Cellulose production based on hemicellulose hydrolysate from steam-pretreated willow

The production cost of cellulolytic enzymes is a major contributor to the high cost of ethanol production from lignocellulosics using enzymatic hydrolysis. The aim of the present study was to investigate the cellulolytic enzyme production ofTrichoderma reesei Rut C 30, which is known as a good cellulase secreting micro-organism, using willow as the carbon source. The willow, which is a fast-growing energy crop in Sweden, was impregnated with 1–4% SO₂ and steam-pretreated for 5 min at 206°C. The pretreated willow was washed and the wash water, which contains several soluble sugars from the hemicellulose, was supplemented with fibrous pretreated willow and used for enzyme production. In addition to sugars, the liquid contains degradation products such as acetic acid, furfural, and 5-hydroxy-methylfurfural, which are inhibitory for microorganisms. The results showed that 50% of the cellulose can be replaced with sugars from the wash water. The highest enzyme activity, 1.79 FPU/mL and yield, 133 FPU/g carbohydrate, was obtained at pH 6.0 using 20 g/L carbon source concentration. At lower pHs, a total lack of growth and enzyme production was observed, which probably could be explained by furfural inhibition.     

Immobilization of glucose oxidase with the blend of regenerated silk fibroin and poly(vinyl alcohol) and its application to a 1,1′- dimethylferrocene-mediating glucose sensor

The structure and properties of the blend of regenerated silk fibroin (RSF) and poly(vinyl alcohol) (PVA) were investigated. The two polymers in the blend are in the state of phase segregation. Infrared (IR) spectra indicate that the RSF in the blend maintains its intrinsic properties, thus, ethanol treatment can transfer silk I structure of RSF to silk II structure. The water absorption property and mechanical property of the blend are improved in comparison with those of RSF. The blend maintains the major merit of RSF, that is, it can immobilize glucose oxidase on the basis of the conformational transition from silk I structure to silk II structure. The properties of the immobilized enzyme are examined. Moreover, the second generation of glucose sensor based on the immobilized enzyme is fabricated and it has a variety of advantages including easy maintenance of enzyme, simplicity of construction, fast response time and high stability.     

Thermal stability and energy of deactivation of free and immobilized amyloglucosidase in the saccharification of liquefied cassava starch

Amyloglucosidase from Novo (Copenhagen, Denmark) was immobilized in controlled pore silica particles with the silane-glutaraldehyde covalent method. Thermal stability of the free and immobilized enzyme (IE) was determined with 30% (w/v) α-amylase liquefied cassava starch, pH 4.5, temperatures from 35 to 75°C. Free amyloglucosidase maintained its activity practically constant for 240 min and temperatures up to 50°C. The IE has shown higher stability retaining its activity for the same period up to 60°C. Half-life for free enzyme was 20.6, 6.44, 2.07, 0.69, and 0.24 h for 55, 60, 65, 70, and 75°C, respectively, whereas the IE at the same temperatures had half-lives of 116.4, 30.88, 8.52, 2.44, and 0.73 h. The energy of thermal deactivation was thus 50.6 and 57.6 kcal/mol, respectively for the free and IE, confirming stabilization by immobilization.     

Stabilization ofEscherichia coli penicillin G acylase by polyethylene glycols against thermal inactivation

The effects of five polyethylene glycol (PEG) compounds of different molecular weight on the thermal stability of penicillin G acylase (PGA) obtained from a mutant ofEscherichia coli ATCC 11105 have been investigated. The molecular weights of PEG compounds were 400, 4000, 6000, 10,000, and 15,000. The thermal inactivation mechanisms of both native and PEG-containing PGA were considered to obey first order inactivation kinetics during prolonged heat treatments. Optimal concentrations of PEGs at molecular weights of 400,4000, 6000,10,000, and 15,000 were found to be 250,150,150,100, and 50 mM, respectively. The greatest enhancement of thermostability was observed with PEG 4000 and PEG 6000, as a nearly 20-fold increase above 50°C. PGA showed almost the same temperature activity profile and optimal temperature values both in the presence and absence of PEG. The addition of PEGs did not cause any change in the optimal temperature value of PGA, but the parametersV _m ,K _m , the activation energy, and thek _cat values of enzyme were markedly decreased because of the mixed inhibition by PEG compounds. The type of inhibition was found to be hyperbolic uncompetitive.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

芯片式流通池顺序注射可更新表面反射光谱法用于酶反应检测

将芯片式流通池顺序注射可更新表面反射光谱法用于酶反应检测。HRP催化H2O2氧化BPR底物的反应用于对H2O2的检测。此反应体系与葡萄糖氧化酶联用，用于对血清中葡萄糖的检测。     

A molecular docking model of SARS-CoV S1 protein in complex with its receptor, human ACE2

The exact residues within severe acute respiratory syndrome coronavirus (SARS-CoV) S1 protein and its receptor, human ACE2, involved in their interaction still remain largely undetermined. Identification of exact amino acid residues that are crucial for the interaction of S1 with ACE2 could provide working hypotheses for experimental studies and might be helpful for the development of antiviral inhibitor. In this paper, a molecular docking model of SARS-CoV S1 protein in complex with human ACE2 was constructed. The interacting residue pairs within this complex model and their contact types were also identified. Our model, supported by significant biochemical evidence, suggested receptor-binding residues were concentrated in two segments of S1 protein. In contrast, the interfacial residues in ACE2, though close to each other in tertiary structure, were found to be widely scattered in the primary sequence. In particular, the S1 residue ARG453 and ACE2 residue LYS341 might be the key residues in the complex formation.     

An optical surface plasmon resonance biosensor for determination of tetanus toxin

Surface plasmon resonance (SPR) has been successfully applied for the simple, rapid, and label-free assay of various biomolecules. This assay evaluates a novel wavelength modulation SPR biosensor for the detection of tetanus toxin. The wavelength modulation SPR biosensor is designed based on fixing the incident angle of light and measuring the reflected intensities in the resonance wavelength range spanning 400-800 nm simultaneously. Tetanus toxin (TeNT), one of the most potent toxins known, is synthesized as a 150 kDa single polypeptide chain. The SPR biosensor has been shown to be capable of directly detecting concentration of tetanus toxin as low as 0.028 Lf ml⁻¹. Under selected experimental conditions, the SPR biosensor has a good reproducibility, sensitivity and reversibility. The results illustrate how wavelength modulation SPR biosensor can be used to detect biomolecular interactions.     

Ascorbate oxidase

Ascorbate oxidase fromCucurbita sp. was isolated by ammonium sulfate precipitation and DEAE-dextran-silochrome column chromatography. The thermal and pH stabilities of the purified enzyme were investigated. TheK _M forl-ascorbic acid (1.5 mM) and chlorohydroquinone (0.37 mM) was determined. Substrate specificity of ascorbate oxidase was investigated and compared with those of laccases fromCoriolus hirsutus andCerrena maxima. Ascorbate oxidase was covalently bound to a polymeric membrane and used in an enzyme electrode for ascorbic acid.     

A microtiter plate assay for the detection of inhibitors of the Na +, K + -ATpase

A rapid microtiter plate assay for the detection of inhibitors of the Na⁺, K⁺-ATPase has been developed. The assay is based on the measurement of inorganic phosphate released from the substrate, ATP, and has been designed to be carried out in the individual wells of a microtiter plate. Since the production of inorganic phosphate is determined colorimetrically, multiple samples can be tested simultaneously using a microtiter plate reader. This microtiter plate assay is particularly useful for screening large numbers of samples, such as microbial culture supernatants.