The activity of immobilized enzymes on different krill chitin preparations

The suitability of krill chitin, prepared by using different concentrations of KOH and HCI for deproteinization and demineralization, respectively, was investigated. The activity of enzymes immobilized on such supports depends on the degree of deproteinization of chitin, availability of amino groups, content of minerals, mesh size, structure of the surface, and conformation of the chitin molecules. It was found that invertase and amyloglucosidase achieved high activity after immobilization on chitin obtained at not too rigorous conditions of deproteinization. However, the activity of immobilized α-amylase and diastase increased significantly with the increase in concentration of KOH used for deproteinization. High content of minerals and proteins in chitin preparation causes a loss of immobilized enzyme activity.     

Immobilization of enzymes on chitin and chitosan

During the last few years, d-glucose isomerase, glucoamylase, β-d-galactosidase (lactase), β-d-glucosidase, d-glucose oxidase, AMP deaminase, urease, pronase, subtilisin, trypsin, papain, alkaline phosphatase, acid phosphatase, pepsin, chymotrypsin and lysozyme have been immobilized on chitin and on some of its derivatives, mainly with glutaraldehyde. The preparation and performances of the immobilized enzymes are described.     

Adsorption and reactions of chitinase and lysozyme on chitin

Summary Isotherms for adsorption of chitinase on chitin and lysozyme on chitin have been determined at two temperatures and rates of hydrolysis of chitin catalysed by these enzymes have been measured at three temperatures and at several enzyme concentrations for each. Ribonuclease, not an enzyme for chitin, and heat-denatured lysozyme and chitinase show reduced or no adsorption to this substrate.Initial hydrolysis rates of chitin by both enzymes are proportional to total enzyme concentrations in the range of concentrations studied. These kinetics cannot, however, be related to the adsorption isotherms because of the non-equilibrium nature of the isotherms.     

Lactase and other enzymes bound to chitin with glutaraldehyde

Acid tolerant lactase (I), α-chymotrypsin (II), and acid phosphatase (III) were immobilized on chitin with glutaraldehyde. Pretreatments of the chit in with acid, alkali, ammonia, and pronase were compared with respect to release of titratable amino groups and ability to retain lactase activity. Shrimp chitin appeared to be more sensitive to pretreatment conditions and so effort was concentrated on crab. An acid-alkali pretreatment was selected as most practical and economical, and the properties of enzymes fixed on crab chitin were studied intensively. The pH optima of the fixed enzymes were shifted about one pH unit; the shift for I was toward more acid pH, for II was toward alkaline pH, and for III was toward acid pH. The retained activity of immobilized I was approximately 60% that of the native enzyme. A column in continuous operation with I on chitin-glutaraldehyde gave an apparent activity half-life of 27 days.     

Anti-inflammatory effect of chemically modified chitin

Anti-inflammatory effects of the three types of chitin derivatives namely phosphated chitin (P-chitin), phosphated–sulfated chitin (PS-chitin), and sulfated chitin (S-chitin) were investigated using a canine model of chitosan-induced pneumonia. After simultaneous administration of chitosan with or without each chitin derivative (chitosan alone: n=6, chitosan and P-chitin: n=6, chitosan and PS-chitin: n=1, and chitosan and S-chitin: n=3), hematological examination and X-ray image processing were performed for up to 24 h. Then the lungs were recovered and were evaluated by softex imaging after inflation and fixation. The hematological findings showed that PS-chitin and S-chitin did not prevent the decrease in white blood cell (WBC) count as seen in dogs administered chitosan, while P-chitin prevented such decrease in WBC count. The surface of the inflated and fixed lung specimens was hemorrhagic in the PS- and S-chitin groups as well as in the chitosan group, while the lung looked like normal in the P-chitin group. The pulmonary blood vessels of the chitosan group showed severe change while the P-chitin group showed no changes with softex findings. Furthermore, the pattern of histogram density obtained with image processing of thoracic X-ray in P-chitin group did not change among pre and post administration while chitosan group showed rightward movement and significant changes on parameters. The cause of which is attribured to an attenuation of X-ray permeability by angiectasis of the lung.     

Determination of glutathione and glutathione disulfide in hepatocytes by liquid chromatography with an electrode modified with functionalized carbon nanotubes

Glutathione (GSH) and glutathione disulfide (GSSG) are important thiols, which provide defence against oxidative stress by scavenging free radicals or causing the reduction of hydrogen peroxide. The ratio GSH/GSSG is often used as a sensitive index of oxidative stress in vivo. In this paper, a direct electrochemical method using an electrode modified with functionalized carbon nanotubes as electrochemical detector (ED) for liquid chromatography (LC) was described. The electrochemical behaviors of GSH and GSSG on this modified electrode were investigated by cyclic voltammetry and it was found that the functionalized carbon nanotubes exhibited efficiently electrocatalysis on the current responses of GSH and GSSG. In LC-ED, both of the analytes showed good and stable current responses. The detection limit of GSH was 0.2 pmol on column and that of GSSG was 1.2 pmol on column, which were low enough for the analysis of real small samples. The method was sensitive enough to detect difference in concentration of GSH and GSSG in hepatocytes from animals with and without introduction of oxidation stress by glucose or hydrogenperoxide.     

Structure and function of enzymes acting on chitin and chitosan

Enzymatic conversions of chitin and its soluble, partially deacetylated derivative chitosan are of great interest. Firstly, chitin metabolism is an important process in fungi, insects and crustaceans. Secondly, such enzymatic conversions may be used to transform an abundant biomass to useful products such as bioactive chito-oligosaccharides. Enzymes acting on chitin and chitosan are abundant in nature. Here we review current knowledge on the structure and function of enzymes involved in the conversion of these polymeric substrates: chitinases (glycoside hydrolase families 18 & 19), chitosanases (glycoside hydrolase families 8, 46, 75 & 80) and chitin deacetylases (carbohydrate esterase family 4).     

Use of chemically modified activated carbon as a support for immobilized enzymes

Loading and activity assays of the enzymes alpha-chymotrypsin, alpha-chymotrypsinogen, and glucose oxidase covalently bound to an activated carbon support are presented. The activated carbon support material was pretreated using either a radio-frequency oxygen plasma or an electrochemical oxidation to maximize the enzyme attachment. Cyanuric chloride or water-soluble carbodiimide linking reactions were used to covalently attach the enzymes to the carbon support. Discussion of the relative merits of each reaction scheme is presented.     

Electrochemical properties of hydrogenase on glass carbon electrodes modified with carbon nanotubes

Hydrogenase (H₂ ase) purified from phototropic bacteriumThiocapsa roseopersicina was coassembled with carbon nanotubes (CNTs) on glass carbon electrodes. Both oxidized CNTs and Nafion-CNT composites were used to modify the electrodes. The pure H₂ are formed dot-like domains, while the oxidized CNT-H₂ ase and Nafion-CNT-H₂ ase composites formed wire-like and large closely packed aggregates, respectively. The reductive potentials for the [4Fe-4S]^2+/1+ clusters of H₂ase were at about −500, −650, and −700 mV (vs Ag/AgCl) for the electrodes modified with pure H₂ase, Nafion-SWNT-H₂ase, and Nafion-MWNT-H₂ase composites, respectively. Potential step chronocoulometry measurements indicated a larger charge-transfer diffusion coefficient between the H₂ase and electrodes when the CNTs were co-assembled with H₂ase, suggesting that the CNTs can not only act as a supporting layer to immobilize enzymes, but also act as a highly conductive wire throughout the films.     

Novel eubacteria able to grow on carbon disulfide

Four eubacterial strains able to grow on carbon disulfide (CS₂) as sole energy substrate were isolated from soil and leaves of the CS₂-producing tree Quercus lobata. Three of the isolates (strains KS1, KS2, and KL1) were gram-negative, facultatively methylotrophic, and heterotrophic, and capable of growth on a wide range of inorganic and organic sulfur compounds. Biochemical and physiological properties differed slightly among the three strains, but all are proposed to be novel thiobacillus species. Growth yields on CS₂ in batch and chemostat culture ranged from 3.3 g dry wt/mol CS₂ (batch) to a maximum growth yield (Y_max) of 11.1 g dry wt/mol (chemostat). Chemostat data for two of the strains growing, autotrophically on thiosulfate gave Y_max values of 7.4 and 7.1 g dry wt/mol, which fall within the range observed with thiobacilli. The three new Thiobacillus strains had DNA containing 39.8 (KS2), 47.8 (KS1), and 50.5 (KL1) mol% G+C. All three were unusual in being able to grow not only on thiosulfate (aerobically or with denitrification), but also on CS₂, carbonyl sulfide and methylated sulfides as sole energy substrates, and one was unique in being able to grow also on substituted thiophenes. They are the first organisms described to be capable, of anaerobic growth with denitrification on CS₂. The fourth isolate (strain KL2) was gram-positive non-motile and nonspore-forming, with 39.0 mol% G+C. It had a restricted range of sulfur-containing growth substrates, could not grow methylotrophically or on autotrophic substrates other than CS₂, and is not yet classifiable These organisms extend the range of eubacteria known to be capable of CS₂ breakdown and demonstrate that several types of facultatively chemolithotrophic bacteria, able to grow exclusively on CS₂, are associated with a CS₂-producing plant.     

A theoretical study on the hydrolysis mechanism of carbon disulfide

The hydrolysis mechanism of CS(2) was studied using density functional theory. By analyzing the structures of the reactant, transition states, intermediates, and products, it can be concluded that the hydrolysis of CS(2) occurs via two mechanisms, one of which is a two-step mechanism (CS(2) first reacts with an H(2)O, leading to the formation of the intermediate COS, then COS reacts with another H(2)O, resulting in the formation of H(2)S and CO(2)). The other is a one-step mechanism, where CS(2) reacts with two H(2)O molecules continuously, leading to the formation of H(2)S and CO(2). By analyzing the thermodynamics and the change in the kinetic function, it can be concluded that the rate-determining step involves H and OH in H(2)O attacking S and C in CS(2), respectively, causing the C=S double bond to change into a single bond. The two mechanisms are competitive. When performing the hydrolysis of CS(2) with a catalyst, the optimal temperature is below 252°C.     

稻纵卷叶螟几丁质合成酶及合成通路相关酶基因的鉴定及表达分析

【目的】稻纵卷叶螟Cnaphalocrocis medinalis(Guenee)是水稻上的四大害虫之一,危害较为严重,近年来以几丁质合成和代谢过程作为害虫防治的标靶研究已成为热点。为阐明几丁质合成酶及合成通路上关键酶的作用,本研究开展了对稻纵卷叶螟几丁质合成酶及合成相关通路上关键酶的克隆及时空表达分析。【方法】本研究基于稻纵卷叶螟转录组,结合PCR及RACE技术,克隆了几丁质合成酶代谢通路上的4条基因的c DNA全长;利用生物信息学软件对序列进行结构预测、序列比对和进化分析;采用实时定量PCR技术研究了4条基因在不同虫态和幼虫的不同组织中的表达情况。【结果】获得了2条几丁质合成酶序列及2条合成通路上的基因序列,包括几丁质合成酶A(Chitin synthase A,CHSA),几丁质合成酶B(Chitin synthase B,CHSB),N-乙酰葡糖胺磷酸变位酶(Phosphoacetylglucosamine mutase,PGM和UDP-N-乙酰葡萄糖焦磷酸化酶(UDP-N-acetylglucosamine pyrophosphorylase,UAP),并分别命名为Cm CHSA、Cm CHSB、Cm PGM和Cm UAP;序列分析显示Cm CHSA序列全长4 868 bp,编码1 564个氨基酸。Cm CHSB序列全长4 651 bp,编码1 525个氨基酸。Cm PGM全长1 934 bp,编码548个氨基酸。Cm UAP序列全长1 837 bp,编码487个氨基酸。实时定量研究表明,Cm UAP和Cm PGM在血淋巴中表达量最高,Cm CHSA在头部和表皮中表达量较高,而Cm CHSB在中肠中表达量最高。【结论】本研究得到了稻纵卷叶螟几丁质合成路径的4个关键酶基因c DNA全长,它们在稻纵卷叶螟的不同组织和虫态中呈现了差异显著的时空表达,本文为进一步探究稻纵卷叶螟的几丁质合成酶的生理功能和几丁质的合成代谢途径奠定了基础。     

Enhanced enzymatic hydrolysis of langostino shell chitin with mixtures of enzymes from bacterial and fungal sources

A combination of enzyme preparations from Trichoderma atroviride and Serratia marcescens was able to completely degrade high concentrations (100 g/L) of chitin from langostino crab shells to N-acetylglucosamine (78%), glucosamine (2%), and chitobiose (10%). The result was achieved at 32 degrees C in 12 days with no pre-treatment (size reduction or swelling) of the substrate and without removal of the inhibitory end-products from the mixture. Enzymatic degradation of three forms of chitin by Serratia/Trichoderma and Streptomyces/Trichoderma blends was carried out according to a simplex-lattice mixture design. Fitted polynomial models indicated that there was synergy between prokaryotic and fungal enzymes for both hydrolysis of crab chitin and reduction of turbidity of colloidal chitin (primarily endo-type activity). Prokaryotic/fungal enzymes were not synergistic in degrading chitosan. Enzymes from prokaryotic sources had much lower activity against chitosan than enzymes from T. atroviride.