Screening and enzymatic activity of high-efficiency gellan lyase producing bacteria Pseudoalteromonas hodoensis PE1

Figures & data

Figure 1. Screening plate pit. Gellan was the coagulant and the sole carbon source; as it was degraded by lyase, obvious pits would be produced on the plate. Thus, high-efficiency gellan lyase producing bacteria could be obtained by isolating and purifying the strains in the pits.

Table 2. Four strains of gellan lyase producing bacteria.

serial number	Bacteria	Similarity（%）	Completeness（%）	NCBI registration number
PE1	Pseudoalteromonas hodoensis	98.95%	100%	MK788321
PE2	Achromobacter piechaudii	99.65%	98.4%	MK909908
PE3	Pseudomonas meliae	98.23%	86.0%	MK909909
PE4	Enterobacter bugandensis	99.73%	99%	MK909914

16S rDNA sequencing was carried out. The PCR products were sequenced and compared with the BLAST program to determine the classification status of strains.

Figure 2. Phylogenic tree of Pseudoalteromonas hodoensis with related species. Neighbor-joining tree based on 16S rDNA sequences shows the position of Pseudoalteromonas hodoensis. The marker bar denotates the relative strain similarity.

Figure 3. Strain streaking and purification plate, scanning electron microscopy and gram-staining. (a) Strain streaking and purification plate, forming a pale yellow and opaque colony size of 2–3.5mm. (b) Strain scanning electron microscopy, the specific morphology of the bacterial strain was observed by scanning electron microscopy. There was no obvious flagellum and the dimension ranges were 2.2–3.1 μm long, 0.4–0.9 μm wide. (c) The result of gram staining which was red under a light microscope was gram-negative bacteria.

Table 3. Physiological activity detection results.

Physiological activity detection	Results
Arginine	+
Ornithine	+
Lysine	+
Citrate	+
Urea	+
Nitrate reduction	-
ONPG	-
Sucrose	-
Glucose	-
Hydrogen sulfide	-
Methyl red	-
MR-VP	-
Indigo matrix	-

Biochemical identification tube for the physiological and biochemical detection of bacterial strains

Table 1. Effect of various ions and inhibitors on gellan lyase activity (unit:U/ml).

compound	0 min	60 min	difference value	relative activity（%
blank	0.212 ± 0.002	0.324 ± 0.003	0.112	100%
EDTA	0.242 ± 0.001	0.327 ± 0.004	0.085	75.89%
KCl	0.156 ± 0.001	0.274 ± 0.005	0.118	105.36%
MnCl2	0.221 ± 0.005	0.376 ± 0.003	0.155	138.39%
CaCl2	0.192 ± 0.001	0.316 ± 0.001	0.124	110.71%
NaCl	0.227 ± 0.003	0.348 ± 0.003	0.121	108.04%
(NH4)2SO4	0.415 ± 0.004	0.532 ± 0.002	0.117	104.46%
urea	0.164 ± 0.002	0.199 ± 0.003	0.048	42.86%

The enzyme was preincubated for 30 min at 30°C in Tris–HCl buffer, pH 7.0, containing different ions and inhibitors at final concentration 1 mM and the activity was then determined at 30°C, pH 7.0. The difference value was calculated by the difference between absorbance at the initial time 0 min and reaction time at 60 min. At the same time, relative activity was determined by comparing the difference between different ions or compounds and the blank control.

Figure 4. The optimum temperature reaction conditions for gellan lyase (unit:U/ml) .The enzyme was preincubated at pH 7.0 in 50 mM Tris–HCl buffer, with different temperature gradients. The lyase activity was determined by spectrophotometry at 235nm every 30 min.

Figure 5. The optimum pH reaction conditions for gellan lyase (unit:U/ml).The enzyme was preincubated at 30°C in 50 mM Tris–HCl buffer, with different pH gradients. The lyase activity was determined by spectrophotometry at 235nm every 30 min.

Figure 6. TLC profile of the gellan degradation product after 48h incubation. Lane 1, gellan (0.05%); lane 2, rhamnose (1%); lane 3, glucose (1%); lane 4, gellan degradation product after 48h incubation.

Figure 7. HPLC analysis of the gellan degradation products. Gellan was converted to a tetrasaccharide (4,5-ene-glucuronyl-glucosyl-rhamnosyl-glucose) by the gellan lyase.