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Project Digestion

Introduction

Streptomyces is a kind of prokaryotic bacteria which decompose bodies in nature. We extract protease and chitinase genes from this bacterium and introduce into Escherichia coli. Secretion-signal sequences are included in these genes so that the proteins coded by them will go out without occurring cell lysis. After assembling all genes, we examined the activity of these two enzymes in both of qualitative and quantitative ways.

Method

Construction

We created following constructions to allow secretion of Serine protease, SAM-P20 and chitinase, chiA1. These genes are regulated by lactose promoter, BBa_R0011. We used Streptmyces’s RBS into these constructions, because reference article [1] used them to allow E.coli to secrete these proteins.

Assay

Serine Protease

Experiment 1 : Skim milk-hydroryzing assay

In order to identify the expression of SAM-P20 gene, a skim milk-hydroryzing assay was performed. A plate containing 2% skim milk, IPTG(final concentration, 0.5mM), and 0.1% yeast extract was used for this assay.

Experiment 2 : Measurement of enzyme activity

In order to measure the activity of serine protease, the fluorescent assay was done, using fluorescein-labeled casein as a substrate.

Chitinase A

Experiment 1

Experiment 2 : 3,5-Dinitrosalicylic acid assay (DNS method)

This assay is based on this fact: 3,5-dinitorosalicylic acid (DNS) is changed into 3-amino- 5-nitorosalicylic acid by reducing saccharide in reaction solution and the absorbance of this liquid increase in direct proportion to the amount of reducing sugar.

We examined the quantitative relation between absorbance and the volume of sugar and then ::expressed it onto a straight line graph (result fig 1:リンク). We led chitinase E.coli had ::secreted disassemble chitin into N-acetylglucosamine and other sugar derivatives in water.  ::After passing enough time (_min), we added this liquid 1 ml into DNS reagent 3 ml (How to prepare) and boiled this solution for 5 min. After cooling this soluion, We diluted with water to 25 ml and assay the absorbance in 550 nm.

The results of this measurement and the fig 1 graph enabled us to calculate the amount of digested chitin, showing the relative activity of chitinase. We did same examination about commercial chitinase derived from Streptomyces and exhibited in result fig 2:リンク

　２－１：Evaluate the accidental change of absorbance

Even though we centrifuge media to remove E,coli, there might be still some E.coli in solution.For this reason, we examined accidental errors in absorbance as the following way before conducting DNS assay. The speed of increasing the number of E.coli would depend on media so that we examined all kind of media which we could have used in DNS assay.

・cultured E.coli overnight in LB medium

・poured the medium (above 1㎕) into each three microcentritube and measured OD

Following operations were conducted to all tubes.

・centrifuge for 5 min at 5,000 rpm and move 1㎕ the supernatant to new microcentritube. (It was ::incubated at 37℃ until this experiment was finished.)

・measured absorbance

・one hour after, we measured absorbance and took 200㎕ the solution to new tube and heated it ::for 3 min in boiling water and then cooled it in water.

・applied 200㎕ DNS reagent and heated it for 5 min in boiling water and then cooled it in water.

・two, three, five hours after, we did above operation, taking supernatant, measured absorbance,heating and cooling, applying DNS reagent and heating and cooling again.

Result

Chitinase A1

1. Standard Measurement for ChiA1.

Fig.1: Absorbance550 vs. glucose concentration. r²=0.98936.

From the result, a strong correlation between glucose concentration and its A₅₅₀ was observed.

1. Consideration of medium and growth of E.coli.

Discussion

Reference

[1] Molecular Characterization of a Gene Encoding Extracellular Serine Protease Isolated from a Subtilisin Inhibitor-Deficient Mutant of Streptmyces albogriseolus S-3253

[2]Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis

[3]Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites

[4]還元糖の定量法 (生物化学実験法)福井 作蔵

[5]Quantitative Analysis　of Cellulose-Reducing Ends