JOSHI SWANAND (IN)
GAIKWAD PRASHANT (IN)
ZINJARDE SMITA (IN)
| US20170174745A1 | 2017-06-22 | |||
| US20140127748A1 | 2014-05-08 | |||
| US20040115790A1 | 2004-06-17 | |||
| US20130137140A1 | 2013-05-30 | |||
| US20170044557A1 | 2017-02-16 |
| Claims We claim, 1 ] A method to obtain significantly enhanced yield of a purified native self assembling protein from liquid culture of Yarrowia lipolytics, comprising- — Providing, in the liquid culture, a set of combinatorial physical and chemical conditions optimized for enhanced secretion and therefore increased yield of the native self assembling protein; and — Collecting, from the liquid culture, cell-free supernatant for subjecting it to a modified protein extraction protocol to thereby obtain the native self assembling protein in purified form. Characterized in that: i) the method involves no genetic amplification techniques otherwise common to art for enhancing secretion of microbial metabolites; ii) flux of secretion of the native self assembling protein is directed to extracellular space, therein simplifying downstream processing for purification of said protein; and iii) the method results in enhancing yield of the native self assembling protein from 20 mgl 1 in basal media to 60 to 80 mgl"1 from application of the physical and chemical conditions optimized for enhanced secretion and therefore increased yield of the native self assembling protein in the liquid culture of Yarrowia lipolytica. 2] The method to obtain significantly enhanced yield of a purified native self assembling protein from liquid culture of Yarrowia lipolytica as claimed in claim 1 , wherein the set of physical conditions optimized for enhanced extracellular section and therefore increased yield of the native self assembling protein include, in combination: a) Arranging incubation for a period of 24 hours; b) Providing aeration at 50 RPM for the period of incubation; c) Introducing inoculum of Yarrowia lipolytica at 10 OD/100ml; d) Maintaining surface to volume ratio of the liquid culture media at 20 %; and e) Maintaining temperature at 25 °C for the period of incubation. 3] The method to obtain significantly enhanced yield of a purified native self assembling protein from liquid culture of Yarrowia lipolytica as claimed in claim 1 , wherein the set of chemical conditions optimized for enhanced extracellular section and therefore increased yield of the native self assembling protein include providing in the culture medium, in combination: a) 1 % of yeast extract; 1 .5% of ammonium sulphate; 1 % of glycerol; 2 % of salt solution; and 0.1 % of sodium chloride The method to obtain significantly enhanced yield of a purified native self assembling protein from liquid culture of Yarrowia lipolytica as claimed in claim 1 , wherein the modified protein extraction protocol comprises execution of a protocol designed to progressively concentrate the native self assembling protein, the protocol comprising: a) Subjecting 1000 ml of cell free supernatant to ultrafiltration, therein allowing frothing, for concentrating the native self assembling protein while other proteins are degraded and carbohydrate-protein complexes are dissociated; b) Subjecting the supernatant of ultrafiltration step to lyophilization to obtain a dry powder, which is dissolved in 200ml Milli-Q water to obtain a solution containing the native self assembling protein; c) Estimating protein content of the solution obtained at step b) by Bicinchoninic Acid method; d) Subjecting the solution of step b) to protein extraction using 100% Triflouroacetic acid, taken at a proportion of 5 ml per milligram of protein estimated in step c), in chilled conditions; e) Subjecting the admixture of step d) to sonication at 53 Hz for 10 min and centrifugation at 30000 G for 60 minutes to obtain a supernatant containing the native self assembling protein; f) Lyophilizing the supernatant of step e) to remove water content, and dissolving the resultant powder in phosphate buffer of physiological pH having 0.8M ammonium sulphate to obtain a further concentrated solution of the native self assembling protein; g) Subjecting the solution obtained at step f) to hydrophobic interaction chromatography, therein causing elution by reduction in salt concentration till water phase; h) Subjecting output of step g) to dialysis to result in a further concentrated solution of the native self assembling protein; i) Subjecting the output of step h) to lyophilization to obtain a dry powder, which is dissolved in 200ml milli-Q water to obtain a solution containing the native self assembling protein; j) Estimating protein content of the solution obtained at step i) by Bicinchoninic Acid method; k) Subjecting the solution of step h) to protein extraction using 100% Triflouroacetic acid, taken at a proportion of 5 ml per milligram of protein estimated in step c), in chilled conditions; and I) Subjecting the admixture of step g) to sonication at 53 Hz for 10 min and centrifugation at 30000 G for 60 minutes to obtain the final purified native self assembling protein. The method to obtain significantly enhanced yield of a purified native self assembling protein from liquid culture of Yarrowia lipolytica as claimed in claim 4, wherein the step of ultrafiltration is 3 kDa AMICON concentrate ultrafiltration performed under 2 bar positive nitrogen pressure. The method to obtain significantly enhanced yield of a purified native self assembling protein from liquid culture of Yarrowia lipolytica as claimed in claim 4, wherein the step of hydrophobic interaction chromatography is performed using Phenyl sepharose 6 F.F where sample volume is 5 times bed volume. The method to obtain significantly enhanced yield of a purified native self assembling protein from liquid culture of Yarrowia lipolytica as claimed in claim 4, wherein the step of dialysis is undertaken using a 3kDa membrane. The method to obtain significantly enhanced yield of a purified native self assembling protein from liquid culture of Yarrowia lipolytica as claimed in either of the claims 4 or 8, wherein the step of protein extraction using Triflouroacetic acid is carried out in chilled conditions, particularly at a temperature of 4°C to allow effective dissociation of protein-carbohydrate complexes present in the cell-free supernatant. |
Priority: This international application is filed pursuant to and claims priority from Indian patent application No. 201621044801 dated 29/06/2017.
Cross references to related applications: None
Reference to sequence listing, a table, or a computer program listing compact disk appendix:
Not Applicable
Statement Regarding Federally Sponsored Research: Not Applicable [001 ] Field of the invention
[002] This invention belongs to the field of biotechnology, and in that, relates generally to the methods, techniques and systems employing microorganisms for production of industrially applicable products.
[003] The present invention specifically relates to underlying aspects, mainly media and growth conditions, for conditioning the enhanced secretion of a self-assembling native protein having surfactant and emulsifier properties through liquid culture of a Yarrowia lipolytica strain.
[004] Definitions and interpretations
[005] Before undertaking the disclosures below, it may be advantageous to set forth definitions of certain words or phrases used throughout this patent document: the terms "include" and "comprise," as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and/or; the phrases "associated with" and "associated therewith," as well as derivatives thereof, may mean to include, be included within, interconnect, with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have, have a property of, or the like; and the term "GRAS" means Generally Regarded As Safe; "BCA" means Bicinchoninic Acid; "PBD" means Plackett-Burman Design; "TFA" means Triflouroacetic acid; "YE" means yeast extract;"HBN" refers the protein isolate of interest / subject hereof obtained by process of the present invention; "HIF" means Hydrophobic Interaction Chromatography.
[006] Background of the invention
[007] Understanding of microbial cell organization, and utility thereof, for engineering the production of desirable structural and/ or functional metabolites has significantly incremented over the recent past. Identification of products that are or can be microbially synthesized, screening for high-yielding microbial strains therefor, process engineering thereupon, and applications thereof have become major drivers of the science, economy and art employing microorganisms viably to result in monetizable assets.
Microbial cells have been widely recognized as efficient cell factories capable of outputting molecules of interest should their biochemical flux be directed and managed in the right manner. State-of-art today refers various techniques including physical, chemical, and genetic interventions for aforesaid objective. However, technical complexities, high demands as to costs, resources and skills remain considerations that limit their global applicability to a large extent.
Among microorganisms widely used for biotechnological applications, ability of the eukaryote Yarrowia, a genus within family Dipodascaceae which contains the species lipolytica, to utilize unusual carbon sources, especially hydrophobic substrates such as hydrocarbons, triglycerides and fatty acids, has garnered distinct interest from industrial microbiologists. (Gancedo C, Flores C-L (2005). "Yarrowia lipolytica mutants devoid of pyruvate carboxylase activity show an unusual growth phenotype". Eukaryotic Cell. 4 (2): 356-64. doi:10.1 128/EC.4.2.356-364.2005). Also, applications of Yarrowia species for production of specialty lipids are documented (Papanikolaou S, Aggelis G (2010). "Yarrowia lipolytica: A model microorganism used for the production of tailor-made lipids". European Journal of Lipid Science and Technology. 1 12 (6): 639-654. doi:10.1002/ejlt.200900197).
Despite being the most widely studied and engineered oleaginous yeast known to habit several polluted environments rich in hydrophobic substrates, alkanes, and fats, the art is silent on realization of true potential of Yarrowia as an industrial workhorse, otherwise discernable from its unique biochemistry and ecosystem associated with its native habitat. Generically, it comes naturally for any industrial application foreseen to be simple, cost- effective yet scalable and efficient in achieving the aforesaid objectives.
Biosurfactants are generally low molecular weight microbial products composed of sugars, amino acids, fatty acids and functional groups such as carboxylic acids. These molecules are amphiphilic in nature and this property allows them to dissolve in both polar and non-polar solvents. Biosurfactants are known for their excellent surface activity which involves lowering the surface and interfacial tension between different phases. They can act as wetting, foaming and solubilizing agents in different industrial processes (Source: . Perfumo A., Smyth T. J. P., Marchant R., Banat I. M. (2009). Production and roles of biosurfactant and bioemulsifiers in accessing hydrophobic substrates, in Microbiology of Hydrocarbons, Oils, Lipids and Derived Compounds, ed Timmis Kenneth N., editor. (Berlin; Heidelberg: Springer- Verlag; ), 1502-1512; Satpute S. K., Banpurkar A. G., Dhakephalkar P. K., Banat I. M., Chopade B. A. (2010). Methods of investigating biosurfactants and bioemulsifiers: a review. Crit. Rev. Biotechnol. 30, 127-144. 10.3109/07388550903427280). The present inventors, through screening, have focused on viable production of protein having surfactant and emulsifier properties through liquid culture of a Yarrowia Iipolytica strain.
As known in the art, bioemulsifiers are higher in molecular weight than biosurfactants as they are complex mixtures of heteropolysaccharides, lipopolysaccharides, lipoproteins and proteins. The isolated protein is a surface active biomolecule which is categorized into surfactants and emulsifiers, while surfactants play the role of surface tension reduction, emulsifiers are involved in formation and stabilization of emulsions. However, some biomolecules possess both surfactant and emulsifying properties which contributes to their unique functions and broad industrial uses. (Source: Chibuzo Uzoigwe, J. Grant Burgess, Christopher J. Ennis, Pattanathu K. S. M. Rahman. (2015). Bioemulsifiers are not biosurfactants and require different screening approaches. Front Microbiol. 2015; 6: 245. 10.3389/fmicb.2015.00245)
As mentioned before, Yarrowia Iipolytica is one of the most extensively studied nonpathogenic yeasts having GRAS status, which is currently used as one of the models for the study of secretion studies, dimorphism, and degradation of hydrophobic substrates. Yarrowia Iipolytica, with its rich potential is considered to be a promising candidate that can be utilized in various biotechnological industries. It has known to produce large number of biomolecules such as bio-surfactant, bioemulsifier growth factors, acids, enzymes etc. The earlier reports explains that the characterized bioemulsifier from Y. Iipolytica is the complex mixture of protein-lipid-carbohydrate, which shows the surfactant and emulsifier activity. But the purification and characterization of the protein showing similar surface active properties was not achieved earlier. (Source: Cirigliano MC, Carman GM. Isolation of a bioemulsifier from Candida Iipolytica. AppI Environ Microbiol 1984;48(4):747-50; Zinjarde SS, Pant A. Emulsifier from a tropical marine yeast, Yarrowia Iipolytica NCIM 3589. J Basic Microbiol 2002;42(1):67- 73; Sarubbo LA, Marc al MC, Neves MLC, Silva MPC, Porto ALF, Campos- Takaki GM. Bioemulsifier production in batch culture using glucose as carbon source by Candida Iipolytica. AppI Biochem Biotechnol 2001 ;95: 59-67)
Prior art, to the limited extent presently surveyed, therefore, does not list a single effective solution embracing all considerations mentioned hereinabove, thus preserving an acute necessity-to-invent for the present inventor/s who, as result of focused research, has come up with novel solutions for resolving all needs once and for all. Work of the presently named inventor/s, specifically directed against the technical problems recited hereinabove and currently part of the public domain including earlier filed patent applications, is neither expressly nor impliedly admitted as prior art against the present disclosures. [015] A better understanding of the objects, advantages, features, properties and relationships of the present invention will be obtained from the description set forth hereinunder which outlines few illustrative yet-preferred but non-restrictive embodiments.
[016] Objectives of the present invention
[017] The present invention is identified in addressing at least all major deficiencies of art discussed in the foregoing section by effectively addressing the objectives stated under, of which:
[018] It is a primary objective to provide a method for achieving enhanced secretion of a self- assembling native protein having surfactant and emulsifier properties through liquid culture of a Yarrowia lipolytics strain.
[019] It is another objective further to the aforesaid objective(s) to use statistical design for optimizing physico-chemical parameters that help increase yield of protein in liquid culture of a Yarrowia lipolytica strain.
[020] It is another objective further to the aforesaid objective(s) to increase protein yield in liquid culture of a Yarrowia lipolytica strain without using traditional genetic amplification tools and techniques.
[021 ] It is another objective further to the aforesaid objective(s) that the method so implemented is not unduly costly, does not include complex or obscure ingredients, is easy to execute and scale up with least if not none localizations and does not include undue downstream processing costs.
[022] The manner in which the above objectives are achieved, together with other objects and advantages which will become subsequently apparent, reside in the detailed description set forth below in reference to the accompanying drawings and furthermore specifically outlined in the independent claims. Other advantageous embodiments of the invention are specified in the dependent claims.
[023] Brief description of drawings
[024] The present invention is explained herein under with reference to the following drawings, in which:
[025] FIGURE 1 is a micro-photograph of foam produced by HBN obtained as per the present invention.
[026] FIGURE 2 is a photograph of test for foaming undertaken using HBN obtained as per the present invention. [027] FIGURE 3 showcases surfactant properties as determined by hanging drop method undertaken using HBN obtained as per the present invention.
[028] FIGURE 4 includes microphotographs, particularly of various substrates modified via coating with HBN obtained as per the present invention.
[029] FIGURE 5 is a comparative of water droplets on various substrates in untreated versus when treated with HBN obtained as per the present invention.
[030] FIGURE 6 is a graph illustrating vortexing time required to achieve a stable emulsion with
HBN obtained as per the present invention.
[031 ] FIGURE 7 showcases concentration dependent variation in the particle size with HBN obtained as per the present invention.
[032] The above drawings are illustrative of particular examples of the present invention but are not intended to limit the scope thereof. The drawings are not to scale (unless so stated) and are intended for use solely in conjunction with their explanations in the following detailed description. In above drawings, wherever possible, the same references and symbols have been used throughout to refer to the same or similar parts. Though numbering has been introduced to demarcate reference to specific components in relation to such references being made in different sections of this specification, all components are not shown or numbered in each drawing to avoid obscuring the invention proposed.
[033] Summary
[034] The present invention attempts to resolve the wants of art, by meeting the objectives stated hereinabove. Specifically, principles of the present invention are generally directed to optimization of media and growth conditions, for the enhanced secretion of a self-assembling extracellular self assembling native protein from a Yarrowia lipolytics strain through liquid culture. Further disclosed are applications of said protein based on its surfactant and emulsifier properties.
[035] Attention of the reader is now requested to the detailed description to follow which narrates a preferred embodiment of the present invention and such other ways in which principles of the invention may be employed without parting from the essence of the invention claimed herein.
[036] Detailed description
[037] Principally, general purpose of the present invention is to assess disabilities and shortcomings inherent to known systems comprising state of the art and develop new systems incorporating all available advantages of known art and none of its disadvantages.
[038] Accordingly, the disclosures herein are directed towards an inventive method of achieving enhanced production of a self-assembling protein having surfactant and emulsifier properties secreted by a native Yarrowia lipolytica strain arranged to so manifest in liquid culture. The resultant product, its downstream processing and applications form integrative aspects of the present invention.
[039] Under an evolutionary point of view, the inventors named herein reason that the unique biochemical signature of the Yarrowia species, including its extracellularly-expressed chemical species, is configured for conquering its unusual habitat. In continuum to this approach, synthesis of glycolipids is expected as part of the biochemistry responsible for breakdown of unusual carbon sources including aliphatic and aromatic hydrocarbons listed hereinabove. Biochemical flux studies primarily indicate extracellular secretion of protein-lipid- carbohydrate complexes which may have emulsifier and/ or surfactant properties, but their purification and characterization find no mention in the presently studied prior art.
[040] On this background, the inventors named herein have studied the growth characteristics of a Yarrowia lipolytica strain isolated from Scottish coastal water, (culture obtained from National collection of Industrial Microorganisms NCIM3590/ MTCC35/ NCYC789), and furthermore, the effects on secretion of metabolites in diverse growth conditions typified by variations in sources of carbon, nitrogen, and micro-nutrients in the growth medium; and/ or pH, temperature, time of incubation; specifications of inoculum, agitation and aeration (active/ passive); presence of stress conditions; and surface to volume ratio considerations for deciding reactor geometry and scale of operation matrices.
[041 ] Against backdrop of the research undertaken, the inventors named herein have identified certain physico-chemical conditions that enable and enhance extracellular secretion of a self- assembling protein having surfactant and emulsifier properties in liquid culture of the Yarrowia lipolytica strain mentioned above. These parameters were statistically processed using response surface methodology to direct flux towards extra-cellular secretion of the aforementioned self-assembling protein. Accordingly, optimized media composition and growth conditions established are listed in Table 1 below.
Parameter/ Factor Low (-) High (+)
1) Yeast Extract 0.1 % 5%
2) Peptone 0.1 % 5%
3) Glycerol 1 % 20%
4) Detergent 0% 1 %
5) Incubation Time 24 hrs 120hrs
6) Aeration 50 rpm 200 rpm
7) Inoculum Size 1 OD/100ml 10 OD/100ml
8) Temperature 20 U C 30 U C
9) Surface/Volume ratio 0.2 0.5
10) Salt Solution 0.1 % 2.0% Parameter/ Factor Low (-) High (+)
1 1) NaCI 0.1 % 2.0%
Table 1
[042] Reference is now made to certain examples by way of reference by which the present invention may be implemented as a best mode thereof.
[043] Example 1 : Scheme of practicing the present invention-
1) Selection of components- a. Media components - Carbon source, Nitrogen source, chemical components b. Physico-chemical parameters - Temperature, inoculum size, Aeration, surface to volume ratio, incubation time
2) Process optimization- a. Short listing the components using design of experiments
b. Range of experiments of components at shake flask level - Plackett-Burman model c. Identification and selection of significant factors influencing production process.
d. Evaluation of results at shake flask level - F test, p test, Annova
e. Evaluation of core factors
3) Modified protein purification protocol- 1000 ml cell free supernatant is subjected to ultrafiltration (3 kDa AMICON concentrate ultrafiltration under positive nitrogen pressure of 2 bar). Frothing usually destroys protein content, but as the protein of interest (HBN) is resilient, frothing is purposefully allowed in this step so that other proteins get degraded, and also the HBN is dissociated from extracellular polysaccharides. Resultant supernatant is lyophilized and the dry powder so obtained is re-dissolved in 200ml Milli-Q water. Protein content is then estimated using BCA to therefore decide volume of TFA later in the procedure. The dry powder re-dissolved in 200ml milli-Q water is lyophilized again to remove water content. Resultant powder is subjected to TFA extraction using appropriate amount of TFA as determined above, (maintaining 5ml 100% TFA per 1 mg protein) in chilled conditions (<4°C). The resultant solution is sonicated at 53 Hz for 10 min and centrifuged at 30000 G for 60 minutes. Pellet is discarded, and supernatant is collected and lyophilized to remove water content. The resultant powder is dissolved in buffer (phosphate buffer of physiological pH having 0.8M ammonium sulphate) and subjected to HIC (Phenyl sepharose 6 FF, where sample volume is 5 times bed volume), causing elution by reduction in salt concentration till water phase. Output of HIC is dialyzed (3kDa screen) and subjected to another cycle of lyophilization, TFA Extraction (5ml TFA per 1 mg protein) in chilled (<4°C) condition (100% TFA), sonication (53 Hz for 10 min) and centrifugation (30K g for 1 hour). The supernatant at this stage is lyophilized for the last time to give the purified protein of interest (HBN). Table A below shows stepwise purification of protein obtained. Purification of protein from basal media
Total Total
Steps Volume Protein Carbs
protein Carbs
(ml) (mgml 1 ) (mgml 1 )
(mg) (mg)
Cell free supernatant 1000 0.133 1 13 120 120
Ultra filtration 45 0.52 23.4 4.95 4.95
HIC 70 0.08 5.6 0.7 0.7
Purified protein (HBN) 12 0.27 3.25 0.18 0.18
Table A
[044] Example 2: Optimization of media and growth conditions
[045] The optimization of media conditions for self assembling production was carried out using statistical design of experiments. In one step screening of factors likely to be important for protein production was done by Plackett-Burman Design. Design Expert (Stat-Ease, Inc. , USA) was used to design the experiments in each step and also to analyze the obtained data.
[046] Aim of media optimization was to improve the yields for protein production. To study biophysical and biochemical properties, large quantity of the protein was required. A statistical experimental designs and surface response methodologies were used to improve the protein yield. Response surface methodology is a collection of statistical and mathematical techniques useful for developing, improving and optimizing processes, which also helps in understanding the effects of individual variables and their interactions in the final response. The influence of system aeration, agitation speed , temperature and carbon and nitrogen sources were evaluated on the production of protein. Plackett-Burman design was adopted to determine most important medium components that affect protein production In this optimization step, a Plackett-Burman design was used to determine the likely effects of different media components on protein production. Plackett-Burman design was created using Design Expert (from Stat Ease Inc, USA).
[047] Selection of significant variables by Plackett-Burman design: PBD allows evaluation of N variables in Λ/+1 experiments with each independent variable studied at two levels, i.e., the low and high factor level and coded as -1 and +1 , respectively (Plackett and Burman 1 946). In the present PBD study, 10 factors and dummy variable were chosen for a twelve run design. The experimental range and levels of the parameters used are listed in Table 2 below.
Levels
Sr. No Variables
Low (-1 ) High(+1 )
Fi Yeast Extract (%) 0.01 1
F 2 Ammonium sulphate (%) 0.5 1 .5
Table 2: The actual experimental design is given in Table 3 (table is split into three sections a, b, and c due to space constraints of this document - these three sections should be read together in order of rows representing the 12 trail runs) accompanying this document as generated by the software. The factors chosen for PBD are glycerol as the carbon source, Yeast extract and ammonium sulfate as nitrogen sources, NaCI, salt solution along with five other physical parameter viz., incubation time, aeration, inoculum size, temperature and surface /volume ratio of the media. Parameter optimization was undertaken across 12 runs as under.
Table 3(a) Factor 6 Factor 7
Factor 8 Factor 9 Factor 10
Run Inoculum Surface:
Temp °C Salt solution % NaCI %
OD/100ml Volume %
1 1 20 20 2 0.1
2 1 20 25 0.1 2
3 10 20 25 2 2
4 10 50 20 0.1 0.1
5 10 50 25 0.1 0.1
6 10 50 20 2 2
7 1 50 20 2 2
8 1 20 20 0.1 0.1
9 1 50 25 0.1 2
10 10 20 20 0.1 2
11 10 20 25 2 0.1
12 1 50 25 2 0.1
Table 3(b)
Table 3(c)
[049] Example 3: Results
[050] Screening of medium components was carried out using the Plackett-Burman design to determine their effect on protein production. The media composition was chosen on previous studies carried out. Ten medium components were examined. The protein yield obtained was 60 mg/l (run 1 1). The protein yield obtained using basal medium viz., 0.7% YNB, 1 % glucose was 20 mg/l. A 3 fold increase (mg/l) was obtained using Plackett Burman model (Table 3). On analysis of regression coefficient of ten medium component, Yeast extract, ammonium sulphate, incubation time, aeration, salt solution inoculum size and sodium chloride showed a positive effect for protein production, whereas glycerol, inoculum size and surface to volume showed negative effect in the tested range of concentration. ANOVA result for protein yield is shown in Table 4 below.
Table 4
[051 ] Table 4 represents the effect of each variable along with the mean squares, F-values, and p- values. The observed protein yield varied from 20 mg/l to 60 mg/l, reflecting the importance of medium optimization to attain higher yields. Variables having a probability value (p-value) less than 0.05 were considered significant. The analyzed data in Table 3 suggests that protein production was affected by yeast extract surface to volume ratio and temperature which had p-values of 0.0004, 0.0353 and 0.0006, respectively. There is significant effect of yeast extract as a nitrogen source for protein production by Y. lipolytica. Thus, Yeast extract and temperature were chosen and their possible interactive effects on protein production were evaluated. Statistical analysis of above results is shown in Table 5 below.
Table 5
[052] The Model F-value of 33.71 implies the model is significant. There is only a 0.01 % chance that an F-value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. mln this case A, G, H, AH are significant model terms. [053] Values greater than 0.1000 indicate the model terms are not significant. Table 5 shows the R- squared for the model is 0.9506. The "Pred R-Squared" of 0.8337 is in reasonable agreement with the "Adj R-Squared" of 0.9224; i.e. the difference is less than 0.2. "Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. The ratio of 15.473 indicates an adequate signal.
[054] Production level of self assembling protein secreted in the culture medium is usually low. By way of example, the crude protein yield obtained using standard medium (sterile Yeast nitrogen base with 1 % glucose) was observed to be 20 mgl "1 . For increasing production, the inventors herein have implemented response surface methodology, that is, a statistical model based on predicted growth and media parameters, by which yield of crude protein with the modified media and growth conditions was increased three-fold to 60 mgl "1 , and yield of purified protein obtained after the standardization of the purification procedure was 20 mgl "1 . It shall be appreciated that this increase in the production of the protein was achieved without using traditional genetic amplification tools and techniques.
[055] Characterization:
[056] The small molecular weight protein (8 to 9 kDa) obtained as above is a natural, surface active, bio-compatible molecule with dual properties of bioemulsifier as well as surfactant. The purified protein so obtained was observed to have low molecular weight along with following characteristics: a) Assembly at air-water interfaces - The purified protein (HBN) was able to reduce the surface tension showing the surfactant like activity as determined by capillary method (measured value of surface tension (γ) = 60.82076 mN/m). Accompanying FIGURE 1 is a microphotograph of foam produced by HBN. The accompanying FIGURE 2 is a photograph showing difference in foaming obtained using blank water (control), 10% SDS (positive control / next peer for comparison) and 50u.gml "1 HBN (test). Accompanying FIGURE 3 showcases surfactant properties as determined by hanging drop method using blank water (control), 1 % SDS (positive control / next peer for comparison) and 0.05mgml "1 HBN (test), thereby prove excellent surfactant properties. b) Required in small amounts for surface modification and adsorption applications -
The accompanying FIGURE 4 includes microphotographs, particularly in which is (a) glass coated with 50u.gml "1 HBN seen via light microscopy; (b) glass coated with 50u.gml "1 HBN seen via SEM; (c) silicon coated with 50u.gml "1 HBN seen via light microscopy; and (d) teflon coated with 50u.gml "1 HBN seen via light microscopy. c) Assembly on solid surfaces - Hydrophobicity of the surfaces was reversed by change in water contact angle by 30° upon coating with HBN. The accompanying FIGURE 5 is a comparative of water droplets on various substrates in untreated versus when treated with HBN. These results are listed in Table 6 below.
Table 6 d) Assembly at oil-water interfaces -Self-assembling nature resulted micelles formation. It also helped entrapment of the hydrophobic drug molecule into the micelles. In independent experiments conducted by the inventors named herein, the partially (3kda— > TFA extracted) isolated protein (HBN) was observed to exhibit good emulsification of oils, and hydrocarbons. The accompanying FIGURE 6 is a graph illustrating minimal time required for vortexing to achieve a stable emulsion (observed time was 10 minutes, which emulsion was stable till at least 144 hours). Stability of emulsions were as illustrated in the Table 7 below.
Table 7 e) Assembly in aqueous solution - HBN was observed to show concentration dependent variation in the particle size. Self assembly is exhibited with optimized media - higher molecular sizes can be observed. The accompanying FIGURE 7 showcases concentration dependent variation in the particle size, particularly (a) is a graph showing particle size distribution in electrophoresis run where lane 1 has 7.5 μ9 HBN and Lane 2 has 5 μ9 HBN; and (b) is a compilation of graphs showing vortexing dependent variation in the particle size.
[057] Industrial Applicability
[058] The present invention has been reduced to practice by the present inventors, which has been successful in production of the target self-assembling protein (that is, HBN) which has been so isolated and purified from the crude bioemulsifier and characterized for its bio-material property. It shall be appreciated that said protein is isolated from cell free supernatant of the organism and not extracted from cell lysate or cell wall extract, which makes it more suitable for large scale applications in industries by minimizing the downstream processing cost.
[059] As will be further appreciated from the foregoing disclosures, the present invention is identified in having the following salient features-
1) Production of emulsifier and surfactant which is effective at low concentrations.
2) Enhanced yield of target protein by changing media and growth conditions without using any genetic modification.
[060] Though disclosures of the present invention above are stated for shake flask level upto 1 liter capacity, this can further easily be optimized and scaled up to industrial levels without undue considerations.
[061 ] It shall also be appreciated that as Yarrowia lipolytics is identified as a GRAS resource, and ability of the protein, being secreted by it as per foregoing narration to change the surface property upon adsorption and its assembly at the interface it is evident that said protein is a promising molecule as a surface active bio-material in food, environmental, cosmetic industry, in biosensors and as scaffolds in tissue engineering applications as well as an efficient drug delivery vehicle (independent trials conducted by the present inventors confirmed proof of concept by able imbibing of curcumin in HBN micelles with paraffin oil and ethanol as co- solvent).
[062] As will be realized further, the present invention is capable of various other embodiments and that its several components and related details are capable of various alterations, all without departing from the basic concept of the present invention. Accordingly, the foregoing description will be regarded as illustrative in nature and not as restrictive in any form whatsoever. Modifications and variations of the system and apparatus described herein will be obvious to those skilled in the art. Such modifications and variations are intended to come within ambit of the present invention, which is limited only by the appended claims. It shall be generally noted that at least a major portion of the foregoing disclosures of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in files or records of the receiving Patent Office(s), but otherwise reserves all copyright rights whatsoever.