxyAgu115A for xylan recovery and processing.

High-level expression of a biologically active staphylokinase in Pichia pastoris

ABSTRACTStaphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis. SAK variant of SAKфC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310 mg/L of the culture medium) after 48-hr stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37°C. It was highly active at temperatures 20–37°C and pH ranges of 6.8–9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK were measured as 9,002 and 21,042 U/mg for the nonpurified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720 µg/mL with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6 kDa. It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.

Expression and biochemical characterization of a multifunctional glycosidase from the thermophilic Bacillus licheniformis SR01.

A gene (gkdA) (741 bp) encoding a putative protein of 247 amino acids was cloned from the Bacillus licheniformis SR01. The protein was expressed in Escherichia coli BL21 with a molecular mass estimated by SDS-PAGE of approximately 28.03 kDa and showed a calculating isoelectric point (pI) of 6.42. Structure analysis and function identification showed that the enzyme was a multifunctional glycosidase. Its specific activity was 0.013 U/μg. The recombinant glycosidase showed a maximum activity at 50°C and pH 7.0. It was very stable below 90°C and may have heat activation at higher temperatures. The relative residual activity was still more than 80% after 120 min at pH 5.0-10.0. The enzyme activity was inhibited by Cu2+, Fe2+, Ca2+, Mg2+, Co2+, Li+, SDS and EDTA, activated by Ca2+, and not affected by Mn2+ and K+. Under simulated stomach, and in vitro intestine, conditions, the enzyme retained 80%, and more than 100%, activity, respectively, after incubation for 90 min. The excellent properties of this enzyme, specifically its thermal stability and multifunctional abilities, give it potential application in the field of feed processing and other high-temperature processing industries.

Asymptomatic Dysprothrombinemia (Prothtombin Himi) with p.M380T and p.R431H Shows Severely Reduced Clotting Activity, Moderate Antithrombin Resistance and Severe Thrombomodulin Binding Defect

Background and purpose:Congenital prothrombin (PT) deficiency is categorized into two phenotypes, true hypoprothrombinemia and dysprothrombinemia. A strong discrepancy between observed values from coagulation tests and bleeding severity can be found in some forms of dysprothrombinemia. Some dysprothrombinemias, such as the PT Himi that we reported previously (Morishita E, et al: Blood 1992), are asymptomatic despite low PT activity. PT Yukuhashi disturbs the interaction between thrombin and antithrombin (AT), causing a paradoxical thrombotic phenotype, called "AT resistance (ATR)". To investigate the mechanisms by which patient with PT Himi is asymptomatic, mutant PTs reflecting each of the two different gene sites identified in gene analysis were prepared, and functional analyses of fibrinogen (Fbg) degradation, ATR, and thrombomodulin (TM)-binding capacity were conducted. This study was approved by the ethics committee at Kanazawa University School of Medicine.Case report: The proband was a 26-year-old Japanese woman who had been diagnosed with congenital dysprothrombinemia at the time of first pregnancy, based on PT activity and PT antigen levels of 10% and 88%, respectively. Although cesarean section was performed for the second delivery, abnormal bleeding did not develop. Genomic DNA analysis revealed compound heterozygosity for two missense mutations in the F2gene (c.1139T>C, p.M380T and c.1292G>A, p.R431H).Methods: We established stable transformants of Chinese hamster ovary cells that expressed wild-type and two mutant recombinant PTs (rPTs), M380T PT and R431H PT. Conditioned media of stable transformants expressing rPTs in serum-free medium were collected, concentrated, and stored at -80°C until use. 1) Fbg clotting assays: We performed a one-stage clotting assay and a chromogenic assay. 2) Kinetic analysis of thrombin inactivation using AT and formation of thrombin-AT complex (TAT): After incubation with PT activator components, samples were treated with excess AT in the absence of heparin for various durations and changes in absorbance per minute were measured. Formed TAT was measured using an enzyme-linked immunosorbent assay (ELISA) kit. 3) TM-binding assay: To evaluate the ability of wild-type and mutant rPTs to bind to TM, after activating PT, we detected TM-binding thrombin using a TM ELISA method.Results:1) M380T PT activity in the one-stage assay was below the threshold of sensitivity (clot not detected), whereas R431H PT activity was decreased to 10% of that of wild-type rPT (100%). On the other hand, both mutant PT activities in the chromogenic assay were slightly decreased compared to wild-type rPT. 2) After 30 min without heparin, relative residual thrombin activity of M380T PT (74%) was higher than that of wild-type (10%), indicating severe ATR, whereas that of R431H PT was slightly increased (21%), indicating mild ATR. TAT formation by wild-type PT increased over time. TAT formation by R431H PT was about 40% of that of wild-type PT, but that by M380T PT was almost negligible after 120 min. 3) TM-binding thrombins of both mutant rPTs were barely detected.Discussion: M380T PT has high ATR and fails to bind with TM, so a large amount of thrombin with greater clotting activity than normal is thought to be present. With this abnormal thrombin, however, fibrin-forming ability is greatly decreased and almost no clotting ability is thought to exist in the body. Hence, R431H PT, which made up about half of the PT protein in the proband, was considered responsible for hemostasis in the body. The mutant thrombin activated from the R431H PT shows decreased clotting activity, but is resistant to inactivity from AT and thus maintains activity longer than ordinary thrombin. At the same time, the activation of protein C is also decreased due to failure to bind with TM, so ultimately the clotting ability necessary for hemostasis was conjectured to have been maintained, resulting in the proband being asymptomatic.Conclusion:Although neither PT position M380 nor R431 are AT binding sites, M380T PT and R431H PT showed ATR. These mutants are the first AT-resistant PTs seen from mutations other than at the Arg596 site. In recent years, TM resistance has been newly proposed as a mechanism producing thrombotic tendencies, and may also apply to the present patient. The reason the proband did not show a bleeding tendency may have been the combined contributions of AT and TM resistance. DisclosuresNo relevant conflicts of interest to declare.

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance.

Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.

Production and Characterization of Highly Thermostable β-Glucosidase during the Biodegradation of Methyl Cellulose by Fusarium oxysporum.

Production of β-glucosidase from Fusarium oxysporum was investigated during degradation of some cellulosic substrates (Avicel, α-cellulose, carboxymethyl cellulose (CMC), and methylcellulose). Optimized production of β-glucosidase using the cellulosic substrate that supported highest yield of enzyme was examined over 192 h fermentation period and varied pH of 3.0–11.0. The β-glucosidase produced was characterized for its suitability for industrial application. Methyl cellulose supported the highest yield of β-glucosidase (177.5 U/mg) at pH 6.0 and 30°C at 96 h of fermentation with liberation of 2.121 μmol/mL glucose. The crude enzyme had optimum activity at pH 5.0 and 70°C. The enzyme was stable over broad pH range of 4.0–7.0 with relative residual activity above 60% after 180 min of incubation. β-glucosidase demonstrated high thermostability with 83% of its original activity retained at 70°C after 180 min of incubation. The activity of β-glucosidase was enhanced by Mn2+ and Fe2+ with relative activities of 167.67% and 205.56%, respectively, at 5 mM and 360% and 315%, respectively, at 10 mM. The properties shown by β-glucosidase suggest suitability of the enzyme for industrial applications in the improvement of hydrolysis of cellulosic compounds into fermentable sugars that can be used in energy generation and biofuel production.

Liquid–liquid extraction of lipase produced by psychrotrophic yeast Leucosporidium scottii L117 using aqueous two-phase systems

Aqueous two-phase systems (ATPS) have been used in biomolecules separation and as an efficient alternative to traditional purification systems for lipases extraction. Here, we investigated the partitioning and recovery of lipase derived from Leucosporidium scottii L117 using ATPS and aqueous two-phase micellar systems (ATPMS). Thus, we evaluated three ATPS: (i) polyethylene glycol (PEG)/phosphate salts and (ii) PEG/polyacrylic acid (NaPA) in different molecular weights (1500, 4000 and 8000 g/mol). (iii) Triton X-114 (TX-114)/McIlvaine buffer pH 7.0 in different conditions (2.0% (w/w) of TX-114 at 25.0 and 28.0 °C). The PEG/phosphate and PEG/NaPA systems resulted in a great loss of enzymatic activity; thus these systems do not represent viable alternatives for these lipase extraction. The micellar systems yielded the best results for lipase extraction with enzyme activity balances ranging between 84.7% and 113.05%. After optimizing the micellar system by experimental design of the partition coefficient of lipase increased by 10.3-fold (0.75–7.76). Lipase preferentially partitioned into the micelle-rich phase with KLip = 7.76, %RECBot = 93.85% and PF = 1.2 at 25.03 °C, 5.1 pH and 10.38% TX-114 and KLip = 4.77, %RECBot = 73.53% and PF = 1.97 at 28.00 °C, 4.5 pH and 8.0% TX-114, indicating that the ATPMS represents an alternative to purification/extraction of lipase L. scottii L117. A crude lipase extract was also evaluated to define the optimum pH and temperature. Lipase reached optimal activity at 40 °C, and remained stable in pH values ranging from pH 3.0 to 8.0 and temperatures from 20.0 to 45.0 °C, with relative residual lipase activity above 80% after 30 min of incubation.

Enzymatic Manufacture of Deoxythymidine-5'-Triphosphate with Permeable Intact Cells of E. coli Coexpressing Thymidylate Kinase and Acetate Kinase.

A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25°C for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'- triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.

Enhancement of Non-thermal Treatment on Inactivation of Glucoamylase and Acid Protease Using CO2 Microbubbles

Thermal treatment is usually used for food pasteurization and enzyme inactivation. However, it has an adverse effect on the quality of thermal-sensitive food such as fruit juice, Japanese sake, milk, yogurt and jam. In this study, we presented an alternative method for a non-thermal treatment with at 45 and 50°C for glucoamylase and protease inactivation using pressurized carbon dioxide (CO2). Twelve liters of enzyme solution (0.004% glucoamylase or 0.015% protease) was fed into a low pressure (2 MPa) CO2 mixing vessel. CO2 microbubbles (MB-CO2) were generated by introducing the mixture through a swiveling microbubble generator. The mixture containing MB-CO2 was flowed to incubate in a heating coil at various conditions (temperature at 45 or 50°C and pressure 2, 4, or 6 MPa). After incubation, the mixture was sampled at 10, 20 and 30 min from the sampling valve. The relative residual activities of glucoamylase and acid protease were measured by a spectrophotometer at the absorbance of 400 nm (Abs400) and 660 nm (Abs660), respectively. Relative residual activity of glucoamylase with MB-CO2 treatment at 50°C and 4 MPa was 15.01% whereas 74.83% of glucoamylase activity was found from treatment without MB-CO2 at same temperature. For acid protease, relative residual enzyme activity with MB-CO2 treatment at 45°C and 4 MPa was 2.29% whereas that without MBCO2 treatment at 45°C was 81.25%. These results suggested that glucoamylase and acid protease could be inactivated effectively at 45 and 50°C present of MB-CO2.

Preliminary Study towards Enhanced Crude Oil Biodegradation Reveals Congeneric Total Peroxidases with Striking Distinctions

Peroxidases (POXs) are the key extracellular enzymes produced by crude oil degrading microbes. Knowledge of optimum conditions for POXs activity is crucial for providing effective environment for bioremediation. In this study, physicochemical properties of POXs produced by Actinomyces israelii and Actinomyces viscosus during growth on crude oil were studied. The POXs exhibited similarities in activity and stability with striking differences in response to two divalent metal ions. The POXs from both species had optimum pH of 7.0 and were very stable over a narrow pH range (6.0 - 8.0). The POXs demonstrated similar thermostability exhibiting relative residual activity of 62% at 50°C after 30 min incubation and 45% residual activity at the same temperature after 60 min despite the fact that POXs from A. viscosus and A. israelii had optimum temperatures of 50°C and 40°C, respectively. The POXs from A. viscosus and A. israelii were greatly activated by Fe2+ at 5.0 and 10.0 mM. The enzymes were both strongly inhibited by Cu2+, Mg2+ and Hg2+. Surprisingly, these congeneric POXs demonstrated striking differences in their response to Ca2+ and Mn2+. POX from A. viscosus was activated by Ca2+ and Mn2+ exhibiting relative activity of 136% and 106% at 5 mM, respectively. In contrast, POX from A. israelii was strongly inhibited by Ca2+ and Mn2+ exhibiting 62.5% relative activity in the presence of 5 mM of each metal ion. Increasing the concentration of Ca2+ and Mn2+ led to further activation of POX from A. viscosus and inhibition of POX from A. israelii. Results provide deeper insights into functional properties of studied POXs from closely related microbes. The physicochemical properties are very similar; however, notable differences provide a strong basis for structural characterization of these congeneric enzymes.