Preparation of esterified derivatives of α-lactalbumin and investigation of their antibacterial activity
Negar Ghorbani1, Abdol-Khalegh Bordbar1,2, Asghar Taheri-Kafrani1, Akbar Vaseghi1
1University of Isfahan, Faculty of Advanced Sciences and Technologies, Department of Biotechnology, 81746-73441, Isfahan, Iran
2University of Isfahan, Department of Chemistry, 81746-73441, Isfahan, Iran
Corresponding author: Abdol-Khalegh Bordbar, Email: firstname.lastname@example.org, email@example.com.
Citation: Ghorbani N, Bordbar AK, Taheri-Kafrani A, Vaseghi A. Preparation of esterified derivatives of α-lactalbumin and investigation of their antibacterial activity. J Biother, 2014, 1(1): e1. doi:10.15383/jbt.1.
Funding: This work was funded by research council of Isfahan University.
Competing interests: The authors have declared that no competing interests exist.
Conflict of interest: None
Copyright: 2014 By the Editorial Department of Journal of Minimally Invasive Orthopedics. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract: α-lactalbumin (α-La) is one of the major milk proteins that in addition of nutritional value it can get many other useful novel functions such as antiviral and tumoricidal activities via structural modifications by enzyme or chemical methods. Several researches show antiviral activity of esterified milk proteins because of their new functional properties followed by structural modifications. In this research, the hypothesis based on antibacterial activity of esterified α-La was examined. The esterification reaction as an important tool for modifying food proteins was employed in this study by using different alcohols under strong acidic condition at 4 °C for 3 days. α-La was esterified to the extent of 53.8%, 34.95%, 32.2% , 6.07% and 46.8% with methanol, ethanol, 1-butanol, 2-pentanol and 1-hexanol, respectively, showed the influence of molar ratio, type of used alcohol and time of reaction on the reaction yield. Then the effects of esterified derivatives of α-La were tested on the growth of some positive and negative gram bacteria (including Bacillus cereus, methicylin resistant Staphylococcus aureus (MRSA), Escherichia coli, and Klebsiella) by disc assay. After 24-48 h incubation, no inhibition zone surrounding the discs was observed which represents the fail of any antibacterial activity of α-La and its esterified derivatives.
Keywords: α-lactalbumin; protein esterification; antibacterial activity; milk protein
α-lactalbumin is a small (14.2 KDa containing 123 amino acids) acidic (pI 4-5) calcium binding milk protein found in almost all milk of mammalians (Permyakov, 2005) and its main known biological role is regulation of lactose biosynthesis as a component of lactose synthase system (Neville, 2009).
There are several reports based on novel functional properties of α-La resulted from modification of its structure. For example partial unfolded α-La and oleic acid combine to form a stable tumoricidal complex was first named HAMLET (Human α-lactalbumin Made Lethal to Tumor cells) (Hakansson et al., 1995). Due to the HAMLET specificity in induce apoptosis in cancerous cells without side effects on healthy, differentiated cells, it is suggested in many experiments as a therapeutic agent (Fischer et al., 2004, Gustafsson et al., 2004, Mossberg et al., 2007, Puthia et al., 2013).
In other study it has been shown limited hydrolysis of α-La by a specific bacterial protease produces the self-assembly of the peptides in nanotubular structures (Otte et al., 2005; Graveland-Bikker and De Kruit, 2006). Pellegrini et al. (1998) identified antibacterial peptides containing disulfide bounds, from α-La digested by trypsin and chymotrypsin. Induction of antiviral activity in milk proteins such as α-La was performed through chemical modification of their functional groups. For example α-La could inhibit HIV replication after acylation of the amino function of the lysine residues (Neurath et al., 1995; Swart et al., 1996; Berkhout et al., 1997).
The esterification reaction was applied as an important tool for modifying food proteins such as α-La. Esterification with different alcohols leads to the blocking of free carboxyl groups in protein, thus enhancing the net positive charge, making the modified protein more basic (Halpin and Richardson, 1985; Bertrand-harb et al., 1991; Chobert, 2003). In several research, esterified milk proteins including methylated and ethylated α-La displayed antiviral activity against a broad range of viruses because of the electrostatic and or hydrophobic interactions between modified protein and vital components (nucleic acid and proteins involving viral replication) in that viruses (Sitohy et al., 2001a,c; Sitohy et al., 2002; Sitohy et al., 2005; Chobert et al., 2007; Sitohy et al., 2007; Taha et al., 2010).
It has been found esterified legume proteins were capable to reduce bacterial growth via interaction with some sectors of bacterial membranes characterized by the presence of negatively charged structural constituents, e.g. phospholipids, leading to their disruption and therefore affecting the bacterial growth (Sitohy and Osman 2010).
Therefore, it is expected esterified milk proteins show antibacterial activity because of the increased net positive charge and hydrophobicity arising esterification. Due to the absence of any reports corresponding to the investigation of antibacterial activity of esterified milk proteins and because of the increasing requirement to discover new and safe antibiotics because of raising the number of antibiotic-resistant bacteria, we decided to produce various esterified derivatives of α-La and investigate their likely antibacterial activity in the present study.
2. Materials and Methods
All of the chemical reagents, alcohols (methanol, ethanol, 1-butanol, 2-pentanol, 1-hexanol), Hydrochloric acid and culture media (nutrient agar and nutrient broth) were obtained from Merck Chemical Co. Microorganisms including Bacillus cereus, methicylin resistant Staphylococcus aureus (MRSA), Escherichia coli, and Klebsiella were obtained from Microbial Biotechnology lab, University of Isfahan, Isfahan, Iran.
2.2.1 Protein preparation
Isolation of α-La from whole raw caw’s milk was performed based on salting out method with 26.4% ammonium sulfate (Armstrong et al., 1967). Protein preparation was assessed by Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Reverse Phase High Performance Liquid Chromatography (RP-HPLC).
SDS-PAGE was performed based on the method of Laemmli (1970) in 3% acrylamide stacking gel containing 0.5 M Tris-HCl, pH 6.8 and 12% acrylamide resolving gel containing 2.0 M Tris-HCl, pH 8.8. The migration was conducted at 10mA for the stacking gel and 20mA for the resolving gel.
RP-HPLC carried out according to the method of Kong et al., (2012) with C18 column (250 mm×4.6 mm×5 μm) that was purchased from MACHEREY-NAGEL GmbH & Co. KG.
2.2.4 Protein esterification
The reported methods by Sitohy et al. (2000, 2001b) was carried out for esterification of α-La using different alcoholos as follows: native α-La was dispersed (3%, v/w) in cold alcohols (methanol ≥ 99%, ethanol ≥ 99%, 1-butanol 96%, 2-pentanol 96% and 1-hexanol 96%). Amounts of stock hydrochloric acid equivalent to 70 molar ratio (mole H+/ mole COOH groups) were added drop-wise at the begining of reaction. All the reaction mixtures were kept at 4 °C under continuous stirring. After 72 h the samples were centrifuged at 10,000 g for 10 min. The resulting supernatant was discarded and the remained sediment was dispersed in a volume of alcohol equal to that of the discarded supernatant and mixed well before re-centrifuge under the same conditions. The washing step was repeated 3 times. The final sediment was dissolved in a proper amount of distilled water and kept at -80 °C until lyophilization. Then the lyophilized samples were kept at -20 °C until later analysis.
2.2.5 Extent of esterification
The extent of α-La esterification was determined by the color reaction using hydroxylamine hydrochloride as described, previously (Bertrand-Hurb et al., 1991).
2.2.6 Antibacterial activity of esterified derivatives of α-La
Traditional disc assay was performed to test antibacterial activity as follows (Sitohy and Osman, 2010): sterile paper discs (5 mm in diameter) were dipped into different protein concentrations (0.1 mg/mL, 1.0 mg/mL and 10.0 mg/mL) and placed on the surface of nutrient agar media inoculated by the tested bacteria suspensions that were already prepared in McFarland 0.5 standard . Then the sample plates were incubated at 37 °C for 24-48 h.
3. Results and Discussion
3.1 Protein purification
According to the results that are shown in figure 1, α-La was purified to 80-85%.
Figure 1. SDS-PAGE patterns (A) of α-La standard (1) and isolated α-La from raw cow’s milk by salting out (2). Chromatogram detection of α-La standard (B) and isolated α-La from raw cow’s milk by salting out (C) obtained by RP-HPLC.
3.2 Extent of esterification
The esterification yields of α-La (3%, w/v) based on the methods of Sitohy et al. (2000, 2001b) with different alcohols under strong acidic conditions and 4ºC after 72 h are presented in table 1. The observed results for low molecular weight alcohols are similar to previous report (Sitohy et al., 2001b).
Table 1. The extent of esterification of α-La (3%, w/v) with different alcohols under acidic conditions (molar ratio = 70), at 4ºC, after 72 h
Types of alcohols
Methanol (99% ≤)
Ethanol (99% ≤)
Degree of esterification (%)
The interested results for esterification with high molecular weight alcohols, could describe due to the high molar ratio and prolonged reaction time which promised conformational changes of protein under acidic condition to interact with hydrophobic alcohols efficiently.In this study differences in the yield of esterification were influenced by the protein structure and nature of used alcohols such as polarity and structural hindrance which is especially evidenced in 2-pentanol as they have been mentioned in previous studies (Sitohy et al., 2000, 2001; Chobert, 2003).
3.3 Antibacterial activity
The inhibition effect of esterified α-La on the growth of E.coli (A) and Klebsiella (B) as negative gram bacteria was shown in Fig. 2 and no clear zone of inhibition is observed surrounding each one of paper discs . The similar result was obtained for positive gram bacteria (the results have not been shown). According to obtained results from previous researches which reported antimicrobial activity of hydrophilic and cationic proteins and peptides (Sitohy et al., 2010; M. Yin et al., 2012) it would has been expected that esterified α-La displayed antibacterial activity against tested bacteria. However, the lack of any inhibition zone rejected this hypothesis.This result may be related to the structural properties of bacteria and their inhibition mechanism which caused resistance to esterified derivatives of α-La. Moreover, the randomized protein structures that resulted from esterification may be also reduced the antibacterial activity (Matarella et al., 1983). Both of these mentioned mechanisms could be encountered in our observations.
Figure 2. The ineffectual esterified derivatives of α-La on the growth of E.coli (A) and Klebsiella (B) as negative gram bacteria.
Legands: Native α-La (1,4) in all samples was as a control. 2: methylated α-La, 3: ethylated α-La, 5: Butylated α-La, 6: pentylated α-La and 7: hexylated α-La.
The yield of α-La esterification depends on the molar ratio, type of used alcohol and time of reaction. However, the esterification of α-La with high molecular weight alcohols has been carried out in this study for the first time. The lack of any antibacterial activity for these prepared esterified α-La, rejected the using of these modified α-La as antibacterial agents. However the nature of protein and type of randomized protein structure arising from esterification, play an important role in the induction of new functions such as antimicrobial activity that can be taken as the objective of future studies.
The financial support of research council of Isfahan University is gratefully acknowledged.
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