Isolation, Purification and Characterization of Pectinase enzyme from Streptomyces thermocarboxydus

Pectinases, commonly referred to as pectic enzymes, are an important class of enzymes for their uses in industries like wine, paper, and food for the processing of fruits, vegetables, tea, coffee, and can be extracted from actinomycetes, a group of bacteria popular for research for such products as the penicillin and pectinases. Such pectinase enzymes that will work at high temperatures and at appropriate pH conditions can be of benefi t at high temperatures, which can be used to speed up these processes. Speed of processing gives an economic gain in commercial applications. This study involved the isolation, purifi cation and characterisation of pectinase enzyme from Streptomyces thermocarboxydus. Soil bacterial isolation resulted in seventeen different types of colonies and these from petri were transferred to agar and cultured on pectin containing media at 37° C temperature, and the strain showing maximum pectin clearing detected with iodine vapour. The selected isolate was fi rst sent for sequencing using rRNA technology and phylogenetic analysis. Experiments for culture optimization and enzyme characterization were carried out. The optimum pH and temperature for culture was found out to be 4 and 50  ̊C, and for the partially purifi ed enzyme, maximum activity, at neutral and alkaline pH and 60  ̊C. Hence, this enzyme proves to be a promising candidate for commercial applications. Research Article Isolation, Purifi cation and Characterization of Pectinase enzyme from Streptomyces thermocarboxydus Priyanka S Bharadwaj* and Prajna M Udupa Department of Biotechnology, BMS College of Engineering, Visvesvaraya Technological University, Bangalore, India Received: 15 March, 2019 Accepted: 30 March, 2019 Published: 02 April, 2019 *Corresponding author: Priyanka S Bharadwaj, Department of Biotechnology, BMS College of Engineering, Visvesvaraya Technological University, Bangalore, India, E-mail:


Introduction
Pectin is a naturally occurring substance (a polyscaccaride) found in berries, apples and other fruits. When heated together with sugar, it causes a thickening that is characteristic of jams and jellies. It is a structural heteropolysaccharide contained in the primary cell walls of terrestrial plants. It was fi rst isolated Commonly referred to as pectinases, pectic enzymes, are an enzyme that breaks down pectin. They are an important class of enzymes for their uses in industries like wine, paper industries and food industry for the processing of fruits, vegetables, tea, coffee and so on.

Pectinases from fungi and bacteria
Pectinases act on pectin substances. It is naturally present in higher plants and microorganisms. In plants, it hydrolyses the cementing pectin in cell wall to allow remodeling and growth during development. In microorganisms, such fungi, they help to break the pectin to saprophyte, while the mushrooms hyphae use these enzymes to do the same as it grows and absorbs the nutrients. Commercially, these pectinases are used extensively in a number of different processes that include fruit juice clarifi cation, juice extraction, manufacture of pectin free starch, refi nement of vegetable fi bers, degumming of natural fi bers, wastewater or sewage treatment. They also fi nd their use in cocoa and tobacco industry towards their endproduction, and as an analytical tool in the assessment of plant products [1]. In tea and coffee, pectinase treatment accelerates tea fermentation and destroys pectin the foam forming property, and remove mucilaginous coat from coffee beans [1].
Pectinolytic enzymes can be applied in various industrial sectors wherever the degradation of pectin is required for a process. Several microorganisms have been used to produce different types of pectinolytic enzymes [2]. Microbial pectinases account for 25% of the global food and industrial enzyme sales [3] and their market is increasing day by day.
These are used extensively for fruit juice clarifi cation, juice extraction, manufacture of pectin free starch, refi nement of vegetable fi bers, degumming of natural fi bers, wastewater treatment, and cocoa and tobacco and as an analytical tool in the assessment of plant products [4]. Pectinase treatment accelerates tea fermentation and also destroys the foam forming property of instant tea powders by destroying pectins. They are also used in coffee fermentation to remove mucilaginous coat from coffee beans [5].
Due to the importance of the pectinase enzyme in biotechnology, lot of researchers have attempted isolation of species that express pectinase, and that are hence, of benefi t.
One such a study was conducted with the aims of screening pectinolytic microorganisms from coffee pulp and identifying molecularly using 16S rRNA. Another study records the studying of such species enriched in fruit material.
Actinomycetes: primer Actinobacteria are Gram-positive bacteria with high G+C DNA content. The majority of the Actinobacteria are free-living organisms that are widely distributed in both terrestrial and aquatic (including marine) ecosystems [6]. Actinomycetes hold a prominent position as targets in screening programmes due to their diversity and their proven ability to produce novel antibiotic and nonantibiotic lead molecules of pharmaceutical interest.

Actinomycetes -their products
Since the discovery of actinomycin, the fi rst antibiotic from an actinomycete, many commercially important bioactive compounds and anti-tumor agents have been produced using In this present study, our aim is two-fold. To determine the culture optimization to obtain a pectinase producing bacteria that works at high temperatures and to determine Biochem Technol 5(1): 001-006. DOI: http://dx.doi.org/10.17352/jcmbt.000031 enzyme characterization. In order to do this, we aim to isolate culture soil bacteria on agar, and then grow the isolates on petri with agar containing pectin. The isolate with the largest pectin clearance is then to be sent for rRNA technology and phylogenetic analysis. Culture optimization and enzyme standardization are also to be carried out on this isolate and also on partially purifi ed enzyme.

Discussion
The selected 'isolate' was fi rst sent for sequencing using rRNA technology and phylogenetic analysis. Experiments for culture optimization and enzyme characterization were carried out. The optimum pH for the culture was found to be 4 and temperature was found to be 50 ˚C. For the partially purifi ed enzyme, maximum activity was at neutral and alkaline pH, and the optimum temperature was 60 ˚C. Hence, this enzyme proves to be a promising candidate for commercial applications, where high temperatures may be benefi tting faster reaction and hence more economically viable processes can be achieved to meet the increasing demand of the products.
The focus of this study was to obtain a pectinase that could withstand higher working temperatures for commercial applications. Soil bacteria were isolated, and the isolates plated on pectin containing agar, which resulted in giving IS-1, an isolate with the highest pectin clearance. This selected isolate was fi rst sent for sequencing using rRNA technology and phylogenetic analysis. The result of this sequencing and analysis are discussed later below. Culture optimisation and enzyme characterisation were carried out for pH and temperature. Graph, in Figure 1. results, shows relation between 'pH vs culture growth' and 'pH vs enzyme activity'. The culture conditions were optimized and the optimum pH for the culture was found to be 4. The partially purifi ed enzyme shows maximum activity at both neutral and alkaline pH. The optimum pH for the culture is determined to be acidic while that for the partially purifi ed enzyme is neutral and alkaline. Culture refers to the growth of the whole organism. The pH for this culture growth when optimised is acidic because the organism has evolved to live in the acidic environment. The optimum pH for the enzyme is neutral or alkaline, meaning that internal environment of the bacteria must be maintained at neutral or alkaline conditions. During growth at acidic pH values, Streptococcus can increase production of their F-ATPases. By pumping protons out of cells, these proton pumps can function to assist in maintaining or even increasing internal pH values [9]. Respiration by the bacteria, under aerobic conditions releases carbon dioxide, and while reacting with water turns its surrounding acidic. The Streptococcus can also be facultative anaerobic.
The partially purifi ed enzyme was found to have its optimum activity at 60 ˚C. The culture conditions were optimized and the optimum temperature for the culture was found to be 50 ˚C, ten degrees less than for the partially purifi ed enzyme. The culture, consisting of the whole organism has other enzymes such as the respiratory enzymes, that may be sensitive at higher temperatures, and hence a temperature above 50 ˚C would begin to degrade it. Moreover, the sampling was done in a tropical country and hence the higher temperature tolerance evolution is evident. Of course, it must be noticed that the enzyme has its optimum activity at 60 ˚C, and this is unusually higher as we would have expected the optimum temperature to have been around the region of the soil temperature, say about 35 to 40 ˚C. The organism must be a result of evolution and gain mutation, which enables to work at such a high temperature of 60 ˚C.
The result is that we identifi ed a species that produces pectinase that is tolerant and optimal at higher temperatures, of about 60 ˚C. The main use of this particular enzyme is that, this high temperature tolerant enzyme can be of immense use in industrial applications where speed of process may be necessary for increased economics.
16s rRNA technology based sequencing and phylogenetic analysis.
The isolate that is found to give the largest pectin clearance was sent for identifi cation by rRNA sequencing technology and phylogenetic analysis. The isolate is found to have 99% homology or sequence match with the species Streptomyces thermocarboxydus strain, NBRC 16323. The phylogenetic analysis further tells us the closeness of this species-strain to another species-strain of this same species, the Streptomyces, with the bootstrap value, of course, the closeness or distance to a number of closely-related species.

The Phylogenetics -the bootstrap explained
The bootstrap value gives the chance value of this species strain occurring in its closeness to the neighboring species or species strain under study. The closest Streptomyces strain to this identifi ed strain is , NR 041102.1 Streptomyces variabili, is strain NBRC 13046 with a bootstrap of 97% chance of it

Conclusion
The identifi cation of the other species-strains closes to the Streptomyces thermocarboxydus strain under analysis will help fi nd other species-strains that may be close to this species-strain under analysis and hence there is a possibility of getting a mutant enzyme pectinase, that maybe more resistant to higher temperatures as compared to the enzyme that is found in this study. The uses of the pectinase enzyme in commercial application can be seen in everyday life and can be seen in the production lines including fruit juices and wine brewing. To conclude, it must be noted that our study and such studies involving phylogenetics, culture optimization and enzyme standardization can help fi nd enzymes that have a more useful temperature withstanding abilities to the one in hand, either for this application or for others. It can also be taken a step further, and controlled mutations of this enzyme can also help produce a sequence and hence fi nd enzyme that is slightly different and more effective than, probably, all that may be present in the close existing species.