Identification and Characterization of Bovine (Bos taurus) Pancreatic Bile Salt Activated Lipase for Potential Halal Alternative.

doi:10.21203/rs.3.rs-4945296/v1

Download PDF

Research Article

Identification and Characterization of Bovine (Bos taurus) Pancreatic Bile Salt Activated Lipase for Potential Halal Alternative.

https://doi.org/10.21203/rs.3.rs-4945296/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Introduction: Bile salt-activated lipase (BSAL) is one of the pancreatic lipases that plays a critical role in the digestion and absorption of dietary fats.

Objective: This study aimed to purify bile salt-activated lipase (BSAL) from bovine (Bos taurus) pancreatic tissue.

Methods: Bovine pancreas was freshly collected from Abattoir Complex, Department of Veterinary Services, Shah Alam, Malaysia. The fats were removed by trimming grossly visible fat, and extraction was performed using organic solvents. The BSAL was further purified by anion exchange chromatography and sent for protein identification by liquid chromatography-mass spectrometry (LC-MS/MS).

Results: Successful purification of bovine BSAL was visualized as a single protein band on sodium dodecyl sulfate-polyacrylamide (SDS) gel, which LC-MS/MS confirmed as a bovine BSAL (Accession number – P30122) with a molecular mass of 65.12 kDa and calc pI of 5.57. Peptide identification based on the MS spectrum found 200 predictive peptides, of which ten sequences with bovine BSAL peptide characteristics. The selected predictive peptide sequences have a molecular mass of 1104.60 - 3378.94 Da with Qvality q-value greater than 0.01 and XCorr Sequest HT value ranging from 2.6 to 6.8. The specific lipolytic enzyme activity of bovine BSAL was comparable with the positive control, as measured using lipase assay.

Conclusion and Recommendations: In conclusion, the results of this study indicate the effectiveness of bovine BSAL purification by anion exchange chromatography from fresh pancreatic tissue and have the potential for further Halal pharmaceuticals and medical applications.

Pancreas

Pancreatic enzyme

Lipase

Bile Salt-Activated Lipase (BSAL)

Bile Salt-Stimulated Lipase (BSSL)

Purification

and Halal enzyme.

Enzymes are one example of a biological catalyst. Three major classes of digestive enzymes are protease, amylase, and lipase (1). Lipases are the third most utilized among available enzymes, followed by proteases and amylases (2). Essential enzymes, including chymotrypsin, trypsin, amylases, and lipases, are produced in the pancreas to facilitate food digestion (3, 4). Lipases are responsible for catalyzing the hydrolysis of triacylglycerols to glycerol and fatty acids (1). Mainly, lipases are essential for lipid digestion to provide the highest dietary source of calories (3). Lipase has become of remarkable interest to many researchers as most nutritional problems are secondary to the improper digestion of dietary fats (5). Two major lipase clusters working synergistically are gastric and pancreatic lipases. Several lipolytic enzymes are synthesized and secreted by the exocrine pancreas, which works with gastric lipase to achieve complete dietary fat digestion and absorption (6). Approximately 10–30% of lipolytic activities are contributed by the gastric lipase, which participates in the early stage of lipolysis to release fatty acids in the stomach alongside bile salt-dependent lipase and pancreatic colipase-dependent lipases (3).

Bile salt-activated lipase (BSAL, EC 3.1.1.13) is one of the pancreatic lipases that plays a critical role in the digestion and absorption of dietary fats. It is a lipase secreted from the vertebrate exocrine pancreas into the intestine and is essential for lipid digestion (7). The presence of bile salt is substantial for the hydrolysis of water-insoluble substrates (8). It is a bio-surfactant present in the gastrointestinal tract that has a significant role in the digestion and absorption of nutrients (9). Bile salt will facilitate solubilization and help to transport fat-soluble nutrients to the mucosa of the small intestine (9). Since there is a discrete interaction between the enzyme and bile salt for enzyme activation, it is commonly known as bile salt-activated lipase (10, 11). Alternatively, BSAL is also known as bile salt-stimulated lipase, carboxyl ester lipase, cholesterol esterase, pancreatic lysophospholipase, and bile salt-dependent lipase (7, 10, 12–14).

BSAL can be extracted from various sources, including microorganisms, humans, and animals. Mainly, BSAL has been isolated from pancreatic tissues. However, BSAL is also expressed by lactating mammary glands and secreted in milk in some species, including humans (6, 7, 14–17). BSAL was also reported to have considerable lipase activity in human milk, significantly contributing to milk lipid utilization in newborns (18). Pancreatic BSAL purification has been studied from several sources, including humans (19–21), porcine (22–24), bovine (25), rats (26, 27) and fish (28). The isolation of BSAL from the pancreatic tissue is more complicated due to proteolytic digestion during the purification process (10). Thus, it is more convenient to isolate BSAL from milk as no proteolysis will occur. High BSAL content in breast milk has significantly increased the fat digestion capacity of newborns during the first few months after birth due to immature pancreatic development and limited pancreatic lipase secretion (29, 30).

Although the study on BSAL purification has previously received considerable attention, there has been no recent update on the methods related to the extraction and purification of BSAL as well as the peptides sequence analysis by LC-MS/MS from bovine pancreas. Infact, the research interest in BSAL has switched from purification to its application to induce smooth muscle cell proliferation (31), as well as in vitro angiogenic effects suggesting potential implications in vascular pathophysiology (32).

The Bovidae family, including domestic cattle, is widely distributed in Malaysia for the livestock agriculture sector. Beef is Malaysia's most popular red meat source (33). Nevertheless, higher meat production contributes to large amounts of waste as some animal organs are not fully utilized and are finally disposed of as waste. This includes the pancreas, as it is a non-edible organ produced in significant quantities by the meat industry and represents waste disposal and potential pollution (34). Thus, the bovine pancreas, a by-product of the meat industry, has the potential to be utilized as an alternative source of Halal digestive enzymes, particularly for lipase applications. In addition, the premier quality and Halal-certified products, including medicine and health supplements, are gaining much attention from both Muslim and non-Muslim consumers (35). Furthermore, Halal pharmaceutical products are considered cleaner and healthier (36), with a projected market value of $3.2 trillion by 2024 (37).

BSAL has a comprehensive specificity and hydrolyzes a variety of different substrates (15, 38), including major lipids; dietary fat, and triacylglycerol, as well as minor lipids; fat-soluble vitamin esters, and cholesterol esters digestion (19, 39). A sound lipolytic system is needed for BSAL, along with other pancreatic lipolytic enzymes and gastric lipase, to act in concert for complete dietary lipid digestion (40). Thus, due to the concerted action of BSAL and other pancreatic lipolytic enzymes, the impairment of BSAL activity may disrupt complete intestinal dietary fat digestion processes (12, 39). Drastic reduction of BSAL leads to the development of many diseases, such as pancreatic cancer, necrotizing pancreatitis, and chronic pancreatitis, affecting the gastrointestinal tract in the digestive system (12).

Bile salt is also crucial for activating the BSAL mechanism and contributes to the pharmaceutical industry, particularly in addressing low solubility and transport for drug delivery (9, 41). In this study, the BSAL was successfully isolated and purified from fresh bovine pancreatic tissue, subsequently confirmed by ten predictive peptide sequences with bovine BSAL characteristics identified through LC-MS/MS and followed by determination of BSAL lipolytic activity. In the pharmaceutical industry, lipases have been utilized as modulators (activators and inhibitors) to treat lifestyle diseases such as obesity (5) and show great promise in therapies (42). Microbial lipase from bacteria, fungi, and yeast is the current primary source and is pivotal in various industries, including pharmaceuticals (2, 43). Thus, the purification of BSAL is significant. Therefore, a successful purification of BSAL from bovine pancreas can be suggested as an alternative substitute for porcine enzymes for Halal medical and pharmaceutical demands.

Extraction and Purification of Bile Salt Activated Lipase. The bovine pancreas was firstly washed with tap water and then dissected free from adherent fat and connective tissues with a scalpel blade (44). The pancreas was cut into smaller pieces on ice, and the tissue slices were washed with a 50 mM Tris pH 7.4 buffer containing 0.1 M NaCl to remove blood (45). Approximately 160 g of pancreas samples were homogenized. Bovine pancreatic tissues were suspended in 20 ml of extraction buffer (50 mM Tris pH 7.4, 2 mM EDTA pH 8, 10 mM HEPES pH 7.4, 1% Triton X-100, and 150 mM NaCl) and vortexed for 10 mins before centrifugation at 4000 rpm for 2 mins. The supernatants were collected and added with 70% (v/v) ethanol, bringing a total of 25 ml. The mixture was vortexed for 10 secs, and 2 ml of

5 µg/ml of aprotinin (a protease inhibitor) was added to the samples. The samples were centrifuged at 14,000 rpm for 20 mins before collecting the supernatant. The supernatant was evaluated for absorbance at 280 nm and stored at -20^oC until further use.

Bovine pancreatic BSAL were further purified by anion exchange chromatography on a pre-packed HiTrap Q Fast Flow column (bed height 1.6 cm, column diameter 2.5 cm, column volume 5 ml) using AKTA Avant™ chromatography system (GMI – USA). A 20 mM Tris pH 8 was used as the start buffer, and 20 mM Tris 1 M NaCl pH 8 was used as the elution buffer. The sample was filtered using 0.45 µm and 0.25 µm cellulose acetate pore membrane filters (Merck – Germany). The ethanol system was initially replaced with distilled water and then washed with the start buffer. A total of 200 ml filtered sample was applied to the column in buffer A, and the column was washed until the absorbance monitored at 280 nm returned to baseline. The BSAL pancreatic enzymes were eluted using an elution buffer at a 2.4 ml/min flow rate, and 1.5 ml fractions were collected. The elution buffer composition was set up as a step gradient at 5%, 10%, 20%, 30%, 40%, 50%, and 100% sodium chloride (NaCl) to elute the targeted BSAL. Each fraction representing different peaks was run on 1-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) for molecular weight determination. Fractions containing lipolytic activity based on the molecular weight were pooled and concentrated using a 30,000 MW and 50,000 MW pore-size concentrating device (MERCK).

Bile Salt Activated Lipase Protein and Peptide Identification. Protein concentration was measured at 280 nm using the NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, US). The molecular mass of purified bovine pancreatic BSAL was estimated by 1D SDS-PAGE stained with Coomassie Brilliant Blue stain (Invitrogen, US). Protein bands were further identified using liquid chromatography-mass spectrometry (LC-MS/MS) (46). Before the LC-MS/MS procedure, trypsin gel digestion was carried out. Then, a total of 5 µl sample was injected into Dionex Ultimate 3000 RSLCnano Orbitrap Fusion (Thermo Fisher Scientific) with EASY-Spray Acclaim PepMap™ C18 100 A⁰ (2 µm particle size, 50 µm id x 15 cm) separation column. The sample was eluted with H₂O (LC-MS grade), 0.1% formic acid (A), and ACN, 0.1% formic acid (B), at a flow rate of 250 nL/min. The elution gradient was 5%-40% B for 91 min, 2 min to 85% B, 3 min at 85% B, 1 min at 5% B, and 4 min at 5% B. The full scan spectra were collected by orbitrap MS (OTMS) scan mass ranging from 310–1800 m/z with 50 ms maximum injection. The spectra were analyzed by ion trap MS (ITMS) with 250 ms maximum injection. The LC-MS/MS data was analyzed using Thermo Scientific™ Proteome Discoverer™ Software Version 2.1. The results were matched with the database downloaded from UniProt. The variable modifications were oxidation (M) and acetyl (N-terminal) with fixed modification of carbamidomethyl (C). All peptides were validated using the percolator® algorithm, based on a q-value of less than 1% false discovery rate (FDR). The LC-MS/MS analysis was performed at the Proteomic Laboratory, Malaysian Genome and Vaccine Institute, Bangi, Malaysia.

Lipolytic Enzyme Assay. Lipase activity was evaluated through the conversion of 4-nitrophenyl Palmitate (pNPP) into p-nitrophenol (pNP) formation (47). The lipolytic activity of crude extracts and purified fractions from anion exchange chromatography were determined using the modified method from previous studies (47, 48) alongside porcine pancreatic lipase (Sigma Aldrich, Germany) as a positive control. The pNPP calibration standard curve was prepared using serial dilution. The substrates were prepared by mixing pNPP with acetonitrile (ACN). The standard assay contained 10 µl of lipase sample, 150 µl of substrates, and 40 µl of 25mM sodium phosphate buffer pH 7. The 25mM sodium phosphate buffer pH 7 and ACN mixture was used as blank. The lipolytic assay was performed in a 96-well microplate with 200 µl reaction volume incubated at 25°C. The experiments were repeated three times. One unit of lipase is defined as the amount of enzyme that will generate 1.0 µmole of pNP from the pNPP reaction per minute at 25°C as measured at 405 nm using a SpectraMax iD5 Multi-mode Microplate reader (Molecular Devices, USA). The readings were taken periodically at 1, 5, 10, 15, 30, 45, 60, 90, 120 and 150 minutes.

Bovine pancreatic BSAL was successfully purified from the fresh pancreatic tissue by pancreatic tissue delipidation, protein extraction, and anion exchange chromatography. Figure 1a illustrates the elution pattern of anion exchange chromatography on the prepacked HiTrap Q Fast Flow column, developed by sodium chloride (NaCl) step gradient. A total of 17 fractions (25.5 ml) containing bovine BSAL were eluted at 10% NaCl concentration and labeled as peak C. This fraction was observed as a single protein band estimated at 60kDa (Fig. 1b, Lane 3) with confirmed lipase activity. Next, the band was excised and destained for LC-MS/MS protein identification, which confirmed it as a bovine pancreatic BSAL with a molecular mass of 65.12kDa and calc pI 5.57.

Figure 1. a) Anion-exchange chromatography of bovine pancreatic aqueous crude extract.

The chromatogram represents the absorbance of crude extract at 280nm. The arrows marked peaks that showed the presence of bovine BSAL on the 1D SDS Page with lipolytic activity. Tubes were pooled at 10% NaCl concentration, indicating the presence of active BSAL. Column: Hi Trap Q Fast Flow column (pre-packed); flow rate: 2.4ml/min; mobile phase: 20mM Tris pH8, 20mM Tris NaCl pH8.

Figure 1b) 1D SDS-PAGE (Bis-Tris 12%) of pure BSAL stained with Coomassie blue.

Well 1: BenchmarkTM Unstained Protein Ladder; Well 2: Crude extract; Well 3: Purified BSAL obtained after Hi Trap Q Fast Flow chromatography.

For the protein identification by LC-MS/MS, the predictive peptide sequences related to bovine BSAL were identified and validated using the percolator® algorithm, based on a q-value of less than 1% false discovery rate (FDR). The identification results showed that 199 predictive peptides were obtained (data not shown). Ten predictive peptides showing similar characteristics to the same master protein of bovine pancreatic BSAL and having low FDR with Qvality q-value (> 0.01), XCorr Sequest HT (> 2.0), and percolator q-value Sequest HT value (> 0.01) (Table 1). Most of the predictive peptides have low molecular weight ranging from 1104.60–3378.94 Da and net charge of 2 to 3. Six of ten predictive peptide sequences undergo modifications during the sample handling process, with frequent variable deamination and oxidation modifications and less frequent fixed carbamidomethyl modifications.

Table 1

Protein identification and peptide sequence prediction of Bovine Pancreatic BSAL *via* LC-MS/MS
No	Annotated Sequence	Confidence	Modifications	Charge	Position in Master Protein	Theo. MH+ [Da]	Qvality q-value	XCorr Sequest HT	Percolator q-Value Sequest HT
1	[R].TGDPNTGHSTVPANWDPYTLEDDNYLEINK.[Q]	High	1x Deaminated [N] 2x Deaminated [N14; N24]	2	492–521	3378.49	0	6.54	0
2	[R].VGPLGFLSTGDSNLPGNYGLWDQHMAIAWVK.[R]	High	1xOxidation [M25] 1xDeamidated [N/Q] 2xDeamidated [N17; Q/N]	3	163–193	3361.62	0	6.64	0
3	[R].AISQSGVGLCPWAIQQDPLFWAK.[R]	High	1xDeamidated [Q] 1xCarbamidomethyl [C10]	3	234–256	2572.30	0	5.76	0
4	[R].CLQATLTQDSTYGNEDCLYLNIWVPQGR.[K]	High	1xDeamidated [Q/N] 2xCarbamidomethyl [C1; C17]	3	82–109	3315.54	0	3.56	0
5	[K].TMVDLETDILFLIPTK.[I]	High	1xOxidation [M2]	2	401–416	1864.99	0	4.25	0
6	[R].GNVIVVTFNYR.[V]	High	-	2	152–162	1281.70	0	3.75	0
7	[K].LSLFGDSIDIFK.[G]	High	-	2	38–49	1354.73	0	2.64	0
8	[K].LPLGSTEYPK.[L]	High	-	2	290–299	1104.60	0	2.73	0
9	[K].SANTYTYLFSQPSR.[M]	High	-	2	428–441	1634.78	0	4.62	0
10	[K].QDVTEEDFYK.[L]	High	-	2	355–364	1273.56	0	2.64	0
Description of Bovine Pancreatic BSAL peptide sequence prediction. The bold symbol represents the possible site of fixed and variable modifications. The confidence level for the identified protein was determined by the false discovery rate (FDR) and interpreted as high-confidence hits, which were 99% accurate. The charge is the sum of positive (basic) and negative (acidic) charge residues in neutral pH. Position in Master Protein is the location of the annotated predictive peptide sequence in the protein. Theo. MH+ [Da] is the protonated monoisotopic mass of the peptides in Daltons. Qvality q-value and Percolator q-Value Sequest HT are calculated from the number of target and decoy proteins, and the minimum FDR is required for a hit to be considered correct (Qvality q-values and Percolator q-Value Sequest HT > 0.01 are considered high-confidence hits). XCorr Sequest HT indicates the number of fragment ions that are common to two different peptides with the same precursor mass and calculates the cross-correlation (XCorr) for all candidate peptides in search (XCorr value > 2 is considered favourable).

Figure 2. Standard curve of 4-nitrophenyl Palmitate as the substrate for the lipolytic enzyme assay.

Absorbances at 405 nm were recorded at 25°C. Each point represents the mean value of duplicates.

Table 2

Sequential Purification of Bovine Pancreatic BSAL
Purification stage	Volume (ml)	A₂₈₀	Protein content (mg)	Specific lipolytic activity (U/mg)	Total lipolytic activity (U)	Yield (%)
Aqueous crude pancreatic extract	200.0	57.30	11460.00	0.207	2372.22	100.00
Anion-exchange chromatography (Peak C – 10% NaCl)	25.5	6.83	174.16	0.222	38.66	1.63
Note: Specific lipolytic activity of porcine pancreatic lipase (1 mg/mL) = 0.223 U/mg

The lipolytic activity was measured using the described lipase assay for all the crude and anion exchange chromatography samples. The pNPP standard curve was constructed to determine the lipase activity of bovine BSAL at different sequential purification stages, with the R² value of 0.99 (Fig. 2). Table 2 summarizes the purification of bovine BSAL. The protein concentration of purified bovine pancreatic BSAL, as calculated from A₂₈₀, was 6.83 mg/ml. The specific lipolytic activity was increased after anion exchange chromatography. This postulated that there was very little loss of lipase during the delipidation process. Lipase activity was also compared using a commercially available porcine pancreatic lipase as a positive control. The concentration of the porcine pancreatic lipase was standardized to 1 mg/ml and yielded 0.0223 U/mg of the specific lipolytic activity. This suggests that the final purification of bovine BSAL is comparable to that of the porcine origin with specific BSAL lipolytic activity of 0.0222 U/mg. However, the total lipolytic activity of the aqueous crude extract was recorded higher based on the starting material volume and the protein concentration. Hence, the pancreatic bovine BSAL can be an excellent alternative for Halal enzyme replacement, specifically for pancreatic lipase.

This study employed a one-step purification of anion exchange chromatography, 1D SDS-PAGE, LC-MS/MS, and lipase activity assay to characterize bovine pancreatic BSAL. Firstly, the bovine pancreatic crude extract was subjected to anion exchange chromatography, and a single peak was eluted at 10% NaCl concentration (peak C), which contained active BSAL lipolytic activities. The elution profiles of the BSAL enzyme are shown in Fig. 1a. A summary of the BSAL purification results is presented in Table 2. After the one-step purification, purified fractions yielded higher BSAL lipolytic activity than the crude extract. Previously, pancreatic lipase was reported to have more significant similarity in molecular weight, similar amino-acid composition, and common antigenic determinants among mammalian lipases such as human, porcine, bovine, and ovine (49). On top of that, the specific lipase activity of bovine BSAL is comparable with that of porcine pancreatic lipase, which has been discovered in this study, despite some arguments that lipases will have distinct lipolytic activity and specificity between different biological sources (50). This discovery proves that purified bovine BSAL can be suggested as an alternative to porcine BSAL, considering that its specific lipolytic activity is similar to commercial pancreatic porcine lipase.

The molecular weight of bovine BSAL is 63.83 kDa, as reported in the protein database system (PDB). In this study, the molecular weight of purified BSAL was determined by gel electrophoresis. As shown in Fig. 1b, the purified product revealed a single band on the 1D SDS-PAGE with a molecular weight of approximately 65.12 kDa, confirmed with the LC-MS/MS. This finding was consistent with the previous study, whereby the 1D SDS-PAGE of the purified BSAL from defatted pyloric caeca pancreatic tissue was seen as a homogenous band at 60 kDa (28). In contrast, the molecular weight of purified porcine pancreatic BSAL was reported to be slightly larger in the range of 70 to 85 kDa (22, 23). Meanwhile, the molecular weight of purified rat pancreatic BSAL was reported to be 70 kDa (27). Several mammalian BSALs were reported to have similar specificities and kinetic properties. However, the inconsistency of the physical properties and heterogeneity, including pI and molecular weight, have been controversial findings for BSAL from different animal sources (23). It is speculated that although they may have similar protein cores but not in their N-terminal sequences or amino acid compositions, they also contribute to different degrees of glycosylation in various hosts, which leads to variation in BSALs' molecular weight (20). In addition, glycans may contribute up to 30% of the molecular weight of glycoproteins; therefore, the expected size of the protein of interest is influenced by post-translational glycosylation modification (51).

This work aims to identify the bovine pancreatic BSAL that could be beneficial in pharmaceutical and medical applications, especially to cater for Halal requirements. Thus, the LC-MS/MS proteomic analysis showed a strong protein identification, resulting in ten predictive peptide sequences showing bovine pancreatic BSAL characteristics. Mass spectrometry helps detect the peptide as the amino acid sequence is unique to the candidate protein and provides better selectivity (52, 53). Previously, the bovine pancreatic BSAL was only limited to the purification and the determination of the N-terminal amino acid sequence study (25).

To the best of our knowledge, the literature on bovine BSAL purification is limited. The recent finding on BSAL extraction and purification was two decades ago, reporting on the affinity binding sites of pancreatic BSAL in pancreatic and intestinal tissue of rats (40). Although there have been scientific reports on successful BSAL purification from bovine, our study presents a single-step purification of bovine pancreatic BSAL. Previously, bovine BSAL was isolated in a pure form by lyophilization of fresh bovine pancreas, sucrose solution enzyme extraction, ammonium sulfate, and acetone fractional precipitation followed by gel filtration chromatography on Sephadex G-100 (50). In another study, the bovine BSAL was purified from a defatted pancreas with 50 mM sodium phosphate pH 7.5 containing 0.5 mM PMSF and 2 mM benzamidine as an extraction buffer (25). The extraction buffer made up of 0.1 M sodium acetate buffer pH 4.8 containing 0.9% NaCl, 0.2% Triton X-100, 3 mM sodium taurocholate, 2 mM benzamide hydrochloride, 0.2 mM phenylmethylsulfonyl fluoride, 2mM hydrocinnamic acid, and 0.5 mM N^α-benzoyl-D, L-arginine were added with proteinase inhibitors were used during the BSAL extraction from frozen porcine pancreas (23). In the same study, the ammonium precipitation was done before running on Sephacryl S-200, followed by TSK 3000 SW columns to purify the porcine pancreatic BSAL (23). Another study also used the frozen pancreas, followed by Sephadex G-150 gel filtration, to obtain purified porcine BSAL (22). Instead of the fresh frozen pancreas as the starting material, the pancreatic juice was also collected from a dog using a cannula, followed by ammonium precipitation, and applied to a DEAE-cellulose column and concentrated utilizing a PM-10 membrane filter (54). The cannulation process from the pancreatic duct for the pancreatic juices collection raises animal concerns, especially when using a non-invasive alternative (55). A quick method using affinity chromatography of cholate-derivatized Sepharose successfully purified BSAL from bovine and porcine commercial crude and human milk whey has also been reported (11). Despite the various approaches, the findings are surprisingly consistent, with the molecular weight obtained for bovine pancreatic BSAL being approximately 60kDa. In summary, there are various purification methods, as well as the choice of starting material, buffer selection for extraction, and different kinds of columns used to purify pancreatic BSAL successfully. However, the study herein offers a more straightforward approach with minimal purification steps and directly using a fresh bovine pancreas. This results in an improved yield of BSAL with the specific lipolytic activity of purified bovine BSAL comparable to that of commercial porcine pancreatic lipase.

In summary, BSAL was successfully purified and characterized from Bos taurus fresh pancreatic tissue. The BSAL yielded the highest after the anion exchange chromatography and was comparable to porcine lipase. Overall, the results of this study indicate that LC-MS/MS analysis followed by peptide prediction allows rapid identification of peptides with bovine pancreatic BSAL. This enables the extraction and purification process to obtain purified BSAL to be carried out effectively. Therefore, future studies on the current topic are recommended to determine the properties of bovine BSAL as a possible alternative to porcine, focusing on the Halal pharmaceutical and medical industries.

Ethics Approval

No ethics approval is required.

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.

Author Contributions.

NHR, DUL, ZZA, and UMAH conceived, conceptualized, and planned the experiments. NHR conducted and investigated the experiments. MAJ advised and strategized the purification methodology of the experiment. NBA assisted in the BSAL purification process. MAJ, DUL, ZZA, and UMAH supervised the project. NHR drafted and edited the original manuscript. MAJ, DUL, ZZA, and UMAH critically reviewed and provided feedback on the manuscript. All authors read and approved the final manuscript for publication.

Funding.

This study was funded by the Ministry of Higher Education FRGS grant (FRGS/1/2018/STG04/UITM/02/6). The funders had no role in the study design, data collection, analysis, or decision to publish the manuscript.

Acknowledgements.

The authors also acknowledge the Faculty of Applied Sciences, Universiti Teknologi MARA, and Miss Farahayu Binti Khairuddin of the Malaysia Genome and Vaccine Institute for technical support during the lipase activity measurement process.

Conflicts of Interest.

The authors declare no conflict of interest. None of them are members of the Applied Biochemistry and Biotechnology editorial board.

Availability of Data and Materials

The data supporting this study's findings are available from the corresponding author, [UMAH], upon reasonable request.

Hou CT, Shimada Y. Lipases. Encyclopedia of Microbiology, Third Edition. 2009. 385–392 p.
Vardar-Yel N, Tütüncü HE, Sürmeli Y. Lipases for targeted industrial applications, focusing on the development of biotechnologically significant aspects: A comprehensive review of recent trends in protein engineering. Int J Biol Macromol. 2024;273(132853):1–16.
de Oliveira BH, Bourlieu C, Lecomte J, Villeneuve P, Nascimento VMG. d. Lipolysis of Burkholderia lata LBBIO-BL02 lipase in simulated human digestive environments: A candidate for enzyme replacement therapy. Food Biosci. 2024;58(103737):1–14.
Peter L, Jutta K, Lankisch PG. Pancreatic enzyme replacement therapy. Curr Gastroenterol Rep. 2001;3(2):101–8.
Szkopek D, Pierzynowski SG, Pierzynowska K, Zaworski K, Kondej A, Wychowański P et al. A review: Pancreatic enzymes in the treatment of chronic pancreatic insufficiency in companion animals. J Vet Intern Med. 2024;(April):1–8.
Susanne. XL, L, Mark. L, Laila. N, Olle H. Bile salt-stimulated lipase and pancreatic lipase-related protein 2 are the dominating lipases in neonatal fat digestion in mice and rats. Pediatr Res. 2007;62(5):537–41.
Wang X, Wang CS, Tang J, Dyda F, Zhang XC. The crystal structure of bovine bile salt activated lipase: Insights into the bile salt activation mechanism. Structure. 1997;5(9):1209–18.
Caruso MA, Sheridan MA. Gut Anatomy and Morphology | Pancreas. In: Encyclopedia of Fish Physiology: From Genome to Environment: Volume 1–3. 2011. pp. 1276–1283.
Maldonado-Valderrama J, Wilde P, MacIerzanka A, MacKie A. The role of bile salts in digestion. Adv Colloid Interface Sci. 2011;165:36–46.
Wang CS, Hartsuck JA. Bile salt-activated lipase. A multiple function lipolytic enzyme. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1993;1166:1–19.
Moore AJ, Vakos H, Neville TM, Tonge P, Arya P, Burton G. A quick method for purifying bile salt-activated lipases. Biotechnol Tech. 1996;10(7):523–8.
Lombardo D. Bile salt-dependent lipase: Its pathophysiological implications. Biochim Biophys Acta - Mol Cell Biol Lipids. 2001;1533:1–28.
Hui DY, Howles PN. Carboxyl ester lipase: Structure-function relationship and physiological role in lipoprotein metabolism and atherosclerosis. J Lipid Res. 2002;43:2017–30.
Lindquist S, Wang Y, Andersson EL, Grebe ST, Alenius GM, Rantapää-Dahlqvist S, et al. Effects of bile salt-stimulated lipase on blood cells and associations with disease activity in human inflammatory joint disorders. PLoS ONE. 2023;18(8):1–15.
Lindquist S, Andersson EL, Lundberg L, Hernell O. Bile Salt-Stimulated Lipase Plays an Unexpected Role in Arthritis Development in Rodents. PLoS ONE. 2012;7(10):1–9.
Nilson J, Blackberg L, Carlsson P, Enerback S, Hernell O, Bjursell G. cDNA cloning of human-milk bile‐salt‐stimulated lipase and evidence for its identity to pancreatic carboxylic ester hydrolase. Eur J Biochem. 1990;192(2):543–50.
Hernell O, Olivecrona T. Human milk lipases II. Bile salt-stimulated lipase. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1974;369(2):234–44.
Christie DL, Cleverly DR, O’Connor CJ. Human milk bile-salt stimulated lipase Sequence similarity with rat lysophospholipase and homology with the active site region of cholinesterases. FEBS Lett. 1991;278(2):190–4.
Wang CS, Kloer HU. Kinetic properties of human pancreatic carboxylesterase. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1983;754(2):142–9.
Abouakil N, Rogalska E, Bonicel J, Lombardo D. Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1988;961(3):299–308.
Aho HJ, Sternby B, Kallajoki M, Nevalainen TJ. Carboxyl ester lipase in human tissues and in acute pancreatitis. Int J Pancreatol. 1989;5(2):123–34.
Momsen WE, Brockman HL. Purification and characterization of cholesterol esterase from porcine pancreas. Biochim Biophys Acta - Lipids Lipid Metab. 1977;486:103–13.
Rudd EA, Mizuno NK, Brockman HL. Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1987;918(2):106–14.
Labow RS, Adams KAH, Lynn KR. Porcine cholesterol esterase, a multiform enzyme. Biochim Biophys Acta - Protein Struct Mol Enzymol. 1983;749(1):32–41.
Tanaka H, Mierau I, Ito F. Purification and characterization of bovine pancreatic bile salt-activated lipase. J Biochem. 1999;125(5):883–90.
Abouakil N, Mas E, Bruneau N, Benajiba A, Lombardo D. Bile salt-dependent lipase biosynthesis in rat pancreatic AR 4 – 2 J cells: Essential requirement of N-linked oligosaccharide for secretion and expression of a fully active enzyme. J Biol Chem. 1993;268(34):25755–7563.
Calame KB, Gallo L, Cheriathundam E, Vahouny GV, Treadwell CR. Purification and properties of subunits of sterol ester hydrolase from rat pancreas. Arch Biochem Biophys. 1975;168(1):57–65.
Gjellesvik DR, Lombardo D. Pancreatic bile salt dependent lipase from cod (Gadus morhua): Purification and properties. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1992;1124(2):123–34.
Koh J, Victor AF, Howell ML, Yeo JG, Qu Y, Selover B, et al. Bile Salt-Stimulated Lipase Activity in Donor Breast Milk Influenced by Pasteurization Techniques. Front Nutr. 2020;7(November):1–7.
Manson WG, Weaver LT. Fat digestion in the neonate. Arch Dis Child Fetal Neonatal Ed. 1997;76(3):206–11.
Augé N, Rebaï O, Lepetit-Thévenin J, Bruneau N, Thiers JC, Mas E, et al. Pancreatic bile salt-dependent lipase induces smooth muscle cells proliferation. Circulation. 2003;108:86–91.
Rebaï O, Le Petit-Thevenin J, Bruneau N, Lombardo D, Vérine A. In vitro angiogenic effects of pancreatic bile salt-dependent lipase. Arterioscler Thromb Vasc Biol. 2005;25(2):359–64.
A OM, S NY. Status of beef industry of Malaysia. Malaysian J Anim Sci. 2015;18(December):1–21.
Li JL, Yu QL, Zhang L, Liu LL, Guo Z, Bin. Purification and characteristics of trypsin from the pancreas of tibetan sheep. J Food Biochem. 2012;36(1):122–8.
Aziz NA, Ibrahim I, Raof NA. The Need for Legal Intervention within the Halal Pharmaceutical Industry. Procedia - Soc Behav Sci. 2014;121:124–32.
Sudarsono H, Nugrohowati RNI. Determinants of the Intention to Consume Halal Food, Cosmetics and Pharmaceutical Products. J Asian Financ Econ Bus. 2020;7(10):831–41.
American Halal Foundation. The Untapped Market For Halal Pharmaceuticals [Internet]. 2024. pp. 1–11. https://halalfoundation.org/the-untapped-market-for-halal-pharmaceuticals/
Hernell O, Bläckberg L. Human milk bile salt-stimulated lipase: Functional and molecular aspects. J Pediatr. 1994;125(5):56–61.
Wang C, Dashti A, Downs D. Bile Salt-Activated Lipase. Lipase and Phospholipase Protocols. New Jersey: Humana; 1999. pp. 71–80.
Bruneau N, Lombardo D, Bendayan M. The affinity binding sites of pancreatic bile salt-dependent lipase in pancreatic and intestinal tissues. J Histochem Cytochem. 2000;48(2):267–76.
Mukhopadhyay S, Maitra U. Chemistry and biology of bile acids. Curr Sci. 2004;87(12):1666–83.
Chandra P, Enespa, Singh R, Arora PK. Microbial lipases and their industrial applications: A comprehensive review. Microb Cell Fact. 2020;19(169):1–42.
Vivek K, Sandhia GS, Subramaniyan S. Extremophilic lipases for industrial applications: A general review. Biotechnol Adv. 2022;60(108002):1–16.
Elamin BA, Al-Maleki A, Ismael MA, Ayoub MA. Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius). Saudi J Biol Sci. 2014;21(6):574–81.
Al-Ajlan A, Bailey GS. Purification and some properties of camel carboxypeptidase B. Mol Cell Biochem. 1999;201:105–10.
Steiner JM, Wilson BG, Williams DA. Purification and partial characterization of feline pepsinogen. Comp Biochem Physiol - B Biochem Mol Biol. 2002;134:151–9.
Vo C-VT, Luu NVH, Nguyen TTH, Nguyen TT, Ho BQ, Nguyen TH, et al. Screening for pancreatic lipase inhibitors: evaluating assay conditions using p-nitrophenyl palmitate as substrate. All Life. 2022;15(1):13–22.
Yel NV. Investigation of The Activity of Lipase Variants on Different 4-Nitrophenyl Esters by Spectrophotometric Assay. Cauc J Sci. 2021;8(2):292–303.
Caro ADE, Bonicel J, Guy O. Comparative studies of human and porcine pancreatic lipases: N-terminal sequences, sulfhydryl groups and interfacial activity. Biochimie. 1981;63:799–801.
Shahani KM, Khan IM, Chandan RC. Bovine Pancreatic Lipase. I. Isolation, Homogeneity, and Characterization. J Dairy Sci. 1976;59(3):369–75.
Puranik A, Saldanha M, Dandekar P, Jain R. A comparison between analytical approaches for molecular weight estimation of proteins with variable levels of glycosylation. Electrophoresis. 2022;43(11):1223–32.
Rauh M. LC-MS/MS for protein and peptide quantification in clinical chemistry. J Chromatogr B Anal Technol Biomed Life Sci. 2012;883–884:59–67.
Chiu HH, Tsai IL, Lu YS, Lin CH, Kuo CH. Development of an LC-MS/MS method with protein G purification strategy for quantifying bevacizumab in human plasma. Anal Bioanal Chem. 2017;409(28):6583–93.
Lee PC. Comparative studies of canine colipase and lipases from bovine, porcine, canine, human and rat pancreases. Comp Biochem Physiol. 1978;60(4):373–8.
Steiner JM, Williams DA. Purification of classical pancreatic lipase from dog pancreas. Biochimie. 2002;84(12):1243–51.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Identification and Characterization of Bovine (Bos taurus) Pancreatic Bile Salt Activated Lipase for Potential Halal Alternative.

Status:

Version 1

Abstract

Figures

Introduction

Materials/subjects and Methods

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1