Isolation, purification and characterization of proteinaceous fungal α-amylase inhibitor from rhizome of Cheilocostus speciosus (J.Koenig) C.D.Specht

Accepted Manuscript Isolation, purification and characterization of proteinaceous fungal α-amylase inhibitor from rhizome of Cheilocostus speciosus (J.Koenig) C.D.Specht

Abinaya Balasubramanian, Manish Bhattacharjee, Meenakumari Sakthivel, Munusamy Thirumavalavan, Thirumurthy Madhavan, Santhosh Kumar Nagarajan, Velusamy Palaniyandi, Pachaiappan Raman PII: DOI: Reference:

S0141-8130(17)34797-9 https://doi.org/10.1016/j.ijbiomac.2017.12.158 BIOMAC 8820

To appear in: Received date: Revised date: Accepted date:

4 December 2017 27 December 2017 29 December 2017

Please cite this article as: Abinaya Balasubramanian, Manish Bhattacharjee, Meenakumari Sakthivel, Munusamy Thirumavalavan, Thirumurthy Madhavan, Santhosh Kumar Nagarajan, Velusamy Palaniyandi, Pachaiappan Raman , Isolation, purification and characterization of proteinaceous fungal α-amylase inhibitor from rhizome of Cheilocostus speciosus (J.Koenig) C.D.Specht. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), https://doi.org/ 10.1016/j.ijbiomac.2017.12.158

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Isolation, purification and characterization of proteinaceous fungal α-amylase inhibitor from rhizome of Cheilocostus speciosus (J.Koenig) C.D.Specht

Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur-603203, Tamilnadu, India

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Abinaya Balasubramanian1, Manish Bhattacharjee1, Meenakumari Sakthivel1, Munusamy Thirumavalavan3*, Thirumurthy Madhavan4, Santhosh Kumar Nagarajan4, Velusamy Palaniyandi1 and Pachaiappan Raman1,2*

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Graduate Institute of Environmental Engineering, National Central University, Chungli, Taoyuan County, 320, Taiwan. Department of Genetic Engineering, School of Bioengineering, SRM University, Kattankulathur-603203, Tamilnadu, India

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Metabolomics, Proteomics and Mass Spectrometry Core Facilities, EEJMRB, 15 N Medical Drive East RM A306 (Basement), University of Utah, Salt Lake City, UT 84112-5650, USA.

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Corresponding author E-mail: [email protected] (R. Pachaiappan), Phone: +91-9486433614, Fax: +91-44-27453903 *

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Corresponding author E-mail: [email protected]; (M. Thirumavalavan), Phone: +886-3-4279455, Fax: +886-34279455

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ABSTRACT As the aim of this present study, a proteinaceous α-amylase inhibitor has been isolated from the rhizome of Cheilocostus specious (C. speciosus) and was purified using DEAE cellulose anion exchange chromatography followed by gel filtration using Sephacryl-S-200

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column. The purity and molecular mass of the purified inhibitor was determined by SDS-PAGE

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and LC-MS respectively. The molecular mass of the purified inhibitor was determined to be 31.18 kDa. Protein-protein docking was also carried out as molecular model. Model validation

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methods such as Ramachandran plot and Z-score plot were adopted to validate the structural description (sequence analysis) of proteins. The inhibitory activity was confirmed using

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spectrophotometric and reverse zymogram analyses. This 31.18 kDa protein from C. speciosus

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inhibited the activity of fungal α-amylase by 71% at the level of ion exchange chromatography and 96% after gel filtration. The inhibition activity of the α-amylase inhibitor was stable and

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high at optimum pH 6 (52.2%) and temperatures of 30-40 C (72.2%). Thus it was suggested that

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the main responsible for the versatile biological and pharmacological activities of C. speciosus is

KEYWORDS

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due to its primary metabolites (proteins) only.

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Cheilocostus speciosus; α-amylase inhibitor; reverse zymography

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1.

INTRODUCTION α-amylase (1,4-α-D-glucan glucanohydrolases; E.C. 3.2.1.1) is an important hydrolytic

enzyme responsible for breakdown of α-1,4-glycosidic linkage in soluble starch or amylose. αamylase is basically involved in the breakdown of starch to glucose and maltose. Since almost all

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living organisms need glucose for their energy requirements, it indirectly comes to the point how

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efficiently stored food in the form of starch is broken down to glucose via α-amylase, thus αamylase stands at a branching point for the activation of most of the energetic pathways in living

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systems. Right from complex polysaccharide digestion in animals to seed germination in plants, α-amylase plays an essential role. Generally α- amylases are metallo-enzymes, which contain at

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least one Ca2+ ion. α-amylases have been found to have a wide range of molecular weight in

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different organisms starting from 10 kDa to as high as 230 kDa [1]. In the case of microorganisms like fungi, α-amylase is one of the major extracellular enzymes to be secreted.

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In fact the fungus Aspergillus oryzae is such a strong secretor of α-amylase and it is very

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extensively used in the industries for mass extraction of α-amylase. The optimal temperature of

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fungal amylase is around 50 ºC; however it is stable in a range between 40 and 60 ˚C. Its activity is higher at a pH range from acidic to neutral and very mild alkaline pH [2].

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In spite of all these benefits of α - amylase, it has certain demerits as well. For plant seeds to germinate, water has to be added to the system which leads to the activation of α-amylase gene which in turn is responsible for the breakdown of stored starch to glucose to fulfill the immediate energy requirements for germination, but in many cases, especially in under developed and developing countries due to improper storage facilities, when germination is not favored, the natural activity of α-amylase leads to huge commercial crop loss. Also one more case where the natural activity of α-amylase is a major cause for commercial crop loss is in case

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where pests and pathogens like fungi use their α-amylase to digest plant parts [3]. Also in humans α-amylase activity leads to hyperglycemia and dental plaques [4, 5]. So with all these issues the solution lies in the usage of a specific α-amylase inhibitor that can inhibit the natural activity of α-amylase to a significant extent in order to overcome these issues, especially in the

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field of agriculture. These inhibitors can be in the form of both secondary metabolite as well as

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primary metabolite like proteins [6, 7]. In this context a primary metabolite like protein that can inhibit amylase is more beneficial than a secondary metabolite inhibitor because the former will

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be coded by only a single gene and can be also easily used in r-DNA technology as its immediate application.

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Many plants contain substances that can inhibit enzymes, especially hydrolases. Most of

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these compounds are proteins by nature, which could specifically inhibit enzymes by forming complexes that block the active site or alter enzyme conformation, eventually reducing the

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catalytic function. Many proteinaceous α-amylase inhibitors have been isolated from various

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plants [8-10] and based on their molecular weight and number of disulphide bridges they have been identified into 5 types [11, 12]. Although many plants have been found to be good source of

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proteinaceous α-amylase inhibitors, Cheilocostus speciosus (C. speciosus) has not been reported

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as a source of proteinaceous α-amylase inhibitor. Costus speciosus (J.Koenig) Sm. native of Southeast Asia is a synonym of Cheilocostus speciosus (J.Koenig) C.D.Specht and belongs to family Costaceae. C. speciousus or crepe ginger. The species reproduces vegetatively by rhizomes and is cultivated in India for its medicinal uses. The rhizome part of C. speciosus is bitter tasting and yet it is considered to be the most valuable part of this plant from the medicinal point of view, as it has been used for the treatment of fever, hyperglycemia [13], rash, asthma, bronchitis, and intestinal worms and useful in relieving burning sensation, constipation, leprosy,

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anemia and other skin ailments [14]. The rhizomes of C. speciosus have been reported to possess steroid–diosgenin [15, 16] which is anti-diabetic in nature. Essential oil from rhizome has been found to have antimicrobial activity [17]. Steroid saponins and sapogenins from C. speciosus have been seen to exhibit antifungal activity [18]. Japanese used the rhizome extract in control of

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syphilis. Thus, pharmacological studies showed that the rhizomes of C. speciosus possess anti-

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lipidemic, cardiotonic, hydrochloric, diuretic and Central Nervous System (CNS) depressant activity [19]. Hence, the rhizome was chosen as the plant part for the extraction of proteinaceous

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α-amylase inhibitor. With this background, the present task was aimed to report the isolation, purification and characterization of proteinaceous α-amylase inhibitor from Cheilocostus

MATERIALS AND METHODS

2.1.

Materials

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speciosus for its potential applications in r-DNA technology and also against plant pathogens.

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Fungal α-amylase (E.C. 3.2.1.1), agar powder, dinitrosalicylic acid and sodium potassium

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tartarate were obtained from HiMedia Laboratories, India and soluble starch was obtained from SRL, India. DEAE cellulose resin was purchased from Sigma Aldrich, USA and it was activated

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according to the company’s specifications. Sephacryl-S-200 column material was purchased

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from GE Healthcare Bio-Sciences, USA. Plant materials were obtained from local market. The rhizomes were air dried completely and ground to fine powder using an electronic mixer. 2.2.

Protein Extraction The extraction buffer that supported maximum yield of protein from the rhizomes was

initially optimized. The various solutions used for optimization studies were 10 mM Tris-HCl, (pH 7.6) containing 1% (w/v) 500 mM NaCl, and 10 mM Tris HCl, (pH 7.6) with 1 % (w/v) 500 mM NaCl containing 1% (v/v) 2-mercapto-ethanol, 0.1% (v/v) Triton-X-100 [20, 21]. For initial

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screening five grams of the rhizome powder was homogenized using three volumes of extraction buffer (10 mM Tris HCl, pH 7.6 containing 1% (w/v) of 500 mM NaCl, 1% (v/v) 2-mercaptoethanol, and 0.1% (v/v) Triton-X-100) followed by filtration using four layers of cheese cloth and centrifugation at 15,000 rpm for 15 min at 4 °C. The supernatant was collected and stored at

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-20 °C. This was used as crude extract for further analysis. Further 375 g of Cheilocostus

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speciosus rhizome powder was extracted using the extraction buffer for purification of the αamylase inhibitor. Protein Estimation

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2.3.

The protein content in the crude extract was quantified following Bradford’s dye binding

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method [22]. The standard curve was plotted using different concentrations of bovine serum

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albumin (BSA). Protein estimation was done after each step of purification. Spectrophotometric analysis for α-amylase inhibitory activity

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α-amylase inhibitory activity was measured according to the method prescribed by

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Ishimito [23] with slight modifications. 10 μl of enzyme containing 0.45 μg of fungal α-amylase and 40 μl of crude extract of each sample were mixed and pre-incubated for 90 min at 37 °C

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prior to the addition of 400 μl of 0.5% soluble starch solution in millipore water. After 10 min,

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the reaction was stopped by the addition of 250 μl of di nitro salicylic acid solution followed by boiling in a water bath for 10 min. The samples were then cooled to room temperature and the volume was made upto 3 ml using millipore water. 40 μl of millipore water was used as control. Then absorbance was measured at 540 nm using Amersham UV-Vis spectrophotometer. The α-amylase inhibitory activity was calculated as follows: Inhibition (%) = {[Absorbance (c) – Absorbance (s)] / Absorbance (c)} × 100 Where c = control and s = sample

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2.5.

Purification of α-amylase inhibitor The crude extracts were subjected to 80% ammonium sulphate fractionation [24] and

incubated overnight at 4 °C followed by centrifugation at 15,000 rpm, at 4°C for 15 minutes. The pellets were collected and dissolved in minimum amount of millipore water followed by dialysis

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against millipore water using a dialysis membrane (Mr exclusion limit < 12 kDa) at 4 °C for

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48 h. The dialyzed samples were screened for inhibitory activity using spectrophotometric assays. The dialyzed protein of C. speciosus was lyophilized then stored at -20 °C for further use.

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DEAE cellulose was rinsed with millipore water till a neutral pH was attained and then the activated resin was packed into a XK 16/40 column (GE Healthcare Bio-Sciences, USA). The

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column was equilibrated using 10 mM phosphate buffer pH 7.6 followed by which 25 mg of

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lyophilized sample of Cheilocostus speciosus was loaded and initially the unbound fractions

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were collected at a flow rate of 1 ml/min .After the complete collection of unbound fractions a linear gradient of 0-2 M NaCl was provided and the bound fractions were collected [25].

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The unbound and bound fractions were pooled respectively according to the protein

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peaks in the chromatogram .The pooled unbound fractions were lyophilized and stored at -20 °C for further analysis. The pooled bound fractions were dialyzed in order to remove the salt and

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then subjected to lyophilization and stored at -80 °C for further analysis. Fractions corresponding to α-amylase inhibitory activity were then pooled and applied to Sephacryl S-200 material was packed in XK 26/100 column (GE Healthcare Bio-Sciences, USA) and the proteins were eluted using 10 mM phosphate buffer (pH 7.6) at a flow rate of 1 ml/min. Fractions obtained after gel filtration were lyophilized and re-suspended in 1 ml of 10 mM phosphate buffer (pH 7.6) and were quantified for protein content, enzyme activity and characterization. 2.6.

Spectrophotometric analysis for α-amylase inhibitory activity

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Spectrophotometric analysis was performed according to Ishimito’s method [23] with slight modifications to identify the active fraction and to determine the percentage inhibition of the purified inhibitor. 2.7.

Analysis of α-amylase inhibitor by tricine SDS PAGE

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The purified amylase inhibitor was subjected to Tricine SDS PAGE in a vertical slab gel

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electrophoresis apparatus on a 10% polyacrylamide gel containing 6M urea [26, 27]. Low molecular weight marker was used as standard [ Bio-Rad laboratories, USA]. The gel was then

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subjected to silver staining [28]. Reverse zymography

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The α-amylase inhibitory activity was confirmed using reverse zymogram on SDS-PAGE

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copolymerized with starch. The purified inhibitor was incubated with amylase (10:1 w/w)

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respectively for 90 min. The incubated sample was loaded in lane 2 and the enzyme alone was loaded in lane 1 on a starch polyacrylamide gel (12%, w/v) containing 0.2% of soluble starch

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[29]. The electrophoresis was carried out using a vertical slab gel electrophoresis apparatus and at 4 °C at a potential of 100 V. The gel was then transferred to 0.1 M citrate phosphate buffer for

Effect of temperature on the activity of the amylase inhibitor

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2.9.

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2 h followed by staining using lugol’s iodine solution.

The purified inhibitor (40 μg) was mixed with 0.45 μg of the amylase and incubated at various temperatures ranging from 10 °C to 70 °C for 90 min. The incubated samples were then assayed for inhibitory activity using starch as a substrate by spectrophotometrically [27]. 2.10. Effect of pH on the activity of amylase inhibitor

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The inhibitor (40 μg) mixed with enzyme (0.45 μg) and was incubated with citrate phosphate buffer (pH 3, 4, 5, 6, 7, 8, 9), in a volume of 0.3 ml at 37 °C for 90 min. Aliquots were assayed for inhibitory activity against fungal amylase [30]. 2.11. LC-MS and MALDI-TOF-MS analysis

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The intact mass analysis was performed with purified liquid protein sample. The protein

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fraction after gel filtration column chromatography which shows fungal amylase inhibition was subjected to analyze the intact mass by nano-LC-MS (Impact HD, Bruker Daltonics, Germany).

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The purified protein was loaded onto nano –LC-MS and profile spectrum was recorded for this protein. The profile spectrum of this protein was calibrated with internal standard spectrum and

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at the same time was captured and deconvoluted for neutral mass with Bruker data analysis

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software. The neutral mass of the protein was reported here.

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The proteins fraction showing inhibitory action against fungal α-amylase excised from the SDS polyacrylamide gel were analyzed by MALDI-TOF-MS analysis (ultrafleXtreme,

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Bruker Daltonics, Germany) after digested using trypsin. The peptides obtained upon digestion

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were also subjected to Tandem mass analysis. The molecular masses were submitted to mascot search for protein identification. Homology modeling and validation

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To analyze the structural characteristics of the protein, we have performed homology modeling which is helpful in predicting the structure of a protein, when only sequence information of the protein is available [31]. A search for templates was performed using BLAST (Basic Local Alignment Search Tool) algorithm [32, 33] in protein data bank (PDB) [34]. Two template sequences having low E-value and high sequence percentage were selected (1DHK and 1VIW). At least a 30% sequence identity of the template is required, for developing a good

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homology model [35, 36], which was considered in various studies [37-41]. The templates we have selected have sequence identities > 40% and the query coverage were 75%. We have used three different modeling platforms such as EasyModeller, IntFold and ITasser. Easymodeller 4.0 [42] incorporates modeller in the backend, to develop homology

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models, whereas iIntFold [43] uses a unified server for modeling and to assess the quality of the

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models. Iterative threading assembly refinement, in short I-Tasser is based on a hierarchical method called as LOMETS (Local Meta-Threading-Server) [44]. The generated models were

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analyzed using various model validation techniques. Root Mean Square Deviation (RMSD) of the models with the corresponding templates was calculated, followed by a validation using

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Ramachandran (RC) Plot, ProSA server [45] and QMEAN server [46]. RC plots were provided

Protein-protein docking

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by the RAMPAGE server (http://raven.bioc.cam.ac.uk/rampage) [47].

Protein–protein docking was performed using the ClusPro 2.0 server, which is the best

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online server for protein-protein docking [48, 49]. ClusPro is based on a correlation method

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known as PIPER, which calculates the energy of the docked complex using fast fourier transform (FFT) coupled with pairwise interaction potentials [50]. Much fewer near native structures are

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only retained, because of the more accurate pairwise interaction potential of PIPER. The algorithm clusters the structures by considering pairwise RMSD as the distance measure. 3

RESULTS AND DISCUSSION

3.1

Protein extraction and purification Among the various buffers and solutions evaluated for isolating the α-amylase inhibitor

from Cheilocostus speciosus, the crude extraction made using 10 mM Tris HCl (pH 7.6) containing 500 mM NaCl, 1% 2-mercapto-ethanol, 0.1% Triton-X-100 as the extraction buffer

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resulted in maximum inhibitory activity. The general understanding of these proteinaceous αamylase inhibitors suggest that these are structurally stable and are not sensitive to mild denaturing environment, thus the mode of extraction of the proteinaceous α-amylase inhibitor was done in a reducing condition to destroy any form of endogenous amylolytic activity and to

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also reduce the extracted protein pool for an easier purification of the active α-amylase inhibitor.

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In fact this buffer has been reported as the appropriate extraction buffer for purification of αamylase inhibitor from Vigna sublobata and Cicer areitinum [23, 25]. The yield of proteins at

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every step of purification has been summarized in Table 1. The fold of purification after dialysis, ion exchange chromatography and gel filtration were found to be 35.92, 47.03 and 239.04

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respectively. The protein profile whcih was eluted after DEAE cellulose column chromatography

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is shown in Fig. 1a as a plot of UV absorbance at 280 nm with volume. The eluted protein profile

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on Gel filtration chromatography using Sephacryl S-200 column is shown in Fig. 1b. On ion exchange chromatography as shown in Fig. 1a, the bound fractions eluted with 2M NaCl showed

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a much higher inhibitory activity as compared to the unbound fractions. Bound fraction 1

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showed a significant inhibition of 71% and was subsequently applied to the gel filtration column which resulted in major peak number 5 with amylase inhibition of 96% and shown in Fig. 2. The

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collected fractions were further subjected to SDS-PAGE analysis. Both the dialysed and lyophilized bound and direct unbound fractions and fractions from gel filtration at different concentrations were loaded into SDS-PAGE to verify the fractions for pooling. 3.2

Spectrophotometric analysis for α-amylase inhibitory activity Spectrophotometric analysis was done after every step of purification and an increased

purification fold of 239.04 with increased inhibition was witnessed. An inhibition percentage of 71% was seen at the level of ion exchange chromatography as shown in Fig. 2a and the same

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was found to be 96% after gel filtration as shown in Fig. 2b. This suggests that with the increase in purity the effect of endogenous amylolytic activity and other molecules that stabilize the activity of amylase have significantly reduced from the protein pool thus enabling the inhibitor to express its maximal activity against fungal α-amylase. A similar pattern of increase in inhibitory

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activity on purification of the inhibitor has also been reported previously [51, 52]. Table 2

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represents the activity of purified α-amylase inhibitor proteins from C. speciosus on various α amylases. The inhibition activity was increased in the order as follows; Aspergillus

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oryzae>Porcine pancreatic>Bacillus subtilis>maize>human salivary.

Analysis of amylase inhibitor by Tricine SDS PAGE and reverse zymography

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The molecular mass analysis was estimated based on SDS PAGE analysis. Single protein

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band of molecular mass 31.1 kDa is clearly observed in reducing Tricine SDS PAGE, as shown

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in Fig. 3a and it testified the purity of the fraction. The molecular mass of 31.18 kDa of the active amylase inhibitor may suggest that this inhibitor may be categorized under Lectin type

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inhibitor [53, 54]. Previously reported proteinaceous fungal amylase inhibitors were also found

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to have a similar molecular mass [55]. Results observed for the reverse zymogram analysis on Starch-PAGE (12% w/v) testified the amylase inhibitory activity of the purified fraction

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containing the active amylase inhibitor as shown in Fig. 3b. Upon staining the gel with iodine, inhibition was clearly seen in lane 2 that contained the inhibitor along with amylase as compared to lane 1 which only contained the same amount of amylase alone. The inhibition was qualitatively comparable and this positive result for amylase inhibition also suggested that there exists a strong affinity of the inhibitor for the amylase that could not be electrophoretically separated. 3.4

Effect of temperature and pH on the activity of the amylase inhibitor

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The amylase inhibitor was tested for optimal activity in varying temperatures ranging from 10 ºC to 70 ºC as shown in Fig. 4. The optimum inhibitory activity was observed in the narrow temperature range of 30-40 ºC with the highest inhibition observed at 30 ºC (72% inhibition) as compared to the other temperature ranges in the experiment. There was a drastic

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decline in the inhibitory activity of beyond 40 ºC suggesting that this inhibitor compared to most

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other types proteinaceous amylase inhibitors is highly temperature sensitive [54]. Also the inhibition percentage was low at temperatures below 30 ºC probably due to very low activity of

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fungal amylase at those temperatures itself. Due to high molecular weights of lectin type amylases and comparatively low cystine residues with respect to total amino acid residues the

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thermal stability of these inhibitors is generally on the lower side [56].

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The data represented in Fig. 5 indicates that the amylase inhibitor was active over a mild

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acidic range pH towards neutral pH and optimum pH at which maximum inhibition was observed at pH 6 (52.2% inhibition) compared to that noted at other pH levels. Further it was

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also observed that there was a significant loss in the inhibitory activity in the alkaline range as

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well as in the pH range below 5. This optimal pH range for the inhibitory activity is almost consistent with the previously reported proteinaceous fungal amylase inhibitors. Also this

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optimal pH range for the inhibitor may be advantageous against plant pathogenesis as the extracellular pH of plants is exactly coinciding with the optimal pH range of this inhibitor [12, 55]. 3.5

Mass analysis, homology modeling and validation The molecular weight of the purified α-amylase inhibitor from C. speciosus after gel

filtration column fraction was determined by LC-MS. The proteins showing inhibitory action was excised from the polyacrylamide gel and subjected to MALDI-TOF-MS analysis after

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digested with trypsin. LC-MS spectrum of the purified intact protein of C. speciosus as shown in Fig. 6a confirmed that the molecular weight of protein showing inhibitory action in the corresponding region is same as the molecular weight determined by SDS-PAGE analysis. The intact mass was confirmed as 31,188.163 Da by LC-MS as shown in Fig. 6a. Results of MALDI-

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TOF-MS molecular mass fragments spectrum after trypsin digestion of purified protein as shown

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in Fig. 6b showed the obtained sequence of molecular mass fragments. Based on the molecular ion peak, the spectral data identified the molecular mass of the corresponding obtained peptide

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mass fragmentation is 2807 which is responsible for the activity and both the peptide mass fragmentations 1754 and 1835 were represented as the basal peaks. The observed significant

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peptide fragmentations were 1317, 1371, 1499, 1520, 1878, 1928 and 2697 along with few more

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possible fragmentations.

In the homology modeling phase, actually we looked for an experimentally determined

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structure of high “sequence identity” with the amylase inhibitor. This normally proceeds via a

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series of well-defined steps such as building a model, refining the model, assessing the quality of the model and sequence alignment. Prot search analysis matched with the known data for the

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analysis of protein series match is shown in Table 3. Table 4 shows the alignment of α-amylase

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inhibitor from C. speciosus against other plant proteins. Sequence alignment of the query sequence with the template was performed, which is represented in Fig. 7. Using three different platforms homology models were generated and 37 models were selected - 27 models from EasyModeller, 5 each from IntFold and ITasser servers. The promising applications of generated protein models depend significantly on the quality of the obtained models. The quality of the generated model of amylase inhibitor was checked by

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several methods. To study the placement of the helices of amylase inhibitor, we overlaid the transmembrane regions of the generated model with the template. The models were subjected to validation using RMSD, Ramachandran plot, ProSA and QMEAN. From the obtained RMSD value for amylase inhibitor, we can deduce that there is a

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good agreement between the helices. Model 22 was identified to be the best model after

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validation, and further used in the protein-protein dock. Model 22 has 93.0% of residues in the favourable region, 5.0% of residues in the allowed region and 2.0% of residues in the outlier

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region. It scored a ProSA Z-score of -2.62 and a QMEAN value of -5.78. Model validation statistics of the developed models are tabulated in Table 5. The superimposition of the model 22

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with the template 1DHK is represented in Fig. 8. Fig. 9a represents the Ramachandran plot and

Protein-Protein docking

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Z-score plot obtained from the ProSA server is shown in Fig. 9b.

Docking is the most widespread method to estimate the extent of protein interaction and

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it is an efficient method to predict the potential binding sites on the protein target. The docking conformations were separated into clusters. Crystal structure of human salivary alpha-amylase

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dimer was downloaded from PDB (PDB ID – 1XV8). CLUSPRO 2.0 developed 27 different

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protein-protein clusters. Different types of intermolecular energies can be estimated using auto dock. Among these the electrostatic interaction is the most significant as it can assign the strength of binding in the active sites. The obtained docking results showed that the compounds occupy the space in the binding site. Fig. 10 represents the protein-protein docked structure and the best possible binding in the amylase inhibitor active site is illustrated in Fig. 10. Table 6 represent the clusters along with their weighted scores. was used to Both the hydrophobic and hydrogen bonding interactions were investigated using LIGPLOT software to validate the

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interaction of developed model and the results confirmed the presence of hydrophobic residues suggesting that more hydrophobic interactions around this area should improve the inhibitory activities. Residues which form important interactions between the receptor and the ligand are identified by analysing the docked complex. Residues which forms H bond are represented in the

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Table 7. Residues ARG27, LYS52, PHE54, ALA57, THR111, TYR151, SER163, HIS305,

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GLY306, ALA307, ALA310, GLU349, ASN350 and GLY351 formed H-bond interaction with the protein. Fig. 11a represents the H bonds formed between proteins and Fig. 11b and c

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represent the Ligplot of the complex. CONCLUSION

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Rhizomes of C. speciosus have been recognized as a source of several bioactive

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substances with properties like anti-hyperglycaemic, antipyretic, anthelminthic etc but so far it

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was not reported as a source of amylase inhibitor. In our study the potential of this plant as a source of fungal amylase inhibitor is indicated. A 31.18 kDa protein was isolated from the

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rhizomes of C. speciosus and was purified. Temperature 30-40 C and pH 8.0 are considered to

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be the optimum parameters. The characterization studies conducted on various aspects of this proteinaceous fungal amylase inhibitor strongly advocates the potential use of this inhibitor

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against fungal pathogens especially in the field of agriculture, where fungi have always been one of the major plant pathogens. We conclude that C. speciosus amylase inhibitor is a valuable source of a potential fungal amylase inhibitor with immense application in plant biotechnology. ACKNOWLEDGEMENT This work was supported by a grant and facility from SRM University, Chennai, India. We thank Dr. M. Vairamani, Dean, School of Bioengineering, SRM University, Chennai, India for his timely help.

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CONFLICT OF INTEREST

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The authors declare that no conflict of interest exists.

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ACCEPTED MANUSCRIPT 22

Yield (%)

Specific activity (AIU/mg)

Purification fold

100

114

1.00

Ammonium sulphate precipitation

6364

41.35

4095

35.92

DEAE Cellulose column

5953

38.68

47.03

Sephacryl S-200 column

7.52

0.05

27250

239.04

5361

AC

CE

PT

ED

M

AN

US

Crude extract

CR

Purification step

T

Total Protein (mg) 15390

IP

Table 1: Summary of purification of α-amylase inhibitor from aqueous extract of C. speciosus

ACCEPTED MANUSCRIPT 23

Table 2: Activity of purified α-amylase inhibitor proteins from C. speciosus on various α amylases Source of α-amylase

Inhibition %*

1

Human salivary

28.2 ± 0.3

2

Porcine pancreatic

87.5 ± 0.9

3

Maize

30.1 ± 0.5

4

Bacillus subtilis

5

Aspergillus oryzae

AC

CE

PT

ED

M

IP

51.0 ± 1.6

CR

US

AN

*All the experiments were conducted three times

T

S. No.

96.1 ± 0.8

ACCEPTED MANUSCRIPT 24

Table 3: Peptide mass fingerprint of α-amylase inhibitor from C. speciosus obtained using protein prospector Observed mass Calculated mass

Position

Peptide Sequence

717.2086

29-34

FQLPGR

767.3790

766.3717

1-7

MIMASSK

820.5151

819.4126

127-133

LHLKPGR

861.4213

861.2872

120-126

LGSYEHR

899.5560

899.3613

93-101

LVGDLALAK

913.4625

912.4552

19-26

ITYNSSTK

1094.5339

1094.5266

39-46

1165.6364

1164.6291

49-58

1320.6641

1319.6569

61-72

1322.6950

1321.6878

75-87

1586.7696

1585.7624

104-117

1678.8534

1678.5114

136-149

SLQDIFQEIESELK

1986.8940

1985.8868

153-169

SVPWDVHDYDGQNAEVR

2090.9447

2089.9375

172-190

DSTTGNVASFDTNFTMNIR

2211.0638

2210.0566

193-213

GAFISNFSMTVDGTTFTSSIK

3120.6979

3119.6906

CR

IP

T

717.4042

WEKPFEMK

ED

M

AN

US

AFYSAPIQIR

216-244

SNNVSTTVELEK VFSVSLSNPSTGK

GDTVTVEFDTFLSR

LLSLALFLALLSHANSATETSFIIDAFNK

AC

CE

PT

[Theoretical pI: 9.68 / Mw (average mass): 27724.69 / Mw (monoisotopic mass): 27707.34]

ACCEPTED MANUSCRIPT 25

Table 4: Alignment of α-amylase inhibitor from C. speciosus against other plant proteins Protein names

Organism

Length

Maximum Score

Total Score

Query cover (%)

Expected value

Sequence coverage (%)

AAB42070.1

Alpha-amylase inhibitor-4

Phaseolus vulgaris

244

87.8

200

75

2.00E-23

43

1VIW - B

Chain B, Tenebrio Molitor Alpha-

Phaseolus vulgaris

205

86.7

200

72

3.00E-23

43

amylase-inhibitor Complex Chain B, Structure of Alpha-Amylase

Porcine Pancreatic

223

86.7

Alpha-amylase inhibitor precursor

Phaseolus costaricensis

244

201

4.00E-23

43

201

4.00E-23

43

74

87

198

74

5.00E-23

43

83.6

200

73

1.00E-21

42

65

70.9

70.9

22

2.00E-14

74

65

69.3

69.3

22

7.00E-14

72

alpha amylase inhibitor-1 precursor

Phaseolus vulgaris

244

Alpha-amylase inhibitor-1

Phaseolus vulgaris

244

AAG34483.1

Alpha-amylase inhibitor, partial

Phaseolus costaricensis

AAG34471.1

Alpha-amylase inhibitor, partial

Phaseolus coccineus

US

CR

AAT35809.1 CAD28835.1

subsp. Polyanthus

72

87

IP

1DHK - B CAH60260.1

T

Entry name

Q41114.1

Full=Alpha-amylase inhibitor -2

Phaseolus vulgaris

240

69.3

113

47

3.00E-12

44

AAB50853.1

Alpha-amylase inhibitor alpha subunit,

Phaseolus vulgaris

76

46.6

46.6

10

4.00E-05

91

P16300.1

Full=Lectin/Full=LBL; Flags: Precursor

Phaseolus lunatus (lima

262

45.8

80.1

49

6.00E-04

30

BAA86927.1

Alpha-amylase inhibitor like protein

Phaseolus vulgaris

262

45.4

80.9

20

0.001

92

ABJ16470.1

Arcelin

Lablab purpureus

216

40.8

40.8

51

0.025

29

S70468

Agglutinin (WBA I) - winged bean

Psophocarpus

238

34.7

34.7

12

3.7

58

AAA82181.1

Phytohemagglutinin

276

33.9

33.9

18

6.2

50

AN

PHA-I alpha subunit

ED

tetragonolobus

AC

CE

PT

(fragment)

M

bean)

Phaseolus acutifolius

ACCEPTED MANUSCRIPT 26

Table 5: Homology modelling validation results using RMSD, Ramachandran plot, ProSA and QMEAN Ramachandran Plot Number of Number of Number of residues in residues in residues in favoured allowed outlier region region region

01

17.444

92.0

6.0

2.0

02

17.257

88.4

8.5

3.0

03

16.336

91.5

6.0

04

17.122

86.4

9.5

05

17.514

88.9

7.0

06

16.750

88.9

07

17.877

83.4

08

18.249

09

16.413

10

15.778

11

15.976

12

15.308

ProSA Z score

QMEAN score

-8.10

0.18

-8.17

0.73

-8.76

4.0

1.54

-9.91

4.0

0.59

-8.39

3.5

0.43

-9.12

9.5

7.0

0.39

-9.99

84.4

13.1

2.5

0.56

-7.99

88.4

8.5

3.0

1.27

-10.16

85.9

10.1

4.0

-1.81

-8.24

90.5

6.0

3.5

-1.78

-7.14

89.9

8.5

1.5

-1.81

-7.07

16.359

88.9

7.0

4.0

-1.66

-8.21

CR

AN

US

2.5

M

14

AC

CE

7.5

IP

0.71

PT

T

RMSD

ED

Model

16.167

92.0

5.0

3.0

-1.27

-8.41

15

16.483

93.5

5.0

1.5

-1.94

-6.50

16

16.034

88.4

9.0

2.5

-1.71

-6.87

17

15.607

91.0

7.0

2.0

-1.97

-7.44

18

16.001

87.9

9.5

2.5

-2.21

-7.16

13

ACCEPTED MANUSCRIPT 27

19

2.494

87.9

10.1

2.0

-2.41

-6.30

89.9

7.5

2.5

-2.97

-6.65

90.5

7.0

2.5

93.0

5.0

87.4

8.0

16.365 20

2.664

2.596

-7.32

-2.62

-5.78

4.5

-2.59

-7.43

3.5

-2.74

-6.84

7.0

3.5

-2.64

-6.82

88.4

9.5

2.0

-2.65

-6.82

92.5

4.5

3.0

-2.96

-6.60

23

2.445

AN

16.315

16.297 2.148

91.5

CE

2.739

89.4

PT

3.216 16.110

26

ED

16.495 25

5.0

M

24

2.0

US

2.412

CR

16.362 22

-2.56

IP

21

T

16.328

16.250 2.764

AC

27

16.360

28

NA

61.3

27.6

11.1

-1.68

-14.45

29

NA

58.3

29.1

12.6

-1.58

-14.33

30

NA

57.8

30.7

11.5

-2.89

-14.18

31

NA

60.8

27.6

11.6

-1.38

-15.21

ACCEPTED MANUSCRIPT

NA

57.3

20.1

22.6

-1.33

-18.72

33

NA

86.4

8.5

5.0

-1.07

-9.01

34

NA

86.4

8.5

5.0

-1.07

-9.01

35

NA

79.9

12.6

7.5

1.87

-10.39

36

NA

79.9

12.6

7.5

1.87

-10.39

37

NA

72.9

16.1

11.1

1.63

-16.48

AC

CE

PT

ED

M

AN

US

CR

IP

32

T

28

ACCEPTED MANUSCRIPT 29

Table 6: Weighted scores for the protein-protein complex clusters developed using Cluspro

1

Center

-891.7

Lowest Energy

-948.8

Center

-906.1

Lowest Energy

-973.8

Center

-868.2

Lowest Energy

-1123.4

99

2

92

3

78

Center

48

PT

35

CE

28

28

10

26

9

AC

Lowest Energy

-979.9

Center

Lowest Energy

-832.7

-733.1

Center

-725.4

Lowest Energy

-932.3

ED

35

7

8

-979.9

36

6

11

12

25

24

-849.4

Center

M

5

-849.4

AN

4

US

Lowest Energy

T

112

Weighted Score

IP

0

Members Representative

CR

Cluster

Center

-823.8

Lowest Energy

-953.1

Center

-864.4

Lowest Energy

-941.4

Center

-721.6

Lowest Energy

-837.3

Center

-713.6

Lowest Energy

-978.1

Center

-719.0

Lowest Energy

-821.5

Center

-969.0

Lowest Energy

-969.0

ACCEPTED MANUSCRIPT 30

15

16

Center

-743.1

Lowest Energy

-749.7

Center

-826.9

Lowest Energy

-826.9

Center

-888.1

Lowest Energy

-906.9

Center

-752.2

22

21

20

Lowest Energy

20

CE

22

AC

23

24

25

26

US

-772.2

Center

-755.0

Lowest Energy Center

-771.5

-784.3

Lowest Energy

-771.5

Center

-789.5

Lowest Energy

-789.5

Center

-715.6

Lowest Energy

-777.0

Center

-755.9

Lowest Energy

-755.9

Center

-741.6

Lowest Energy

-741.6

Center

-710.6

Lowest Energy

-780.0

Center

-726.7

Lowest Energy

-744.9

Center

-721.9

Lowest Energy

-744.2

15

13

PT

21

13

9

8

8

7

-762.6

Lowest Energy 19

14

-919.3

AN

19

Center

M

18

20

ED

17

T

14

23

Weighted Score

IP

13

Members Representative

CR

Cluster

ACCEPTED MANUSCRIPT 31

Cluster

6

Weighted Score

Center

-760.7

Lowest Energy

-760.7

AC

CE

PT

ED

M

AN

US

CR

IP

T

27

Members Representative

ACCEPTED MANUSCRIPT 32

Table 7: Residues forming H bond interaction in the protein-protein complex In protein (alpha amylase) THR111

In ligand (inhibitor) HIS122

2

ALA57

ASN53

2.86

3

PHE54

ASN53

2.76

4

LYS52

GLU349

5

LYS52

GLU349

6

ARG27

GLU349

2.92

7

SER163

ARG20

2.93

8

SER163

9

TYR151

10

ALA307

11

GLY306

12

HIS305

13

ALA310

14

ASN350

15

GLU349

18 19

IP

CR

US

2.65

ARG20

2.73

GLY19

2.71

GLY19

2.76

PRO18

3.04

HIS15

3.17

SER11

2.92

ARG6

2.51

ASN350

ARG6

2.70

ASN350

ARG6

2.78

GLY351

ARG6

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Figure Captions Fig. 1: Chromatogram obtained after purification of fungal amylase inhibitor using (a) Ion exchange chromatography using DEAE Cellulose column. The proteins were eluted at a flow rate of 1ml min-1; (b) Gel filtration chromatography using Sephacryl S-200 column. The proteins were eluted at 1ml min-1

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Fig. 2: Spectrophotometric analysis of α-amylase inhibitory activity of bound fractions obtained from ion exchange chromatography

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Fig. 3: (a) Analysis of C. speciosus purified fraction containing the active proteinaceous amylase inhibitor in a 14 % w/v Tricine SDS PAGE, bands were developed by silver staining, Lane 1 contains the standard low molecular weight marker for reference and Lane 2 contains 10 µg of the purified fraction containing the active inhibitor; (b) Reverse zymography: the purified inhibitor was run on 12% (w/v) polyacrylamide gel co-polymerized with 0.2% (w/v) starch. Lane 1 contains amylase alone as negative control whereas lane 2 contains equal amount of amylase along with the purified inhibitor.

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Fig. 4: Activity profile of the amylase inhibitor at different temperatures: Amylase inhibitory assay was conducted in with starch (0.5%w/v) in 10 mM phosphate buffer pH 6

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Fig. 5: Activity profile of the amylase inhibitor at different pH: Amylase inhibitory assay was conducted in with starch prepared in Citrate Phosphate buffers of pH 4 -9 at 37 ºC

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Fig. 6: LCMS spectra of (a) Intact mass of purified protein of C. speciosus; (b) MALDI-TOFMS of purified protein with inhibitor activity after trypsin digestion

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Fig. 7: Sequence alignment between the query and the templates. Asterisk indicates identical amino acid residues. Period (.) denotes conservation between groups of weakly similar properties. Colon (:) means conservation between groups of strongly similar properties. Dash (-) indicates an unknown amino acid residue.

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Fig. 8: Selected model (Model 22) after model validation aligned with the template 1DHK. Model 22 is represented in red color and the template is represented in green Fig. 9: (a) Ramachandran plot of the selected model (Model 22) is represented. (b) Z plot obtained from the ProSA server, which contains the z-scores of all experimentally determined protein chains in the selected model Fig. 10: Protein-protein docked complex. Cyan colour represents the receptor (human alpha amylase) and green colour represents the ligand (alpha amylase inhibitor) Fig. 11: (a) Residues forming H-bond interaction in the protein-protein complex (b and c) Ligplot representing the residues forming H bond and hydrophobic

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