A Study on Marine bacterial invertase enzyme sourced from Bay of Bengal, West Bengal; Its Isolation and Optimization

 

Panchami Jaya, Sandra Suresh, Arya K, Anwesha Sarkar, K V Bhaskara Rao*

Marine Biotechnology Laboratory, Department of Biomedical Sciences, School of Biosciences and Technology,

Vellore Institute of Technology, Vellore-632014, Tamil Nadu, India

 

ABSTRACT:

In this study, we have focused on isolation and characterization of invertase enzyme produced from marine bacteria. Marine bacteria have proved to have excellent potential for producing varied novel natural compounds and nanoparticles that are having a wide range of applications. In the current study, a total of 42 bacterial isolates were found from marine water collected from Bakkhali, West Bengal. A total of 20 Selected isolates were kept for UV-mutation and 15 UV-mutated marine bacterial isolates were screened for invertase enzyme activity. Out of them, bacterial isolate SPAB3 showed highest enzyme activity. For the 3 potential isolates, optimization was done for the production of the invertase enzyme in different environmental conditions. The optimum conditions for the potential isolate SPAB2 were galactose as carbon source, 37°C and pH 7. For SPAB3, the optimum conditions were glucose as carbon source, 20°C and pH 7. For SPAB4, the optimum conditions were mannitol as carbon source, 20°C and pH 7. The potential isolates SPAB2, SPAB3 and SPAB4 were characterized using macroscopic, microscopic and biochemical tests and identified as Bacillus sp., Staphylococcus sp. and Staphylococcus sp. respectively.

 

 

 

INTRODUCTION:

Microbial enzymes are gaining more importance today and they are widely used in bioprocess industries.  Microbial enzymes are used in industries such as leather, pharmaceuticals, textiles, detergents, chemical, paper, food and beverages, animal feed, biofuel etc.^[1] as they have advantages over plant or animal derived enzymes in many aspects^[2]. In recent years, marine microbial enzymes are capturing researcher’s attention because of their unique catalytic properties due to efficient nutrient utilization in oligotrophic waters, distinct physiological and metabolic characteristics^[3]. They are also the source of novel biocatalysts like cold adapted enzymes which are economical in terms of energy savings^[4,5].

 

Most of the 4000 enzymes that are known to be used in these industries, have either bacterial or fungal origin due to immense microbial diversity and the ease of production of enzymes by microorganisms^[6].

 

Some examples of important marine microbial enzymes are protease, lipase, amylase, inulinase, cellulase, invertase, chitinase, L-glutaminase etc. Among the marine microbial enzymes, invertase (β.D. fructofuranosidase, E.C. 3.2.1.26) is an enzyme which cleaves α-1,4 glycosidic linkage between α-D-glucose and β-D-fructose molecules of sucrose through hydrolysis and also releases monosaccharides^[7,8]. From the fructose end, this enzyme attacks β-D-fructofuranoside (sucrose, raffinose, inulin and stachyose)^[9]. Invertase has gained importance in recent times due to its varied biotechnological applications in confectionary and pharmaceutical industries for the preparation of high fructose syrup and invert sugar syrup from sucrose^[10]. It catalyses the sucrose hydrolysis and breaks the sucrose into equimolar mixture of glucose and fructose^[11]. Invert syrup is produced from the sucrose utilization by invertase and they are 1.5 times sweeter than sucrose^[12]. Microbes can use sucrose as their nutrient, and they utilize it by breaking down using this enzyme which is exploited nowadays. Yeasts are the main producers of invertase though there are few reports of marine bacteria like Bacillus producing invertase enzyme^[13].

 

Microorganisms are ubiquitously present in the world’s oceans. Marine microorganisms have some unique properties as they can adapt to the extreme marine environment conditions such as high pressure, high or low temperature, alkaline or acidic water and limited substrate in the deep-sea water^[14]. Marine bacteria are prokaryotic extremophiles which are abundantly present. They produce many novel compounds that are having wide range of applications^[15]. Though isolating and harvesting marine bacteria is difficult task, scientists are getting attracted to marine bacteria because of their broad range pharmacological activities. Marine bacteria are having several biological activities such as antioxidant activity, antifungal activity etc.^[16].  

 

Marine water from Bay of Bengal, Bakkhali, West Bengal is rich in microbial diversity that is unique to its own characteristics including salt concentration, temperature, pH etc. In the Bay of Bengal, heterotrophic bacteria are the main contributors for forming biomass almost throughout the year. Higher abundance in bacteria was found mostly in the Central Bay region during summer monsoon compared to the other seasons ^[17,18].

 

In the present study, we have isolated and characterized marine bacterial isolates from the water of Bay of Bengal, Bakkhali, West Bengal and checked for their invertase enzyme activity.

 

MATERIALS AND METHODS:

Collection of samples:

Marine water samples were collected from the Bay of Bengal, coast of Bakkhali (21.5631° N, 88.2595°E), Sundarbans, West Bengal, India in sterile autoclave bags and containers. The samples collected were transported to Marine Biotechnology laboratory at Vellore Institute of Technology, Vellore, Tamil Nadu and preserved under 4°C for further process^[19].

 

Isolation of marine bacteria:

The marine bacteria were isolated from the water samples on Zobell Marine Agar^[20] by serially diluting the samples up to 10^-6 dilutions and inoculating on the plates containing Zobell marine agar (prepared using 50% distilled water and 50% marine water). Spread plate technique was used and the plates were incubated at 37°C for 24-48 h. The Isolated colonies were counted using coulter colony counter and they were sub-cultured again on Zobell marine Agar. Characterization of the isolated bacterial colonies was done using macroscopic, microscopic and biochemical tests.

 

Characterization of marine bacterial isolates:

a.   Morphology of isolated colonies: Colony morphology of the bacterial isolates was noted with respect to shape, size, elevation, margin/border, surface, opacity and color.

b.   Microscopic observation:

      Microscopic methods like gram staining was done to determine the characteristics of the bacterial isolates.

c.   Biochemical characterization:

      Biochemical tests viz. indole, methyl red, Voges Proskauer, citrate utilization, oxidase, catalase and triple sugar iron tests^[21,22] were done for characterization of the bacterial isolates which showed positive results for the invertase enzyme production.

 

Fermentation:

Selected marine bacterial colonies were further cultivated in bulk amount using modified nutrient broth. After inoculation, they were incubated at 37°C for 24-48 hours in shaker incubator (REMI). After the incubation period is over, the bacterial cultures were centrifuged at 10,000rpm for 10 min and supernatant from all the cultures were collected and stored for further work.

 

Invertase enzyme activity assay for the marine bacterial isolates:

For checking the invertase enzyme activity, the marine bacterial isolates were mutated for strain improvement by keeping them under UV light for 2 minutes. Then they were inoculated on agar plates using spread plate technique and kept for incubation at 37°C for 24-48 hours. The mutated colonies were checked for invertase enzyme activity by using sodium 2.5ml of 50mM acetate buffer (pH 5.5) and 0.1ml 300mM sucrose solution. After pre-incubation for 5 min at 37°C, 0.1ml of the enzyme solution (crude extract) was added. The whole mixture was kept in a boiling water bath to stop the reaction process and the tubes were cooled down to room temperature. DNS (1ml) was added and again the tubes were kept in boiling water bath for 5 min and cooled down to 24°C. Optical density was measured at 546nm using UV spectrophotometer. Invertase enzyme activity was found out in the basic of OD value at 4°C and 24°C (cooling temperature in acetate buffer. The pH of the assay was fixed to be 5.5)^[23].

 

Optimization of invertase activity:

Medium optimization for the better production of invertase enzyme was carried out at various temperature like 0°C, 4°C, 20°C, 30°C, 37°C and 42°C as well as various pH viz. 2.0, 4.0, 6.0, 8.0 and 10.0. To optimize the carbon source for invertase production, 1% of different carbon sources (glucose, xylose, lactose, galactose, fructose, sucrose, mannitol and arabinose) were added to the production medium at 37°C and kept for incubation for 24-48 hours^[24,25].

 

RESULTS:

Isolation of marine bacteria:

A total of 42 marine bacterial isolates were found from the marine water sample collected. According to the colony morphology, 20 isolates were kept for mutation and a total of 15 UV-mutated bacterial isolates were selected based on their distinct morphology for further processing and characterization.

 

Invertase enzyme activity:

Out of the 15 mutated bacterial isolates selected, 4 isolates showed good invertase enzyme activity. The results were statistically analysed using Graphpad prism 7.0 software. From Fig 1, we can see all of the 4 positive isolates showed similar activity. They all have showed more than 80% invertase enzyme activity. But, out of the 4 isolates SPAB3 showed best activity (84%) and SPAB1 showed least enzyme activity (81.5%). So, SPAB2, SPAB3 and SPAB4 were taken for further processing.

 

Fig 1: Invertase enzyme activity of the marine bacterial isolates

 

Characterization of the bacterial isolates:

Almost all the isolated marine bacterial colonies showed similar morphological characteristics (Table 1).

 

Table 1: Colony morphology of the bacterial isolates

The potential 3 bacterial isolates (UV mutated) which showed positive results for invertase enzyme have the following biochemical characteristics describes in Table 4.

 

Table 2: Biochemical tests of potential UV mutated isolates

 

Optimization of invertase enzyme production:

One-way ANOVA was performed for the optimization studies and the study was proved to be significant. From Fig 2, it can be clearly observed that SPAB2 showed highest invertase activity in presence of galactose as carbon source and it showed least activity in presence of starch. On the contrary, we can see that the optimum carbon source for SPAB3 was glucose and it showed least enzyme activity in presence of galactose. The optimum carbon source for SPAB4 was mannitol and it too showed least enzyme activity in presence of starch. Out of the 3 potential isolates, overall SPAB2 showed best activity compared to the other 2 isolates. From Fig 3, we can figure out that the optimum temperature for SPAB2 was 37°C and it showed least activity in 0°C. The other 2 isolates, SPAB3 and SPAB4 showed highest enzyme activity in 20°C. Overall, SPAB2 showed highest activity among the other 3 isolates. The optimum pH for all the potential 3 isolates was pH 7 (Fig 4). SPAB2 and SPAB4 showed least activity in pH 9 and SPAB3 showed least enzyme activity in pH 5.

  

Fig 2: Enzyme activity in different carbon source

 

Fig 3: Enzyme activity in different temperature

 

Fig 4: Enzyme activity in different Ph

 

DISCUSSION:

A total of 42 isolates have been isolated from the marine water samples collected from coast of Bakkhali, West Bengal, India. A total of 15 UV-mutated bacterial isolates were selected for further characterization. In a previous study, they have isolated a total of 4 marine bacterial isolates including 2 actinobacterial isolates as pure culture for their enzyme activity study^[14]. However, we were able to isolate a greater number of isolates. For invertase enzyme production, out of the 15 UV-mutated marine bacterial isolates 4 isolates showed positive result. SPAB2 showed highest enzyme activity off 82.5% while the other isolates also showed good enzyme activity. In a recent study, they analysed 17 isolates and out of them 1 isolate gave maximum activity of 2.03 U/ml/min after 48 hours^[11]. In another study, invertase enzyme activity was checked with 3 different bacteria and among them bacterial isolate HIN-1 produced highest amount of invertase (1.07 IU)^[12].  Microscopic analysis and biochemical characterization were done for 4 the selected bacterial isolates. All of them were found to be gram positive bacteria. Analyzing all the microscopic and biochemical test results through Bergey’s Manual, we are assuming that SPAB2 can be Bacillus sp., SPAB3 can be Staphylococcus sp. and SPAB4 can be Staphylococcus sp.

 

In our study, the optimum carbon source for SPAB2, SPAB3 and SPAB4 was found to be galactose, glucose and mannitol respectively. In a previous study, they have checked the invertase enzyme activity with Streptomyces sp. and it showed highest enzyme activity of 473±9 U/l in presence of sucrose^[10]. The optimum temperature for invertase production from SPAB2 was 37°C and for both of SPAB3 and SPAB4 was 20°C. On the other side, in a previous study invertase enzyme was produced from Pseudozyme sp. and after purification, they found 40°C as the optimum temperature for invertase production^[13]. For all the 3 potential isolates of our study, the optimum pH was found to be pH 7 and for SPAB2 and SPAB4 showed least activity in pH 5. On the contrary, in a similar study, the optimum pH was found to be pH 5 ^[11]. In another report, highest invertase activity was observed in pH 6 which was 1.13 IU^[12].

 

ACKNOWLEDGEMENT:

The authors are very much grateful to management and all of the staffs and technicians of VIT University, Vellore, Tamil Nadu for their immense support to complete this study.

 

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Received on 15.11.2019           Modified on 05.02.2020

Accepted on 28.03.2020         © RJPT All right reserved