Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Hydrolysis of cyclosporin A: Identification of 1,11 seco-cyclosporin A and 4,5 secoisoCyclosporin A by FAB-MS/MS

We have previously reported that treatment of CsA with aqueous HCl gives rise to the formation of a number of water-soluble compounds. Two of these were identified from their FAB-MS/MS spectra as open-chain nona- and decapeptides. We describe here the identification of two other main compounds deriving from the same treatment. Identification was rendered possible from the comparison of their FAB-MS/MS spectra with those of methyl and acetyl derivatives. The two compounds are water-soluble, open-chain undecapeptides corresponding to 1,11 seco-CsA and of 4,5 seco-isoCsA, respectively.     

Properties of Chlorogenic Acid Quinone: Relationship between Browning and the Formation of Hydrogen Peroxide from a Quinone Solution

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7×10³ for 325 nm and 9.7×10² for 400 nm in an acidic aqueous solution. Chlorogenic acid and H₂O₂ were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H₂O₂ spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.     

Stereoselective Synthesis of (S,E)-6-Isopropyl-3,9-dimethyl-5,8-decadienyl Acetate,the (S)-Enantiomer of the Yellow Scale Pheromone

The (S)-enantiomer of the sex pheromone of the yellow scale (Aonidiella citrina), (S,E)-6-isopropyl-3,9-dimethyl-5,8-decadienyl acetate, was stereoselectively synthesized from (R)-(+)-citronellic acid.     

NAD hydrolysis: Chemical and enzymatic mechanisms

The pyridine nucleotide have important non-redox activities as cellular effectors and metabolic regulators [1–3]. The enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond of NAD⁺ and the attendant delivery of the ADPRibosyl moiety to acceptors is central to these many diverse biological activities. Included are the medically important NAD-dependent toxins associated with cholera, diphtheria, pertussis, and related disease [4]; the reversible ADPRibosylation-mediated biological regulatory systems [5,6]; the synthesis of poly (ADPRibose) in response to DNA damage or cellular, division [7]; and the synthesis of cyclic ADPRibose as part of an independent, calcium-mediated regulatory system[8]. As will be presented in this chapter, all evidence points to both the chemical and enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond being dissociative in character via an oxocarbenium intermediate.     

Enzymatic browning reaction of apple juices prepared using a blender and a low-speed masticating household juicer

ABSTRACT

The aim of this study was to investigate the effect of juicer type (blender or LSM household juicer) on the browning reaction of apple juice and evaluate the remaining antioxidant activity in the juice. The blender apple juice showed a darker brown color and 4.5 times higher PPO activity than LSM apple juice. This result suggested that the blender caused severer damage to plastids in cells leading to leakage of PPO into the juice than the LSM juicer. The total polyphenol and flavonoid content of LSM apple juice was approximately 2 times higher than that of blender apple juice because polyphenols and flavonoids can be used as substrates by PPO. The antioxidant activity of LSM juice was higher than that of blender juice. Together, these results suggested that the LSM juicer is superior to the blender for preparation of fresh apple juices due to the minimization of enzymatic oxidation reactions.

Abbreviations: LSM: low-speed masticating; PPO: polyphenol oxidase; ABTS: 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl     

Optimization of integrated alkaline–extrusion pretreatment of barley straw for sugar production by enzymatic hydrolysis

In this work, an integrated one-step alkaline–extrusion process was tested as pretreatment for sugar production from barley straw (BS) biomass. The influence of extrusion temperature (T) and the ratio NaOH/BS dry matter (w/w) (R) into the extruder on pretreatment effectiveness was investigated in a twin-screw extruder at bench scale. A 2³ factorial design of experiments was used to analyze the effect of process conditions [T: 50–100 °C; R: 2.5–7.5% (w/w)] on composition and enzymatic digestibility of pretreated substrate (extrudate). The optimum conditions for a maximum glucan to glucose conversion were determined to be R = 6% and T = 68 °C. At these conditions, glucan yield reached close to 90% of theoretical, while xylan conversion was 71% of theoretical. These values are 5 and 9 times higher than that of the untreated material, which supports the great potential of this one-step combined pre-treatment technology for sugar production from lignocellulosic substrates. The absence of sugar degradation products is a relevant advantage over other traditional methods for a biomass to ethanol production process since inhibitory effect of such product on sugar fermentation would be prevented.     

Enzymatic Resolutions of Heterocyclic Alcohols

The butyrates and acetates of heterocyclic alcohols like 3 - hydroxy tetrahydrofuran and - pyran, 3- and 4 - chromanol as well as the corresponding sulfur heterocycles were hydrolyzed using lipase from Candida rugosa (CRL) and from Pseudomonas cepacia, (PCL). Poor to excellent enantioselectivities were obtained depending on the structure of the substrates. An electrostatic amendment to the steric substrate model for PGL is proposed.     

Sepiapterin reductase: Characteristics and role in diseases

Sepiapterin reductase, a homodimer composed of two subunits, plays an important role in the biosynthesis of tetrahydrobiopterin. Furthermore, sepiapterin reductase exhibits a wide distribution in different tissues and is associated with many diseases, including brain dysfunction, chronic pain, cardiovascular disease and cancer. With regard to drugs targeting sepiapterin reductase, many compounds have been identified and provide potential methods to treat various diseases. However, the underlying mechanism of sepiapterin reductase in many biological processes is unclear. Therefore, this article summarized the structure, distribution and function of sepiapterin reductase, as well as the relationship between sepiapterin reductase and different diseases, with the aim of finding evidence to guide further studies on the molecular mechanisms and the potential clinical value of sepiapterin reductase. In particular, the different effects induced by the depletion of sepiapterin reductase or the inhibition of the enzyme suggest that the non‐enzymatic activity of sepiapterin reductase could function in certain biological processes, which also provides a possible direction for sepiapterin reductase research.     

Peptide Bond Cleavage by Ni(II) Ions within the Nuclear Localization Signal Sequence

Nickel is harmful to humans, being both carcinogenic and allergenic. However, the mechanisms of this toxicity are still unresolved. We propose that Ni(II) ions disintegrate proteins by hydrolysis of peptide bonds preceding the Ser/Thr‐Xaa‐His sequences. Such sequences occur in nuclear localization signals (NLSs) of human phospholipid scramblase 1, Sam68‐like mammalian protein 2, and CLK3 kinase. We performed spectroscopic experiments showing that model nonapeptides derived from these NLSs bind Ni(II) at physiological pH. We also proved that these sequences are prone to Ni(II) hydrolysis. Thus, the aforementioned NLSs may be targets for nickel toxicity. This implies that Ni(II) ions disrupt the transport of some proteins from cytoplasm to cell nucleus.