In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa

Abstract

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The K_m and V_max of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t _1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-024-01334-w.

Introduction

Alginate is a linear polysaccharide composed of β-D-mannuronic acid and α-L-guluronic acid units linked together by 1,4-O-glycoside bonds [1]. Alginate is widely distributed in nature as a constituent of renewable biopolymer resource. Brown algae cell walls and matrix contains alginate as one of the main constituents [2]. Two bacterial genera Azotobacter and Pseudomonas are known to synthesize alginate [3]. Alginate has excellent gel forming ability; therefore, it is widely used as a biomaterial for hydrogels preparation in drug delivery systems and tissue engineering [4]. Besides this, it also possesses several biological properties including antibacterial, antioxidant, neuroprotective and antitumor properties [5]. Alginate lyase degrade alginate through β-elimination reaction of glycoside bonds into monosaccharides or oligosaccharides [6]. Alginate lyase has widespread application in therapeutics, bioethanol and biofuel production, marine carbon recycling and in feed industry. Various bacteria, fungi, bacteriophages, algae, molluscs and viruses are known to produce alginate lyases [7]. Many bacteria Gram-negative bacteria such as Klebsiella aerogenes, K. pneumonia, Azotobacter vinelandii, Pseudomonas maltophilia, Flavobacterium sp. Sphingomonas sp., P. aeruginosa and P. putida. Gram-positive bacteria including Bacillus circulans and Paenibacillus algicola are well known to produce alginate lyase [1]. In plants, it promotes growth, increases yield, inhibit fungal growth and improve stress resistance, thus can be used as bio-fertilizer and bio-pesticide [8]. Lyases can regulate blood sugar and lipids, and can induce cytokine production. Therefore, has potential applications in pharmaceutical and food industries [6].

Pseudomonas aeruginosa forms biofilms, containing large amount of alginate, which shields it from antibiotics and provide high resistance to antibiotics [9]. P. aeruginosa is a gram-negative opportunistic human pathogen, well known to cause lung infection in cystic fibrosis patients and urinary tract infections in catheterized patients [10]. Therefore, it becomes very tough to manage biofilm-associated infections caused by P. aeruginosa [11]. Alginate lyases has ability to degrade the extracellular polymeric substances in biofilms and resulting in better accessibility of antibiotics to the pathogen. Hence, lyases could be used as potential therapeutic adjunct agent along with antibiotics to treat cystic fibrosis [12].

Due to advancement in the field of structural biology, many different families of polysaccharide lyases has been characterized and reported. Based on structure and carbohydrate binding modules polysaccharide-degrading enzyme has been categorized in fourteen families. PL5, PL15, and PL17 families have dissimilar structures but same lyase activities. PL7, PL14, and PL18 families have a β-jelly roll fold structure and PL6 family has a β-helix fold [7]. Further, based on mode of action, lyases is categorized into exonuclease and endonuclease: exonuclease degrades oligosaccharides into oligosaccharides monomers while endonuclease cleaves glycosidic bonds [13]. Enzymatic hydrolysis of alginate yields alginate oligosaccharide (AOS), has great application in healthcare, food and agriculture field [9, 14].

Alginate lyase has fascinated many researchers due to their unique properties and application potential in various fields: agricultural, environmental and bioenergy [1, 6]. Microorganisms appears to be the promising sources for alginate lyase, as they can produce enzyme in large amount at low production cost. Genus Paenibacillus has been reported to possess excellent capacity for degrading a variety of polysaccharides [15]. In the light of the above facts, we have purified and characterized new alginate lyase SG4 + from P. lautus strain SG4 + . RSM was employed for optimization of alginate lyase SG4 + production in shake flask and batch fermenter of 14L. Further, its ability to degrade the extracellular polymeric substances in biofilms was explored for its application in healthcare improvement.

Material and methods

Microorganism

Alginate lyase-producing strain SG4 + , was isolated Sargassum wightii (algae) using enrichment culture technique. The isolate was characterized as P. lautus strain SG4 + based on 16S rRNA gene sequence. Genbank Database accession number of P. lautus strain SG4 + is MN216324 [16]. Enzymatic assay of key enzymes displayed that the strain, is a potent alginate lyase producer. For further studies, culture was maintained on Luria-Broth containing sodium alginate (0.4%, w/v) at 4 °C. All chemicals used in present study were of analytical and bacteriological grade procured from Hi-Media, India unless mentioned otherwise.

Alginate lyase activity assay

P. lautus strain SG4 + was cultured on Luria-Broth containing sodium alginate (0.4%, w/v) for 24 h and incubated at 37 °C. Cells were pellet out by centrifuging the broth at 10,000 g for 15 min at 4 °C. Wet cell pellet of a known amount was placed in an oven at 80 °C overnight and the corresponding absorbance of the cell slurry was measured at A₆₀₀ nm in a spectrophotometer (Shimadzu, Japan). The cell mass was measured from the standard curve and expressed in the terms of dry cell weight (mg/ml).

Alginate lyase activity in the supernatant was determined by measuring the amount of reducing sugar formed by alginate breakdown using 3, 5-Dinitrosalicylic acid (DNS) reagent as per [17] with slight modification. In brief reaction mixture contained supernatant (0.2 ml) and 1% of sodium alginate (0.8 ml) dissolved in 50 mM Tris–HCl buffer (pH 8.5). The reaction was stopped by adding DNS reagent and subsequently mixture was heated in water bath for 5 min at 100 °C. Absorbance of reaction mixture was recorded at 540 nm using UV–VIS spectrophotometer. Enzyme activity was expressed in terms of international Units (IU).

Statistical optimization of SG4 + 

The Plackett–Burman statistical design is very commonly used to study and screen most important media components effecting alginate lyase production [18]. For optimization studies, following eight independent variables: sodium alginate, peptone, NaCl, yeast extract, MgSO₄.7H₂O, incubation time, agitation and inoculum volume were considered. Effect of variables on the alginate lyase was studied. The experiment comprised of 12 run based on the design, each factor was observed at two levels for a low level (-1) and a high level (+ 1). According to the design, the key factors does not have interactions and follow first-order multiple regression models.

$$\text{Y}={\beta }^{\circ }+iix\sum \beta (\text{i}=1,\cdots .\text{k})$$ 1

where Y is the response function (alginate lyase production) and ${\beta }^{\circ }$ is the model intercept.

A four level RSM based CCD was used for process parameters optimization of alginate lyase production from P. lautus strain SG4 + . Based on observations of PBD experiment, four parameters directly associated with fermentation such as inoculum volume, agitation, yeast extract, and sodium alginate were selected. The CCD contains a total of 30 experimental runs. The experiment was conducted and enzyme activity (U/ml) was recorded as a response (Y). The statistical software “Design-Expert 14.0” (StatEase) was used to analyse the experimental data. The collective effect of various variables on alginate lyase yield was analysed. Three-dimensional response curves were plotted against any two independent parameters, holding the other independent parameters at their initial ‘0’ levels. The significance of model and regression coefficients were studied using analysis of variance (ANOVA). The determination coefficient was used to assess the quality of a polynomial equation (R2). To investigate the mutual effects of different independent parameters on alginate lyase yield, the selected parameters (optimum values) 3D plots were plotted. For the validation of statistical model, the production of alginate lyase SG4 + was carried out experiment in flask under shaking conditions using predicted parameters.

Purification of alginate lyase SG4 + 

For maximum production of alginate lyase, P. lautus strain SG4 + was cultured in optimized medium under submerged condition for 48 h. The cell free supernatant was subjected to ammonium sulphate precipitation (30–60% saturation) followed by dialysis against Tris–HCl (0.05 M, pH 8.5). This enzyme was loaded on DEAE-Sepharose column (20 × 1.6 cm), pre-equilibrated with 50 mM Tris–HCl (8.5). Linear NaCl gradient (0.1–0.7 M) at flow rate of 1.0 ml/min was used to elute the bound protein. The fractions of 1.5 ml each were collected and concentrated. Alginate lyase activity of each fraction was estimated. The fractions exhibiting enzyme activity were pooled and molecular weight of purified enzyme was estimated using 10% SDS and Native-PAGE [19].

To identify the active band, zymography was performed [20]. The activity of alginate lyase SG4 + was detected on 10% polyacrylamide gel run under non-denaturing conditions. Once electrophoresis was over, this gel was placed on agarose gel (2%, w/v) containing sodium alginate (1 mg/ml), flooded with 0.05 M Tris–HCl buffer (pH 8.5) and incubated at 30° C for 24 h. Thereafter, native-PAGE gel was discarded and agarose gel was flooded with cetylpyridinium chloride (10%, w/v) to observe the zone of alginate hydrolysis.

Characterization of alginate lyase SG4 + 

The optimal temperature of purified enzyme was determined at different temperature (25–50 °C) in 0.1 M Tris–HCl (pH 8.5). In order to observe the effect of incubation time on the activity of alginate lyase, the reaction was incubated at 30 °C for 55 min and after every 5 min alginate lyase activity was recorded. The alginate lyase activity was measured using variable substrate concentrations, ranging from 0.5–5 mg/ml. Graph was plotted between 1/[S] vs 1/[V] and K_m and V_max values of the purified alginate lyase SG4 + were calculated using Lineweaver–Burk plot. Thermostability was determined by preincubating the enzyme at different temperatures (4, 30 37, 50 and 60 °C). Samples were withdrawn at regular time intervals and activity was determined. All experiments were run at least three times with triplicate data points within each run.

Scale-up studies for alginate lyase SG4 + 

The culture was inoculated in seed medium (pH 7.5), containing (%, w/v): sodium alginate 0.25, yeast extract 0.612, peptone 0.5, NaCl 2.0, MgSO₄.7H₂O 0.002, K₂HPO₄ 0.001 and MnCl₂ 0.002 under submerged condition at 30 °C and 150 rpm. The 24 h old seed culture, (10%, v/v) was transferred into 700 ml culture media and incubated at 30 °C for 24 h at 150 rpm and used as inoculum for batch fermentation. The composition of production medium was same as seed medium. Production was carried out in 14L bench scale-fermenter (Scigenics, India) with working volume of 7L. Fermenter was well equipped with a bladed disc turbine impeller, air sparger and digital control for aeration, agitation, DO, and temperature. The initial medium pH was set 7.5 and was not controlled during fermentation. In situ, sterilization of 7.0 L production medium was carried out at 121 °C for 15 min. Silicone oil (2 ml) was used as antifoam agent and sterilized medium was aseptically inoculated (10%, v/v) using peristaltic pump through inoculation port. Fermentation was carried for 48 h and periodical sampling was done at 4 h interval, samples were used for growth estimation and enzyme assay. DO of the fermentation broth was recorded throughout the fermentation process using DO probe (Ingold, UK). The dependence of alginate lyase SG4 + on independent variables was calculated using Design Expert (Stat Ease 14.0 Inc., Minneapolis, USA). CCD results response was analysed using second-order polynomial equation:

$$\text{Responses}=\text{R}1=+1.00+0.16\mathrm{A}-0.020\mathrm{B}-0.46\mathrm{A}\mathrm{B}-0.26{\text{A}}^2{\text{B}}^2$$

Effect of the agitation and volumetric flow rate on alginate lyase SG4 + production was studied using CCD. Various combinations of agitation speed (59, 100, 200, 300 and 341 rpm) and aeration rate (0.146, 0.25, 0.5, 0.75 and 0.85 vvm) for alginate lyase production was studied in batch fermentation.

Biofilm formation

For biofilm assay, all strains were grown for 24 h at 37 °C in tryptic soy broth (TSB). After 24 h, the bacterial suspension OD was adjusted to 0.5 McFarland (1 × 10⁸ cfu/ml) according to CLSI guidelines. For the development of in vitro biofilm, TSB supplemented with 0.2% (w/v) glucose and culture (10 μl) were added into each respective well. Microtiter plate was incubated for 48 h at 37 °C [20]. After incubation biofilm biomass was quantified by measuring crystal violet solution (0.2%) intensity at OD₅₇₀ nm using UV–Vis spectrophotometer. The experiments were performed in triplicates and repeated thrice.

Determination of minimum inhibitory concentration and Minimum biofilm inhibitory concentration

MICs (amikacin and gentamicin) were determined using the microbroth dilution assay according to CLSI guidelines. Effect of antibiotics on biofilm reduction of test strains was determined using MTP assay [20]. For assay, strains were grown in TSB containing 0.2% w/v glucose and antibiotics (0 to 40 μg/ml). The aliquots (100 μL) was distributed in each well and under static conditions incubated at 37 °C for 24 h. Thereafter, the CV staining was performed and OD₅₇₀ nm was recorded.

Anti-biofilm activity of alginate lyase SG4 + 

Alginate lyase SG4 + ability in combination with antibiotics to eradicate the pre-formed biofilm of P. aeruginosa was studied using micro-dilution assay [21]. For analysis, 25 μl of antibiotics [gentamicin (40 µg/ml) and amikacin (20 µg/ml)], 12.5μL Tris–HCl (0.5 mm) and 3 7.5μL alginate lyase (18 IU) was added in MTP wells containing biofilm. This concentration was chosen as it was sufficient to reduce biofilm formation. The plate was incubated for 24 h at 37 °C. The eradication of biofilm and CFU were determined at each treatment conditions. The MTP’s wells those were not treated with antibiotic and alginate lyase were used as control.

Assessment of biofilm viable cells and scanning microscopy

For viable cell count, test strains were grown in microtiter plate containing standardized combination of alginate lyase and antibiotics. After 24 h incubation, plates were washed trice with 200 μl PBS and resuspended in 100 μl PBS. The suspension containing cells were serially diluted (10^–1 to 10^–5) and five drops of 10 μl each was dropped on TSA plates and incubated for 24 h incubation at 37 °C and CFU was calculated after incubation [22]. All the experiments were performed in triplicates and repeated thrice.

For SEM, biofilms were treated with alginate lyase SG4 + , and with the cocktail of alginate lyase SG4 + and amikacin. The untreated sample was considered as control. After treatment the cell suspensions were washed with PBS and the samples were processed for SEM [23]. The gold coated specimens were visualized under a scanning electron microscope (Jeol, Japan).

Statistical analysis

Each experiment was performed in triplicate and repeated thrice. Data is presented as mean values (n = 3). Statistical analysis was performed with SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) using one-way ANOVA and Tukey's multiple comparison test (P < 0.05). The differences in results were deemed significant at P < 0.05.

Results

Statistical optimization for alginate lyase SG4 + production

The effect of eight independent variables was studied in twelve runs using the PBD and each factor was examined at two levels, low (-1) for and high (+ 1). Influence of eight independent variables on alginate lyase production is displayed in Table 1. A total of twelve experiments revealed alginate lyase activity variations ranging from 0.054 IU to 0.2750 IU. To determine the order of significance of variables on alginate lyase SG4 + production Pareto chart was created (Supplementary Fig. 1). This demonstrated that four of the eight independent variables, namely yeast extract, sodium alginate, agitation speed, and inoculum volume, have a positive effect on alginate lyase production.

Table 1.

Plackett–Burman design for evaluating the influence of various independent variables on alginate lyase SG4 + production

Run	Yeast extract (%, w/v)	Peptone (%, w/v)	NaCl (%, w/v)	Sodium alginate (%, w/v)	Incubation time (h)	Inoculum Volume (%, v/v)	Agitation (rpm)	MgSO4.7H2O (%, w/v)	Response (IU)
1	0.25	0.25	4.0	0.1	72	5.0	300	0.04	0.0622
2	0.25	0.25	1.0	0.4	24	15.0	300	0.01	0.2750
3	1.0	0.25	4.0	0.4	24	15.0	300	0.04	0.1630
4	1.0	1.0	1.0	0.4	72	5.0	300	0.01	0.2634
5	0.25	0.25	1.0	0.1	24	5.0	100	0.01	0.1409
6	1.0	0.25	1.0	0.1	72	15.0	100	0.04	0.1355
7	0.25	1.0	1.0	0.4	72	15.0	100	0.04	0.1584
8	0.25	1.0	4.0	0.1	72	15.0	300	0.01	0.054
9	1.0	0.25	4.0	0.4	72	5.0	100	0.01	0.1127
10	1.0	1.0	1.0	0.1	24	5.0	300	0.04	0.1987
11	1.0	1.0	4.0	0.1	24	15.0	100	0.01	0.1466
12	0.25	1.0	4.0	0.4	24	5.0	100	0.04	0.1098

CCD was used to determine the optimum level and combined effect of four variables with a positive effect (yeast extract, sodium alginate, agitation, and inoculum volume) on alginate lyase production while keeping the rest of the production conditions and medium components constant (Table 2). Out of the thirty experimental trails, the maximum production of alginate lyase obtained was in trail 27, the response was 0.849 IU. Analysis of variance (ANOVA) revealed that the present studied model was very appropriate for optimising alginate lyase SG4 + production, and Design-Expert 14.0″ (StatEase) recommended four process orders. Based on the ANOVA results of CCD, the model F-value (351.66) and the P-value (0.0001) indicate that the model was appropriate and satisfactory (Table 3). Cubic and quadratic process orders produced comparable results, and quadratic process order was recommended for further analysis due to R2 value (0.9102) and low standard deviation (0.1038). The coefficient of determination "R2″ value indicated that this model could explain 98% of the response variability and indicated model accuracy (Table 4). The 3D graphs were created for the regression analysis of the design, which used a pairwise combination of four factors to produce give effect on alginate lyase SG4 + production. The interaction between sodium alginate, yeast extract, agitation, and inoculum volume is depicted in [(Supplementary Fig. 2 (a-f)]. The shape of the response surface indicates the effect of these variables. Lower and higher values result in lower enzyme activity, and similar type of response is observed in all variable combinations.

Table 2.

Central composite experimental design using factors having positive effects on lyase SG4 + production

Runs	Yeast extract (%, w/v)	Sodium alginate (%, w/v)	Agitation (rpm)	Inoculum volume (%, v/v)	Response (IU)
1	1.38	0.25	200	10	0.461
2	0.625	0.25	200	10	0.837
3	0.25	0.40	100	15	0.303
4	0.25	0.40	300	5	0.297
5	1.00	0.10	100	10	0.251
6	0.625	0.55	200	10	0.476
7	0.625	0.25	400	10	0.353
8	0.625	0.25	200	20	0.6268
9	0.25	0.10	300	5	0.314
10	0.0	0.25	200	10	0.464
11	0.625	0.25	200	10	0.839
12	0.625	0.25	200	10	0.830
13	1.0	0.10	100	15	0.248
14	0.625	0.25	0.00	10	0.1764
15	0.25	0.40	100	5	0.303
16	1.00	0.10	300	5	0.435
17	0.25	0.10	300	15	0.301
18	0.625	0.0	200	10	0.0934
19	0.625	0.25	200	10	0.846
20	0.625	0.25	200	10	0.842
21	1.00	0.40	100	5	0.294
22	0.25	0.4	300	15	0.601
23	0.25	0.10	100	15	0.188
24	0.625	0.25	200	00	0
25	0.25	0.10	100	5	0.217
26	1.0	0.40	300	5	0.553
27	0.625	0.25	200	10	0.849
28	1.00	0.40	100	15	0.294
29	1.00	0.40	300	15	0.648
30	1.00	0.10	300	15	0.339

Table 3.

ANOVA for Response Surface

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	1.64	14	0.1171	10.86	< 0.0001	Significant
A-yeast extract	0.0109	1	0.0109	1.01	0.3300
B-sodium alginate	0.1269	1	0.1269	11.78	0.0037
C-agitation rate	0.0934	1	0.0934	8.67	0.0101
D-inoculum volume	0.0980	1	0.0980	9.10	0.0087
AB	9.000E-06	1	9.000E-06	0.0008	0.9773
AC	0.0219	1	0.0219	2.03	0.1744
AD	0.0038	1	0.0038	0.3510	0.5624
BC	0.0245	1	0.0245	2.27	0.1524
BD	0.0196	1	0.0196	1.82	0.1975
CD	0.0008	1	0.0008	0.0780	0.7838
A2	0.2310	1	0.2310	21.43	0.0003
B2	0.5089	1	0.5089	47.23	< 0.0001
C2	0.5470	1	0.5470	50.76	< 0.0001
D2	0.4557	1	0.4557	42.29	< 0.0001
Residual	0.1616	15	0.0108
Lack of Fit	0.1614	10	0.0161	351.66	< 0.0001	Significant
Pure Error	0.0002	5	0.0000
Cor Total	1.80	29

Table 4.

Quadratic fitted model analysis for alginate lyase S4 + production by P. lautus

Std. Dev	0.1038	R2	0.9102
Mean	0.4420	Adjusted R2	0.8264
C.V. %	23.48	Predicted R2	0.4835
		Adeq Precision	9.4042

Validation of model

The maximum alginate lyase SG4 + activity (0.849 IU) was obtained by carrying out the experiments using statistical experiment data which is slightly higher than the predicted value 0.840 IU). A high degree of similarity was observed between the predicted and experimental values. These values are in the range of the operating variables and thus reflect the usefulness of response surface methodology for optimization of alginate lyase production parameters (Supplementary Fig. 3). A regression model was used to predict observations on the response Y (alginate lyase SG4 + activity) associated with specific variable values. (Fig. 4) depicts the deviation of different variables from the reference point in the alginate lyase SG4 + production. The enzyme activity ranged from 0 to 0.849 IU depending on the conditions of the experiments. The effect of various factors using optimum conditions in the design space is compared in the perturbation plot (SupplementrayFig.4). A nearly steep curvature in agitation and inoculum volume indicated that enzyme activity was very sensitive to these variables, whereas relative flat lines in yeast extract and sodium alginate concentration indicated lower sensitivity to change.

Fig. 4.

A Effect of agitation speed and aeration rate on growth in fermenter. (b) Effect of agitation speed and aeration rate on alginate lyase SG4 + production (c) Effect of agitation speed and aeration rate on DO during growth (d) The course of fermentation for alginate lyase SG4 + production at optimum combination 100 rpm and 0.75 vvm

Purification and characterization of alginate lyase SG4 + 

Alginate lyase SG4 + was purified to homogeneity using a single chromatography step. The enzyme was purified 15.07 times with a yield of 29.6%. (Table 5). Purity and molecular mass were analysed using 10% SDS-PAGE. The analysis revealed that the alginate lyase SG4 + had a subunit molecular mass of 55 kDa (Fig. 1a) and a total molecular weight of 110 kDa on native PAGE (Fig. 1b). The results indicate that alginate lyase SG4 + exists in dimeric form.

Table 5.

Summary of purification of alginate lyase SG4 + isolate P. lautus

Procedure	Volume (mL)	Protein (mg/mL)	Total protein (mg/mL)	Enzyme activity (IU)	Total enzyme activity (IU)	Specific activity (IU/mg)	Purification fold	Yield (%)
Crude extract	250	0.11	27.5	1.46	365	13.27	1	100
(NH4)2 SO4 precipitation (30–60% saturation) and dialysis	10	0.23	23	25	250	108.7	8.1	68.5
DEAE-Sepharose	6	0.09	0.54	18	108	200	15.07	29.6

Fig. 1.

Electrophoretic profile of alginate lyase SG4 + a) SDS-PAGE: Lane 1-Standard markers (GeNie™): Lane 2-Dialysed enzyme. Lane 3- Purified enzyme Lane 4–6 same as in Lane 1–3 in silver staining b) Native PAGE: Lane 1-Standard molecular markers (GeNei™): Lane 2-Dialysed enzyme Lane 3- Purified enzyme Lane 4—Zymogram

The biochemical characterization of alginate lyase SG4 + revealed that it displays maximum activity at 30 °C using Tris–HCl buffer (0.05 M, pH 8.5) (Fig. 2 a). The optimum incubation time for alginate lyase SG4 + was 25 min (Fig. 2b). The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 µmol/ml-min, respectively (Fig. 2c). Alginate lyase SG4 + was preincubated at various temperatures, and residual enzyme activity was measured to determine thermostability (Fig. 2d). The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. When the preincubation temperature was raised above 40 °C, the relative activity dropped dramatically. However, alginate lyase SG4 + retained 88% of its activity at 4 °C even after a 7-day preincubation period.

Fig. 2.

A Effect of temperature on alginate lyase SG4 + (b) Effect of incubation time on alginate lyase SG4 + (c) Lineweaver–Burk plot for determining K_m and V_max (d) Thermostability of purified alginate lyase SG4 + 

Scale up of alginate lyase using CCD

Primary goal of fermenter studies, was to investigate the combinatorial effect of the independent variables (aeration rate, and agitation speed), on alginate lyase SG4 + production. CCD experimental design comprised of total thirteen experimental runs; aeration speed, agitation rate and enzyme activity of each trial is displayed in Table 6. Alginate lyase SG4 + maximum production (1.46 IU) was obtained at 100 rpm and 0.75 vvm (Table 6).

Table 6.

CCD table showing the effect of varying rate of agitation speed and aeration

Run	Aeration Speed (vvm)	Agitation Rate (rpm)	Enzyme Activity (IU)
1	0.25	100	0.21
2	0.5	341.421 set at 340	0.11
3	0.75	300	0.12
4	0.5	200	1.07
5	0.5	200	0.90
6	0.146447	200	0.13
7	0.5	200	0.90
8	0.75	100	1.46
9	0.5	58.5786 set as 59	0.67
10	0.853553	200	0.55
11	0.5	200	1.05
12	0.25	300	0.72
13	0.5	200	1.08

Further, CCD results were analysed using ANOVA, and model F-value (Table 7). The model's F-value (17.57) indicates that it is significant. Model P-value (Prob > F) i.e. 0.0008, indicates the model's adequacy. Greater the magnitude of the P, greater the significance of the corresponding coefficient. The F-value of the model is slightly large (0.08%), this might be drowned in noise. Prob > F values less than 0.0500 indicate that model terms are significant. A, B, AB, A2, B2 are practical model terms in this case. The Pred R-Squared of 0.5556 is not as close as expected to the Adj R-Squared of 0.8735. (Table 8).

Table 7.

ANOVA for Response Surface

Source	Sum of Squares	Df	Mean Square	F-value	p-value
Model	2.16	5	0.043	17.57	0.0008	Significant
A-Aeration rate	0.19	1	0.19	7.86	0.0264
B-Agitation rate	0.33	1	0.33	13.37	0.0081
AB	0.86	1	0.86	34.78	0.0006
A2	0.49	1	0.49	19.77	0.0030
B2	0.40	1	0.40	16.21	0.0050
Residual	0.17	7	0.025
Lack of Fit	0.14	3	0.046	5.46	0.0673	not significant
Pure Error	0.034	4	8.450E-003
Cor Total	2.33	12

Table 8.

Quadratic mo del Analysis of variance table

Std. Dev	0.16	R2	0.9262
Mean	0.69	Adjusted R2	0.8735
C.V.%	22.73	Predicted R2	0.5556
PRESS	1.04	Adeq Precision	12.487

Interaction of independent variables on alginate lyase SG4 + production was studied using three-dimensional response surface graphs. The graphs depict the synergistic effect of each independent variable on the response (Fig. 3a). Regression equation was used to determine the optimal levels of both parameters for maximum response. The response surface curvature shows a lower value for agitation speed and a higher value for aeration rate. Alginate lyase SG4 + activity increased, with decrease in agitation speed from 300 to 100 rpm and increase in aeration rate from 0.25 to 0.75 vvm. Actual enzyme units achieved in experimental trail was 1.46 IU, which was very close to the predicted value (1.32 IU) calculated by ANOVA (Fig. 3b). A perturbation graph was created to represent the effect of impeller speed (rpm) and volumetric flow rate (vvm) at the optimum run conditions in the design space (Fig. 3c). A sharp curvature line for both aeration rate (A) and agitation speed (B) strongly was observed, this indicates enzyme activity was directly depend on these factors. Maximum biomass (10.15 mg/ml) production was achieved after 20 h incubation, at 100 rpm and 0.75 vvm aeration (Fig. 4a). Further, less than 2 mg/ml cell mass was obtained when fermentation batches were conducted at 0.5 vvm aeration/58 rpm agitation, 0.25 vvm aeration/300 and 0.5 vvm aeration/341 rpm agitation.

Fig. 3.

A Response surface plots for the production of alginate lyase variable components were agitation speed (rpm) and aeration rate (vvm). (b) Predicted vs. actual plot for different experimental runs in CCD. (c) Perturbation plot showing the optimum value for different variable

The DO profile of P. lautus strain SG4 + during fermentation was studied and the results suggested that DO increases with increase in agitation and aeration rate (Fig. 4b). DO reaches its minimum value (< 50% saturation) after 24 h of fermentation at high agitation (300 and 341 rpm) and aeration rates (0.75 and 0.5vvm). At 100 rpm and 0.75 vvm aeration, DO saturation decreased to 4%, within 40 h of fermentation but increased later. Maximum enzyme activity i.e. 1.46 IU was achieved after 20 h of fermentation (100 rpm, 0.75 vvm) (Fig. 4c), followed by 1.01 IU after 40 h (200 rpm, 0.5 vvm). According to the perturbation curve and fermentation studies, increasing the aeration rate from 0.146 to 0.75 vvm was beneficial to alginate lyase SG4 + production. However, increasing the agitation rate (100 to 341 rpm) decreased alginate lyase activity. In shake flask, maximum biomass (2.28 mg/ml) and alginate lyase production (0.321 IU) was achieved after 40 h of incubation. However, significant achievement in enzyme production (1.460 ± 066 IU) and biomass (10.15 ± 0.57 mg/ml) was achieved, after 20 h incubation of fermentation (Fig. 4d).

Effect of alginate lyase SG4 + and antibiotics on biofilm

Strains formed strong biofilm at 48 h but at 24 h the strength was weak (Table 9). Both gentamicin and amikacin have shown inhibitory action against all three mucoid strains of P. aeruginosa. MIC of amikacin for P. aeruginosa MTCC 2297, P. aeruginosa HSS and P. aeruginosa SSH were found to be 5.3, 5.0 and 4.9 µg/ml, respectively. However, MIC value of gentamicin for P. aeruginosa MTCC2297, P. aeruginosa HSS, P. aeruginosa SSH were 7.8, 7.8 and 7.9 µg/mL, respectively (Table 10). Gentamicin (40 μg/ml) reduced the biofilm of P. aeruginosa MTCC 2297, P. aeruginosa HSS and P. aeruginosa SSH to 5.3, 6.7, 7.2%, respectively. The amikacin (20 µg/ml) reduced the biofilm of P. aeruginosa MTCC 2297, P. aeruginosa HSS and P. aeruginosa SSH to 9.1, 9.8 and 10%, respectively. MBEC for gentamicin was 40 μg/ml whereas for amikacin it was 20 µg/ml.

Table 9.

Biofilm formation after 24 h and 48 h

Organism	Biofilm formation
	24 h	48 h
P. aeruginosa MTCC2297	0.530 < 0.953 ≤ 1.06 (weak)	0.58 < 1.531 ≤ 2.32 (Moderate)
P. aeruginosa SSH	0.530 < 1.0525 ≤ 1.06 (weak)	0.58 < 1.448 ≤ 2.32 (Moderate)
P. aeruginosa HSS	1.06 < 1.174 ≤ 2.12 (Moderate)	0.58 < 2.067 ≤ 2.32 (Moderate)

Table 10.

MIC of antibiotics against P. aeruginosa

Organisms	Antibiotics	MIC (µg/mL)
P. aeruginosa MTCC2297	Amikacin	5.3
	Gentamicin	7.8
P. aeruginosa HSS	Amikacin	5.0
	Gentamicin	7.8
P. aeruginosa SSH	Amikacin	4.9
	Gentamicin	7.9

Alginate lyase SG4 + in combination with amikacin and gentamicin was found to reduce biofilms formed by test strains. Alginate lyase SG4 + alone reduce the biofilm of P. aeruginosa MTCC2297, P. aeruginosa HSS and P. aeruginosa SSH by 38.7, 43.5 and 45.5%, respectively (Table 11). However, gentamicin or amikacin alone could reduce less than 10% of alginate biofilm formed by P. aeruginosa strains. Maximum biofilm reduction was achieved against P. aeruginosa SSH by using alginate lyase SG4 + in combination with gentamicin (52.25%) and amikacin (64.60%).

Table 11.

Disintegration of P. aeruginosa biofilm by co-administration of antibiotics with enzyme

Cultures	Biofilm disintegration (%)
	Antibiotic		Heat denatured enzyme	Enzyme (18.0 IU)	Enzyme (18.0 IU) + Gentamicin (40 µg/mL)	Enzyme (18.0 IU) + Amikacin (20 µg/mL)	Control
	Gentamicin (40 µg/mL)	Amikacin (20 µg/mL)
P. aeruginosa MTCC2297	5.6bx ± 0.12	10.1cx ± 1.20	0.65ax ± 0.14	38.7dx ± 1.03	48.36ex ± 2.29	58.3fx ± 1.09	0a
P. aeruginosa HSS	6.9bxy ± 0.15	9.1bx ± 2.01	0.99ay ± 0.2	43.5cy ± 2.0	51.50dx ± 2.23	61.5ey ± 2.95	0a
P. aeruginosa SSH	7.9by ± 1.03	9.6bx ± 1.78	0.63ax ± 0.10	45.5cy ± 1.21	52.25dx ± 3.02	64.60ey ± 1.32	0a

^a-fMeans in the column with same superscript letter are not significantly different as measured by 2-sided Tukey post-hoc range test between replications

^x-zMeans in the row with same superscript letter are not significantly different as measured by 2-sided Tukey post-hoc range test between replications

Assessment of biofilm viable cells and scanning microscopy

Gentamicin or amikacin in combination with alginate lyase SG4 + reduced the CFU count of all mucoid P. aeruginosa strains. The co-action of amikacin along with alginate lyase reduced the CFU count of P. aeruginosa MTCC2297, P. aeruginosa HSS and P. aeruginosa SSH to 26, 26 and 29%, respectively. Similarly, use of gentamicin and alginate lyase reduced the CFU count of P. aeruginosa MTCC2297, P. aeruginosa HSS and P. aeruginosa SSH to 34, 37 and 41%, respectively (Table 12). SEM images shown a visible change in biofilm integrity with combination of alginate lyase (18.0 IU) and amikacin (40 µg/ml) in comparison to untreated control (Fig. 5).

Table 12.

Inhibition P. aeruginosa growth by co-administration of antibiotics with enzyme

Culture	Inhibition of growth (CFUlog10/mL)
	Antibiotic		Heat denatured enzyme	Enzyme (18.0 IU)	Enzyme (18.0 IU) + Gentamicin (40 µg/mL)	Enzyme (18.0 IU) + Amikacin (20 µg/mL)	Control
	Gentamicin (40 µg/mL)	Amikacin (20 µg/mL)
P.aeruginosa MTCC2297	4.34dx ± 0.21	2.90bx ± 0.22 794.3282	8.45fy ± 0.15 281838293	7.56ey ± 0.10 36307805	3.40cz ± 0.235 2511.8864	1.40ay ± 0.25 25.118864	8.76fy ± 0.14 575439937
P. aeruginosa HSS	5.95dz ± 0.20	2.32bx ± 0.29	8.98fz ± 0.12	7.43ey ± 0.10	2.90cy ± 0.467	1.34ay ± 0.32	9.16fy ± 0.139
P. aruginosa SSH	5.56dy ± 0.18	3.67cy ± 0.24	7.34fx ± 0.121	6.78ex ± 0.10	1.12bx ± 0.49	0.5ax ± 0.39	7.54fx ± 0.129

^a-fMeans in the column with same superscript letter are not significantly different as measured by 2-sided Tukey post-hoc range test between replications

^x-zMeans in the row with same superscript letter are not significantly different as measured by 2-sided Tukey post-hoc range test between replications

Fig. 5.

SEM findings from each treatment of P. aeruginosa MTCC2297 biofilm (a) Control without any treatment (b) Treated with alginate lyase only (c) Treated with alginate lyase and amikacin

Discussion

Microbial enzyme has several advantages such as easy production, cost-effectiveness, product specificity and consistent production. Alginate lyase is very important enzyme, widely reported from marine algae, mollusks and microorganisms [24]. Alginate oligosaccharide plays an important role in plants, animals and humans health [14]. Consequently, alginate lyase has attracted researcher’s attention and interest in optimizing fermentation processes for large scale production [25]. Statistical models are widely practiced to optimize the fermentation processes, due to suitability and applicability [26, 27]. In present study, statistical models were employed to optimize the various fermentation parameters for maximum alginate lyase production. The Plackett–Burman design depicted significance of four factors i.e., yeast extract, sodium alginate, agitation speed and inoculum volume for alginate lyase SG4 + production. Additional, influence of significant factors were optimized by CCD. Statistical optimization increased 8.49-fold alginate lyase SG4 + production. Bacillus sp. TAG8 produced alginate lyase and optimization increased twofold production [28]. Maximum alginate lyase units (145.45 U/ml) and protein (5.26 U/ mg) was achieved using CCD by Pseudoalteromonas tetraodonis and Pseudomonas sp. LB56, respectively [29, 30].

Alginate lyase has been purified and reported from various microorganisms [25]. Purified alginate lyases have been reported to exist in single or multiple forms with molecular masses ranging from 25–60 kDa [5, 9, 14, 30]. The molecular weight of alginate lyase SG4 + was approximately 110 kDa and it belongs to the large group category of alginate lyases [6]. Purified alginate lyase SG4 + has optimal activity at pH 8.0 in Tris–HCl buffer (50 mM). The alginate lyase obtained from the from Microbulbifer sp. SH-1 displayed maximal activity at pH 9.0 [5]. The pH-adaptation property of alginate lyase SG4 + , might provide more scope in the choice of pH application when it came to industrial production. Alginate lyase SG4 + showed maximum activity at 30 °C incubation, and these results are in consonance with earlier reported studies [31]. However, alginate lyases AlgC-PL7 and AlgSH7 showed optimum reaction at temperature 45 °C and 40 °C, respectively [5, 32]. Reaction time for alginate lyase SG4 + was determined and it was observed that maximum activity was recorded when enzyme was incubated for 25 min with substrate concentration (1%, w/v) at 30 °C and pH 8.5 However, alginate lyase from strain FlAlyA and ALW1 showed maximum activity when incubated for 30 min with the substrate [20, 33]

Alginate lyase SG4 + Km and Vmax values were 2.7 mg/ml and 0.84 µmol/ml-min, respectively. The Km and Vmax values of novel alginate lyase AlyH1 obtained from Vibrio furnissii H1 were found to be 2.28 mg/ml and 2.81 U/mg, respectively, using sodium alginate as substrate [33]. The purified alginate lyase SG4 + is stable at 4 °C and the T_1/2 obtained for purified alginate lyase at 37 °C was 180 min. A reasonable half-life at 37 °C indicates that the enzyme is stable at that temperature, which is critical for the manifestation of any clinical applications. Chen et.al 2018 [34] studied thermostability profile of alginate lyase AlgA produced by Bacillus sp. and reported that enzyme activity remains unchanged after 5 h of incubation at 40 °C. However, alginate lyase from Vibrio furnissii H1 exhibited excellent stability at temperature below 30 °C and retained more than 60% of activity after incubation at 40 °C for 30 min [33].

Oxygen is a limiting factor in aerobic fermentation; therefore, fermenter is usually accomplished by correctly adjusting the aeration rate and agitation speed [33]. The combination of agitation speed and aeration rate for alginate lyase SG4 + was studied using laboratory fermenter under statistically optimized conditions, the maximum 1.46 IU activity was achieved at 100 rpm and 0.75 vvm. This type of batch mode cultivation is simple and widely used for large-scale commercial production of biomolecules [35]. This is the first report in which CCD is used for fermenter studies to optimize agitation speed and aeration rate for alginate lyase production (Table 12).

The increase in aeration rate (from 0.146 to 0.75 vvm) aided alginate lyase SG4 + production, whereas increasing the agitation rate (from 100 to 341 rpm) reduced alginate lyase activity. Studies show that at low agitation speed, the viscosity of fermentation broth increases and this subsequently reduces the mass transfer efficiency [36]. However, excess may cause high shear stress, subsequently cell rupture, vacuolation, and autolysis, resulting in decreased enzyme productivity [37]. Our findings show that when DO concentration is not a limiting factor, fermentation time is reduced because the lag phase is minimal, and the incubation period for maximum alginate lyase SG4 + is reduced. It was 48 h in shake flask however, after optimization 20 h at the best combination of agitation speed of 100 rpm and aeration rate 0.75vvm. This is due to a short initial lag period and the achievement of maximum cell mass (10.150.57 mg/ml) after 20 h of fermentation. The optimization of aeration rate and agitation speed for dextransucrase production by Leuconostoc lactis KU665298 in a laboratory fermenter by applying CCD also reduced fermentation time to 10 h [38]. It was reported that below the essential DO level, the microbial metabolic pathways might change [39]. There was a decline in DO from 100% saturation to below 4% during 20 to 40 h of fermentation at 100 rpm agitation and 0.75 vvm aeration and after that DO value started to increase. However, DO at 20% saturation is the minimum DO require for growth and enzyme production [17].

P. aeruginosa is the main cause of chronic bronchial infections in patients with cystic fibrosis [24]. Alginate lyases could be used to dissolve the mucoid biofilm architecture and increase the effectiveness of antibiotics [10, 40]. In present study we have assessed the efficacy of alginate lyases SG4 + in combating P. aeruginosa biofilms. The results revealed that the combination of amikacin and alginate lyase SG4 + disintegrated 58- 65% biofilm produced by various P. aeruginosa strains. Our results indicate that the alginate lyases indeed inhibit biofilm formation. The present data is in consonance with the previous studies on alginate lyase efficiency to disintegrate P. aeruginosa biofilms [41, 42]. Activity of alginate lyases SG4 + is comparable with the glycoside hydrolase, a promising candidate used for biofilm treatment due to its excellent exopolysaccharides disintegrating ability [43].

Scientific investigations have revealed that alginate lyase, a noteworthy enzyme cannot be expressed constitutively due to its inducible nature. Bacillus sp. ATB-1015, Vibrio splendidus 12B01 and Bacillus sp. TAG8 produced alginate lyase when cultured on medium containing alginate, this confirms the inducible nature of the enzyme [28, 44, 45, 46]. However, successful recombinant expression of alginate lyase in Flavobacterium sp. H63, Pichia pastoris and Bacillus subtilis has been reported [47, 48]. Consequently, alginate lyase SG4 + could be successfully recombinantly expressed using a suitable host but could not be expressed constitutively.

Conclusion

In this work, the RSM-based optimisation increased 8.46-fold alginate lyase production by P. lautus SG4 + . An appropriate combination of agitation speed and aeration rate influenced the DO concentration in fermenter and enhanced alginate lyase SG4 + production (1.71-fold). In addition to this, it also reduced production time from 48 to 20 h in fermenter. The reduction in fermentation time favours the bioprocess economics. Alginate lyase SG4 + in combination with gentamicin and amikacin efficiently eradiated alginate biofilm formed by P. aeruginosa strains. The present study proved that alginate lyase SG4 + has excellent biofilms disintegrating ability and may be used as potent therapeutic agent to treat P. aeruginosa biofilms. Further, alginate lyase SG4 + properties could be explored for its use in food processing and agricultural industries.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

RSM

Response Surface Methodology

CCD

Central Composite Design

ANOVA

Analysis of variance

DO

Dissolved Oxygen

Authors Contribution

KK and RB have designed the experiments. KK has optimized, purified and done biofilm studies. KK and RB have done fermenter studies KK and DS has analysed and interpreted the data. DS, KK and HR wrote and edited the manuscript. MP has reviewed the manuscript. WA supervised the project.

Data availability

All the data is available within the manuscript.

Declarations

Ethical issues

The manuscript does not contain experiments using animals and it does not contain human studies.

Conflict of interest

The authors have no conflicts of interest to declare.