Activity Measurement of Chitosanase by an Amperometric Biosensor

Tatsumi, Hirosuke; Katano, Hajime; Ikeda, Tokuji; Kimoto, Hisashi

doi:10.2116/analsci.25.825

Cited by 4 publications

(2 citation statements)

References 7 publications

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“…The reaction mixture, consisting of 0.4 ml of 1.0% substrate in PBS and 0.1 ml of enzyme solution in 1% BSA-PBS, was incubated for 10-120 min at 37 ° C, and the amino sugars released were determined [Blix, 1948;Tatsumi et al, 2009]. One unit of activity was defined as the amount of enzyme catalyzing the production of 1 mol of the amino sugar per min, using glucosamine as the standard.…”

Section: Enzyme and Protein Assaymentioning

confidence: 99%

Discoidin Domain of Chitosanase Is Required for Binding to the Fungal Cell Wall

Kimoto

Akamatsu²,

Fujii³

et al. 2010

Microb Physiol

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Previously, we reported properties of a glycosylase belonging to GH-8 glycosyl hydrolase (GH) and having both chitosanase and glucanase activities. This enzyme (D2), whose molecular mass (86 kDa) was the largest among the GH-8 group, has its catalytic domain at the N-terminal region, and discoidin domain (DD) at the C-terminal region. Although various chitosanases, chitinases and glucanases have been known, DD is unique to the D2 enzyme. Glucanase and chitinase, but not chitosanase, are known to have functional domain such as carbohydrate-binding module, besides catalytic domain. Accordingly, function of the DD of D2 chitosanase was analyzed, using zygomycete cell wall containing chitosan, glucan and chitin as the basic constituents. The DD specifically and tightly bound to chitosan, but did not participate in affinity for glucan and chitin. Deletion of the DD caused marked reduction in absorbability to cell wall and in hydrolytic activity toward chitosan and glucan. These results suggest that the DD is concerned in binding of the enzyme to cell wall and in effective digestion of the insoluble substrate, through hydrolysis of not only chitosan but also coexisting glucan. Thus, this is the first example of chitosan-binding domain among various carbohydrate-binding modules reported thus far.

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Section: Enzyme and Protein Assaymentioning

confidence: 99%

Discoidin Domain of Chitosanase Is Required for Binding to the Fungal Cell Wall

Kimoto

Akamatsu²,

Fujii³

et al. 2010

Microb Physiol

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“…However, there are a few methods to determine glucosamine in different matrixes based on its characteristics as hexosamine, such as Elson-Morgan colorimetric method [2,3]. Other analytical instrumental methods to determine amino sugars have been also implemented: radioisotope dilution [4], capillary electrophoresis [5], amperometric biosensor [6], and high performance liquid chromatography (HPLC) [7][8][9][10], the last one is the most used technique by pharmaceutical laboratories.…”

Section: Introductionmentioning

confidence: 99%

A Comparative Study Between a Flow Injection Method and a Batch Method to Determine Glucosamine in Dietary Supplements

Lorena¹,

Pilar

2011

CAC

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A new, simple and fast automatic spectrophotometric flow injection (FI) method to determine glucosamine in dietary supplements has been developed and compared against a batch method, both based on the same reaction: "Schiff's base" formation as a result of the reaction between ninhydrin and glucosamine. Temperature and flow-rate were the main parameters to optimize the FI method because the reaction kinetic is slow. With the optimized flow-rate and temperature (0.8 ml min -1 flow-rate and 75 °C), the complex was measured in less than three minutes. By the batch method, 30 min of reaction, 20 min for a cooling step and 92.5 °C to measure the complex were needed. The sensitivity of the FI method was much lower than that of the batch method but the linear range was longer: for the FI method was from 0.3-% to 1-%; and for the batch method from 0.01-% to 0.04-%. The FI method allowed a throughput of 24 h -1 with a precision of 1.0-% for repeatability and of 3.5-% for reproducibility. The batch method had a repeatability of 2.1-% and a reproducibility of 4.1-%. The proposed method allowed an on-line determination with a very little human intervention and can be used in routine analysis laboratory. With this method the analysis time decreased 20 times with respect to the batch method demonstrating that the flow methods can be used in slow kinetic reactions improving also the precision.

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A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases

Cruys-Bagger

Badino

Tokin

et al. 2014

Enzyme and Microbial Technology

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A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39-0.40s(-1)) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

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Activity Measurement of Chitosanase by an Amperometric Biosensor

Cited by 4 publications

References 7 publications

Discoidin Domain of Chitosanase Is Required for Binding to the Fungal Cell Wall

Discoidin Domain of Chitosanase Is Required for Binding to the Fungal Cell Wall

A Comparative Study Between a Flow Injection Method and a Batch Method to Determine Glucosamine in Dietary Supplements

A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases

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