2.1 Sample collection and screening of agar-degrading microorganisms 2.1.1 Preparation of C-10 agar(ingredient/liter)
HEPES(N-2-hydroxyethyl- piperazine-N-2-ethanesulfonic acid) 1.2 g MgSO4.7H2O 0.25 g
K2HPO4 0.05 g Ca(NO3)2.4H2O 0.025 g KNO3 1.0 g
Na2-EDTA (Na2-ethylenediaminetetraacetic acid) 0.01 g Fe2(SO4)3.6H2O 0.004 g
Agar (Dfico) 5 % Distilled water 1000 ml Procedure:
For the screening of agarolytic bacteria, city drainer（Taitung, Taiwan）water samples from our university front road side ditch were collected（the ditch was 30 cm wide, 50 cm in depth, with 20 cm deep ordinary house waste water）. Samples (100μl each) were spread on C-10 agar plate with a glass spreader. The plates were incubated at 37℃
several days. Colonies forming depressions or pits and clearing zones on agar surface were picked and re-purified using the same culturing method.
2.2 Idetification of the isolates
Gram’s stain（Difco）MicroScan AS-4 (Dade Behring) Pseudomonas aeruginosa ATCC 27853 E.coli ATCC 25922
Neg Breakpoint Combo Panel Type 12 (Dade Behring) Reagent:
Kovac’s Reagent
40% Potassium hydroxide Alpha Naphthol
0.8% Sulfanilic acid
0.5% N,N-Dimethylalphanaphthylamine 10% Ferric Chloride
Procedure:
Isolates were identified through staining, morphological, motility and physiological properties according to Bergey’s Manual of Systematic Bacteriology (Williams, et al.
1989) and MicroScan AS-4 automatic microorganism analyzing system. Pseudomonas aeruginosa ATCC 27853 and E.coli ATCC 25922 were used as standards. (Bacterial identification is based on measurement of the turbidity or color change in the wells of the MicroScan panel. The automated methods directly match to standard biochemical manual methods).
The 16S rRNA was amplified by using MicroSeq 500 16S rDNA Bacterial Identification PCR Kit. One and half kb length 16S rDNA fragment enhancements were collected. PCR products were checked by electrophoresis. The DNA sequencing was done by using Applied Biosystems Instruments 310（ABI 310）. Sequencing was done by Food Industry Research and Development Institute (FIRDI). The sequence was compared to NCBI GenBank database using BLAST.
2.3 Assay for agarase activity of partially purified enzyme
C-10 medium with 0.1% agar（Difco）0.1 mM phenylmethyl-sulfony fluoride (PMSF) 80% saturation with solid ammonium sulfate 20 mM Tris-HCl buffer pH 8.0
Sephadex G-100 column 50 mM Tris-HCl buffer
Centrifuge (Hitachi Himac CR22G) Procedure:
The isolates were inoculated in 1-liter size Erlenmeyer flasks containing 500 ml of
C-10 medium with 0.1% agar and cultured overnight at 37 ℃ with shaking at 100 rpm. Cultures were supplemented with 0.1 mM phenylmethyl-sulfony fluoride(PMSF) and the cells were centrifuged at 7000 xg for 30 min at 4 ℃ to remove bacterial debries. The supernatants were brought to 80% saturation with solid ammonium sulfate.
The mixture was stirred overnight then centrifuged at 8,000 xg for 45 min at 4 ℃.
Pellets were resuspended in 1 ml of 20 mM Tris-HCl buffer pH 8.0 and dialyzed multiple times against the same buffer at 4 ℃. The purified enzymes were collected after dialyzation.
The dialysate was loaded onto a Sephadex G-100 column (1 x 50 cm) equilibrated with 50 mM Tris-HCl buffer. Elution was carried out with the same buffer. Active fractions were stored at 4 ℃.
2.4 Protein quantification
0.5 N NaClCoomassie Brilliant Blue G-250 reagent (Bio-Rad) Spectronic 20 D
Procedure:
Protein concentrations were determined by Bradford’s method (Bradford, 1976), with a Bio-Rad Protein Assay Kit and using bovine serum albumin as the standard (0 to 25μg). One hundred μl enzyme solution added 2 ml of NaCl and 1 ml of Coomassie Brilliant Blue G-250 reagent mixed and set in room temperature for 5 min, the absorbance were determined at OD595.
2.5 Electrophoresis analysis for agarase
Gradient polyacrylaminde gel (4~20% Tris-HEPES-SDS) Mini-PROTEAN Electrophoresis System (Bio-Rad) Running Buffer100 mM Tris 100 mM HEPES 3 mM SDS, pH 8.0
SDS-PAGE Sample Buffer (4x)
10% SDS 1.6 ml Coomassie Brilliant Blue R-250 reagent
0.25% Coomassie Brilliant Blue R-250 (Sigma) 25% Isopropanol
A purified agarase sample (30 μl) were mixed with 10 μl of 4-fold sodium dodecyl sulfate (4x SDS) loading buffer, denatured at 95 ℃ for 3 min. The sample was loaded onto gradient polyacrylaminde gel (4~20% Tris-HEPES-SDS), Bio-Rad broad range protein marker : Myosine (210 kDa), β-galactosidase (125 kDa), Bovine serum albumin (101 kDa), Ovalbumin (56 kDa), Carbonic anhydrase (36 kDa), Soybean trypsin inhibitor (29 kDa), Lysozyme (21 kDa) and Aprotinin (6.9 kDa) were used as markers. Electrophoresis run for 45 min at 20 ℃（100V）. Protein bands were stained with Coomassie Brilliant Blue R-250 (CBB).
2.6 Assay of agarase activity
DNSA Reagent1% 3, 5-dinitrosalicylic acid (DNSA, Ferak berlin) 0.2% Phenol
0.05% Na-sulfite 1% NaOH
Sodium phosphate buffer pH 7.0 0.3 % agarose
D-galactose(Sigma)
Agarase from Pseudomonas atlantica (Sigma A 6306) Spectronic 20 D
Procedure:
Agarase activity was measured by the relase of reducing ends, using 3,5-dinitrosalicylic acid procedure (Miller, 1959) with D-galactose as the standard assay condition, and
compared to a standard (Agarase from Pseudomonas atlantica).
Enzyme solution (50 μl) was added to 450 μl of sodium phosphate buffer pH 7.0 containing 0.3 % low melting agarose. Make a control that the reaction stopped by the addition of the DNSA reagent and assayed using an identical procedure. After incubation at 45 ℃ for 50 min, 1ml DNSA reagent was added, the reaction was stopped by heating the tube in boiling water for 5 min. Three ml of water were then added and mixed. The absorbance of each tube was determined relative to the blank at 550 nm. One unit of agarase activity was defined as liberating 1 μM of D-galactose per min (Miller, 1959).
2.7 Effect of pH on the activity.
50 mM sodium phosphate pH 3.0、4.0、4.3、4.7、4.9、5.2、5.5 Na2HPO4
NaH2PO4
50 mM citric acid-sodium phosphate pH 5.3、5.5、5.9、6.1、6.5、6.9、7.4、8.4 50 mM glycine-NaOH pH 7.5、8.1、8.5、9.2、10
Procedure:
The effects of pH on purified agarase were determined with 50 mM citric acid-sodium phosphate pH 3.0 to 5.5, 50 mM sodium phosphate pH 5.3 to 8.4, and 50 mM glycine-NaOH pH 7.5 to 10. Same method was used to assay of agarase activity as described.
2.8 Effect of temperature on the stability and activity.
50 mM Sodium phosphate buffer (pH 7.0) Na2HPO4
NaH2PO4
Water bath Procedure:
The optimal temperature for the agarase activity was determined from 25 ℃ to 70 ℃ without prolonged incubation time. For stability assay, enzyme solutions were incubated at different temperature range from 25 ℃ to 70 ℃, each temperature kept for 100 min, then agarase activity was determined. Same method was used to assay of agarase activity as described.
2.9 Plasmid Preparation
NaOH/SDS solution (100 ml)0.2 M NaOH 0.8 g 1%(W/V) SDS
Prepared fresh from stock solution of 10 M NaOH and 10% SDS Potassium acetate solution , pH 4.8
Glacial acetate acid 29.5 ml
Centrifuge (Hitachi Himac CR22G) Water bath
Procedure:
Inoculated 100ml sterile C-10 medium with a single bacteria colony and grew at 37℃.
Thirty ml of cells were centrifuged at 5,000rpm 1 min. The pellet resuspened in 2 ml of GTE buffer and set 5 min at room temperature(RT). Four ml of NaOH /SDS solution were added, mixed , and set on ice 5 min. Three ml of potassium acetate solution were then added, vortexed 10 sec , and placed on ice 5 min. Cells were then centrifuged 13,000rpm 5 min and supernatant transfered to a new tube. Two volumes of 95%
ethanol were added into the supernatant , and set 5 min at RT. The pellet was centrifuged at 13,000rpm 5 min, washed with 2 ml of 70% ethanol, and dried. The pellet was then resuspended in 0.5 ml TE buffer and stored at -20 oC. Two and half μl of the DNA were used for a restriction digestion. RNA contamination was degraded by adding 1μl of a 10 mg/ml RNase solution. The pellet was then resuspended in 0.5 ml TE buffer and stored at -20 ℃(Ausubel, et al. 1999).
2.9.2 DNA electrophoresis
The Plasmid DNA was loaded onto 1% agarose with ethidium bromide and electrophoresised at 50 V for 1 hr at room temperature. Plasmid pRK291 extracted from E. coli HB101 (20 kb) and plasmid pC22 extracted from E. coli MC1061 (17.5 kb) were used as markers (bought from FIRDI, Hsin-Chu, Taiwan. Category No. BCRC 41277 and BCRC 41178 ). All plasmids DNA samples were extracted using the same method.
2.10 Preparation of competent cells
Calcium chloride
Single colony of E. coli HB 101
Centrifuge (Hitachi Himac CR22G) Procedure:
Inoculated a single colony of E. coli HB 101 into 30 ml LB medium. Cells grew overnight at 37℃ with shaking 100 rpm. Ten ml of the culture were inoculated into 400 ml LB medium in 1 liter flask. Cells were then grew overnight at 37℃ with shaking 100 rpm, to an OD590 of 0.375 (about 3-4 hours). This procedure aimed to get cells to grow in early-or mid-log phase. The culture was aliquoted into four 50 ml prechilled, sterile polypropylene tubes and left on ice 5 to 10 min. Cells were then centrifuged 7 min at 1,600 xg at 4℃. The pellet resuspended gently in 10 ml ice cold CaCl2 solution.
Two hundred and fifty μl of cells were aliquoted into prechilled, sterile polypropylene tubes and freezed immediately at -70℃(Ausubel, et al. 1999).
2.11 Transformation of agarase gene (Heat shock)
Modified C-10 agar plate1% agar
0.1% yeast extract 0.5% tryptone 1% NaCl SOC medium
0.5% (w/v) yeast extract
Rapidly thaw competent cells (100 ml) by warming between hands, then immediately dispensed 1μl of plasmid DNA (13.4 ng/μl) into cell tubes, gently swirled, and placed on ice 10 min. Heat shocked cells by placed tubes in 42℃ water bath for 2 min. Five ml of SOC medium containing 0.1% agar were added to each tube.
Cells incubated 1 hr at 37℃ with shacking 100 rpm (Ausubel, et al. 1999). Series dilutions of transformed cells were plated on modified C-10 agar plates and C-10 agar plates separately, and incubated overnight at room temperature. Transformants were screened for agarolytic activity by visually inspecting of the agar plates’ surfaces. Plate surface showed shallow depressions were confirmed to be agarase positive.