4. DESCRIPTION
FIELD OF INVENTION
The present invention relates the process of denaturing of misfolded proteins (amyloids) associated to various disorders such as type II diabetes. More specifically this invention relates to the process for degrading and reducing amyloid fibril formation by using the enzyme serratiopeptidase (SP).
BACKGROUND OF THE INVENTION
In recent times a majority of the worldwide scenario focuses on the diseases related to amyloids. By the year 2020, a large population in India specially South India will be affected with neurodegenerative disorders like CJD, Alzheimer's disease was well as type II diabetes(l). Natural products are also being proposed by many researchers as agents for reducing amyloid deposits in the senile plaques of brain in neurodegenerative disorders (2-3), but no data exists till date exhibiting the process of denaturation of amyloid fibrils using SP.
Serine proteases like SP are very much stable in gastrointestinal tract that can be used for an oral medication and also the structure of amyloids is highly homologous to the structure of this serine protease substrate in case of (3-pleated sheet secondary structure. Thus it may be hypothesized that these serine proteases can be used for the degradation of amyloids. The potential for SP on amyloid degradation is yet to be explored. NK is another serine protease that has been patented for its amyloid degradation properties (4), but it has not been reported so far for SP as an agent for degrading amyloids. In the present study we have exploited the role of this SP on amyloid degradation in comparison to the standard amyloid degrading enzyme NK and our results till date concludes that this enzyme has a potential to denature insulin amyloid.
OBJECT OF THE INVENTION
The present process is denaturation of protein aggregates by SP gives a potential scope for development of agents for dissociation of misfolded proteins.
This potential of SP enzyme is novel that can be used to degrade insulin amyloids as well as amyloids related to neurodegenerative disorders.
This enzyme (SP) is capable of degrading the amyloid fibrils both in vitro and in vivo.
STATEMENT OF INVENTION
The enzyme SP can be used for the dissociation or degradation of misfolded protein aggregates that are harmful to human beings and found in diseased conditions in different neurodegenerative disorders as

well as in Type II diabetes.
SUMMARY OF THE INVENTION
The present invention relates the potential of SP towards the process of denaturation of amyloid aggregates by in vitro as well as in vivo.
In one aspect, the invention relates methods of denaturing insulin amyloid, a model amyloid used for various amyloid related disorders, by using SP enzyme.
SP can be used to digest amyloids present in the human body because it is stable in the gastrointestinal tract and active up to temperature of 60 °C.
In one aspect, the invention relates to the denaturing of pre-formed amyloid aggregates and inhibiting the amyloid formation, deposition or accumulation in amyloidosis.
The therapeutically effective amount of SP may be administered through supplement, aerosol spray or directly through infusion or localized injection.
In another aspect the invention relates to the denaturation or inhibition of amyloids that are formed in vitro. The method comprises the effect of SP by adding to these amyloid fibrils and its potential to denature the amyloids.
In another aspect the invention relates to the denaturing of amyloids that are formed through in vivo environment. The method comprises of the effect of intramuscular injection of amyloid and amyloid with SP and subsequent observation of fluorescent images, depicting amyloid degradation, under in vivo imaging system for a designated time.
BRIEF DESCRIPTION OF DRAWINGS
FIGURE 1 shows (A) Circular Dichroism (CD) spectra of insulin fibrils before and after digestion by'SP at 37 °C, pH 7. (B) Content of (3 sheet in insulin fibrils before and after digestion by SP. (C) Fluorescence intensity by Thioflavin T (ThT) assay of insulin fibrils before and after digestion by SP at 37 °C, pH 7. (D) Fourier Transform Infrared (FT-IR) spectra of insulin amyloid before.and after digestion by SP.
FIGURE 2 shows (A) insulin amyloid degradation ability of SP measured by ThT assay. (B) Turbidity assay of insulin amyloid with or without co-incubation of SP enzyme.
FIGURE 3 shows (A) comparison of SP amyloid degrading ability with standard known amyloid

degrading enzyme NK. (B) The toxicity of insulin amyloid (IA) on PC 12 cells at various concentration of insulin amyloid which is treated with SP (IA+SP).
FIGURE 4 shows particle size distributions detected by dynamic light scattering (DLS). (A) Size distribution of amyloid at designated time (B) Size distribution of amyloid aggregation which is co-incubated with SP. (C) The color map summarized the size distribution of all experimental group measured at indicated time points.
FIGURE 5 shows in vivo live animal imaging by using zebrafishes. (A) Fluorescent images acquired by using in vivo imaging system. (B) Fluorescence intensity of zebrafishes measured by ROI (Region of Interest) tool.
DETAILED DESCRIPTION OF THE INVENTION
Amyloids are insoluble fibrous protein aggregates sharing specific structural traits. Abnormal accumulation of amyloid in organs may lead to amyloidosis, and may play a role in various neurodegenerative diseases. These extracellular protein deposits have some common morphological properties, staining characteristics and x ray diffraction pattern. Amyloids are associated with more than twenty diseases such as neurodegenerative diseases and metabolic diseases and its deposition plays an important role in the pathogenesis of protein misfolding based diseases such as Parkinsons disease, Alzheimers disease, Creutzfeldt-Jakob disease, Hereditary cerebral hemorrhage, and Type II diabetes (5).
Insulin is the pivotal hormone that routinely administered into the subcutaneous tissue for an antidiabetic medication. Thus a localized amyloidosis is commonly found at the site of repeated insulin injection in a diabetic patient and its cytotoxicity is thought to be an early mechanism involved in death of insulin-producing islet (3 cells thus insulin amyloid can be considered as a model protein for amyloid study.
Enzymes play an important role in biotechnological sector because of its specific activity towards its substrate. Protease enzymes have an eminent position in biotechnological sectors as it shares more than 50% sales worldwide. SP is proteolytic enzyme isolated from the non pathogenic enterobacteria Serratia marcenscens. This enzyme found naturally in the intestine of silk worm, which is used by the silkworm to degrade cocoon.
The preliminary result supporting our statement is described in the following examples.
EXAMPLE 1
Materials
Huminsulin 30/70 40 lU/ml, manufactured by Eli Lilly and Company (India) Limited was procured

from local pharmacy. NK plus Astaxanthin & CoqlO from Hawaiian Herbals, USA; Thioflavin T (ThT) from Sigma Aldrich and other chemicals were purchased locally. SP was purchased locally and used for amyloid degradation study.
Normalization of enzyme activity of SP with a standard enzyme NK
To normalize the enzyme activity of SP with NK, in vitro fibrinolytic activity was assayed according to Prasad et.al (2006) (6). The standardization of enzyme activity of the SP with a standard enzyme NK was done using in vitro fibrinolytic assay (data not shown) and caseinolytic assay. Fibrinolytic activity assay showed that NK has 0.96 fold fibrinolytic activity compared to SP. To normalize the activity, 9.6 |al of SP was used for every 10 \il of enzyme NK for degradation of amyloid study. Further, SP protease activity was assayed using casein as substrate and the specific activity of SP was found to be 6.067 unit/mg (data not shown).
EXAMPLE 2
In vitro insulin amyloid formation
Insulin is known to form amyloid fibrils under heat stress. ThT dye can bind specifically to amyloids . and is used for the detection of insulin amyloids. In this study Huminsulin 30/70 40 IU/ml was incubated at 65 °C for amyloid formation (7). The incubation was done in sealed glass vials to avoid evaporation and confirmed the formation of amyloid by ThT fluorescence, FT-IR, CD.
In this work, insulin solution (1.31 mg/ml) was incubated at 65 °C for 2 h, 4 h, 6 h, 12 h, 24 h, 28 h and 32 h, to study the lag, log and stationary phase of insulin amyloid which was further confirmed by ThT assay, turbidity assay and DLS(dynamic light scattering).
To monitor amyloid concentration in solution at respective time, insoluble aggregates (turbidity) were measured at 600 nm using Shimadzu UV-1800: UV-VIS double beam spectrophotometer.
To trace the size of insulin amyloid DLS was performed using a Malvern nano ZS90 zeta sizer. Briefly, In 1 ml PBS 10 pi of samples were added and allowed to equilibrate for 1 min. The hydrodynamic diameter obtained from the DLS data was taken as a measurement for the fibril size!
Enzymatic degradation of pre-formed insulin amyloid fibrils
For enzymatic degradation of insulin amyloid fibrils, the pre-formed insulin amyloid was used at various time intervals (2 h, 4 h, 6 h, 12 h, 24 h, 28 h and 32 h). At each time interval, 9.6 |il of the enzyme SP was added in 190 |il pre-formed insulin amyloid samples and kept for incubation for 37°C for 1 h for enzyme action. After 1 h, the degradation status was measured by Circuar Dichroism (CD) by placing insulin amyloid sample with or without enzyme SP sample was placed in a 1-mm quartz cell. The CD

spectrum is recorded from 200 nm to 250 nm on a J-715 CD spectrometer (JASCO, Japan). For ThT fluorescence assay, ThT solution in PBS (10 mM, pH 7.4) (20 \xM, 190 |il) was mixed with 10 |il samples in quartz cuvette and the meaasurement was carried out using Spectrofluorimeter (Jasco-FP8300 Model). For FT-IR spectra, enzyme treated and non-treated amyloid samples were measured using Bruker alpha FT-IR spectrometer equipped with N2-cooled mercury telluride detector and the area between 1590 cm-1 and 1710 cm*' was considered for [3-sheet indications of amide band.
Structural Evidences of degradation of pre-formed insulin amyloid by SP
To determine whether SP exhibits amyloid degrading activity, its effect on preformed insulin amyloids was assayed. Insulin was incubated at 65 °C for 24 h for the formation of amyloids and to these insulin fibrils SP was added and further degraded at 37 °C (pH 7.2). The degradation of §P was confirmed by a decrease in ellipticity as visible from the CD spectra (Fig. 1A). Post treatment with SP yielded the negative ellipticity at 218 nm which indicates the loss of P sheet structure (Fig; 1A and IB). ThT is a dye that specifically binds to P-sheet rich amyloids and gives enhanced fluorescence. The result obtained by ThT binding assay showed a decrease in fluorescence at 487 nm upon SP treatment compared to insulin amyloid alone, indicating the loss of (3 sheet (Fig. 1C). The FT-IR spectra also revealed the decrease in transmittance at 1635 cm*1 after the treatment of amyloid fibrils with SP (Fig. ID). The FT-IR peak at • 1635 cm-1 indicates the amide bands of amyloid and a decrease of transmittance in this range indicates the loss of P sheet. Thus, the results obtained from CD, ThT fluorescence assay and FT-IR gives an indication that when treated with SP, the amyloid fibrils are reduced giving rise to loss of p sheet structure of the insulin amyloid.
Insulin fibril growth and its degradation by SP
ThT binding assay, turbidity assay, and DLS assay were used to analyze the fibrillation status of insulin
amyloid and its degradation by the enzyme SP at different time after incubation. To observe the growth
of insulin amyloid, time course degradation of insulin amyloid was performed. The insulin sample was
incubated at 65 °C for 2 h, 4 h, 6 h, 12 h, 24 h, 28 h and 32 h. After each time point of incubation, the
insulin fibrils were taken out and SP was added to it and further 1 h (60 min) or 1.5 h (90 min)
incubation was done with this enzyme at 37 °C. The degradation status was confirmed by ThT binding
assay (Fig. 2 A), turbidity assay (Fig. 2B) and DLS (Fig, 3). i
ThT based fluorescence was used to monitor the aggregation process of insulin amyloid at various time j
intervals. There is an increment of fluorescence signals at 479 nm for control insulin sample (without enzyme) and a decrease in the intensity of fluorescence was observed for insulin sample which is treated with enzyme for 1 h at 37 °C (Fig. 2A). The results from 1 h incubation with the enzyme revealed that 1 h is not sufficient for complete clearance of the amyloid fibrils. So we observed the effect SP on insulin amyloids with an incubation time of 90 min at 37 °C temperature in pH 7.0 (Fig. 2 A). There was a significant amount of amyloid clearance observed in insulin sample which is incubated with the enzyme

for 90 min. Control insulin which was not treated with enzyme gave strong fluorescence signal up to 24 h and a consistent fluorescence was observed at 28 h and 32 h.
The ThT fluorescence study was further supported by turbidity assay. The absorbance at 600 nm usually indicates the insoluble aggregates in the sample of insulin (8). An increment in an optical density at 600 nm was observed in control insulin amyloid sample whereas the increase in turbidity was lower for pre¬formed insulin amyloid sample which is treated with enzyme SP (Fig. 2B). The decrease in absorbance at 600 nm in a pre-formed insulin sample which is treated with SP indicates the ability of SP enzyme towards the amyloid degradation.
Nattokinase (NK) is a standard known enzyme for the degradation of various amyloids like insulin, AP, and prion amyloid (4). In our study, NK was used for the comparison of the amyloid-degrading ability of NK and SP. The pre-formed insulin at various designated time was incubated with the enzyme NK and SP at 37 °C at pH 7.0 for 1 h. After enzymatic degradation, the ThT fluorescence assay was performed to observe the possible effect of NK on insulin amyloid maintaining the same conditions as for SP. ThT results (Fig.3) depicted that SP is has higher ability to degrade the insulin amyloid. The degradation of insoluble aggregate may form soluble aggregates, which can be more toxic than the matured insoluble fibrils. So it was very much interesting to see the toxicity of disintegrated particle (product of amyloid digestion by SP) on PC 12 cells (pheochromocytoma of the rat adrenal medulla). At 18 \xM of insulin amyloid cell viability was observed upto 84.19 % whereas insulin amyloid treated with SP at 18 |aM showed cell viability upto 99.72 % (Fig. 3B). MTT assay suggested that there is no toxicity of disintegrated particle compared to control (cells without any treatment).
DLS (DLS) measurements were further performed to monitor the aggregation size of amyloid at each designated time. At 2 h the size of insulin amyloid was observed 460 nm and the size of the aggregates increased up to 3200 nm at 24 h (Fig. 4A). However, the insulin amyloids which were treated with SP exhibited a lower size distribution observed at each designated time compared to untreated insulin amyloids (Fig. 4B) and the maximum size observed after degradation was nearly 1000 nm (Fig. 4C). The results obtained from ThT fluorescence study, turbidity assay and particle size analysis indicated that SP has the capacity to degrade insulin amyloids in vitro.
In vivo degradation potential of SP towards insulin amyloid
Insulin amyloid was formed according to Lee et.al (7) and confirmed by ThT fluorescence assay. For subcutaneous injection in zebrafish, the sample was prepared as follows. 9.6 |il SP from stock solution was added to 190 nl of pre-grown insulin amyloid samples respectively. 10 |al of ThT (20 \xM) was added to 100 \xl sample of the above solution. Finally, 12.5 jo.1 of the solution was injected subcutaneously in the zebrafish using a microsyringe (9). For NK treated fishes the enzyme taken was 10 ^1 from stock and proceeded in the same way as for SP. For control without the enzymes, only insulin amyloid was injected with ThT at the same concentrations mentioned above.- Fluorescence

images were acquired by using IVIS Kinetic from Caliper Life Sciences according to Chen et. al (10) with an excitation at 440 nm (slit width 5 nm) and emission using specific filter up to 2 h post injection. Just after the injection fishes were placed on the stage of an instrument and the image is acquired.
To support the findings of in vitro study, it was necessary to study the in vivo amyloid-degrading ability of SP. Adult zebrafishes were used for the study of in vivo degradation of insulin amyloids. A pre-formed insulin amyloid with or without SP was injected subcutaneously into the zebrafish and the real-time degradation of the amyloid was observed by using small animal live imaging. In this study, we used ThT dye to track the presence of insulin amyloids inside the zebrafishes. In vivo results suggested that the insulin amyloids were distributed throughout the body of the zebrafish till 2 h and there is no significant decrease in the ThT intensity indicating the existence of the amyloid. In the case of SP treated fish, an initial decrease in the fluorescence intensity followed by a sharp decrease was observed compared to the initial stage (Fig. 5A and 5B). Thus, these data clearly depicted a remarkable change in the ThT emission intensity after treatment with the enzyme SP indicating a significant reduction of amyloids. The in vivo study confirms that the SP has a capacity to reduce amyloid fibrils. Thus our data based on CD, FT-IR, ThT fluorescence, turbidity, DLS, MTT assay and in vivo animal imaging provides sufficient information to show that SP has a potential to dissociate amyloid fibrils in vitro and in vivo.
Conclusion
Amyloidosis is the group of several disorders related to the misfolding of normal cellular proteins into highly (3-sheet rich insoluble aggregates and their deposition in various parts of our body. These aggregates share similar traits and are mostly protease resistant and thereby cannot be cleared from our body. Any agent that can dissociate these aggregates can give insight into the development of potential drug candidates for amyloidoses. In the present study, insulin amyloids were preformed and their dissociation by an enzyme SP was studied in vitro and in vivo. The results obtained from in vitro dissociation studies using CD and FT-IR indicated a low amount of P sheets in the SP treated insulin amyloids. The amount of amyloids was also reduced upon SP treatment as indicated by fluorescence and turbidity studies. The sizes of the fibrils were also reduced as visualized from the DLS studies. The cell viability assay shows that insulin amyloid which is treated with enzyme SP is having same cell viability like untreated control cells whereas only insulin amyloid has shown some toxicity. To determine the efficacy of SP in vivo, zebrafish studies were accomplished which indicated a clear reduction of amyloid fibrils after 2 h post injection of SP with insulin amyloids. The data altogether strongly suggests that SP can indeed reduce and dissociate the amyloid fibrils formed by insulin. Further studies are required to be done with other amyloids like Alzheimer's AP and Prion peptide.

1. We claim a method of dissolving or degradation and/or disintegration of pre-formed insulin amyloid fibrils in an in vitro environment, the method comprising: adding to the in vitro environment a composition comprising an effective amount of SP to degrade insulin amyloids.
2. We claim a method of dissolving or degradation and/or disintegration of pre-formed insulin amyloid fibrils in an in vivo environment, the method comprising: adding to the in vivo environment a composition comprising an effective amount of SP to degrade insulin amyloids.
3. We claim a method of subcutaneous insulin amyloid clearance which is associated with, type II diabetes.
4. The method of claim 1, wherein the amyloid associated with type 2 diabetes and can be a model protein for Alzheimer's and/or a Prion disease.
5. The method of claim 1, wherein the pre-formed amyloid comprises the object like surgical instruments, a pharmaceutical product, and animal feed and SP can be used to. sterilize them.
6. The method of claim 2, wherein the in vivo clearance of amyloid by SP comprises the tissue/organ which has been affected with amyloids example kidney, heart and brain.