Comparison of physicochemical properties of recombinant buckwheat trypsin inhibitor (rBTI) and soybean trypsin inhibitor (SBTI)

Highlights

• An effective method for obtaining high purified rBTI was established.

• RBTI has a smaller molecular weight and exhibited better thermal stability than SBTI.

• RBTI is considered to being better suitable for applications comparing to SBTI.

Abstract

The inhibitory activities of buckwheat trypsin inhibitor (rBTI) towards trypsin were compared with soybean trypsin inhibitor (SBTI) in terms of their sensitivities to temperature, pH, salt ions and organic solvents. Both rBTI and SBTI were stable over a broad pH range of 2.0–12.0. rBTI exhibited higher thermal stability than SBTI. The inhibitory activity of rBTI was decreased by Zinc ions (Zn²⁺), KSCN, vitamin C and urea, while that of SBTI remained unchanged. However, H₂O₂, Mg²⁺ and Cu²⁺ ions had no significant effects on the inhibitory activities of rBTI and SBTI. Acetonitrile enhanced the inhibitory activity of rBTI, but had no effect on SBTI, while dimethylacetamide (DMAC) increased the inhibitory effect of both rBTI and SBTI. On the contrary, the inhibitory activities of rBTI and SBTI were reduced by isopropyl alcohol and methanol. The inhibition constants K_i of rBTI and SBTI were calculated to be 7.41 × 10⁻⁹ M and 6.52 × 10⁻⁹ M, respectively.

Introduction

Trypsin inhibitors are substances capable of binding to trypsin and trypsin-like enzymes to reduce their capacity to catalyze the transformation of their substrates into products. Trypsin and trypsin-like enzymes are serine proteases, and trypsin inhibitors cause their partial or complete inactivation by forming enzyme-inhibitor complexes with the enzymes [1]. Trypsin inhibitors are ubiquitous in nature and are widespread in animals, plants and microorganisms. Examples include the Kunitz trypsin inhibitor (KTI), the Bowman-Birk inhibitor (BBI), and potato proteinase inhibitor types I (Pin 1) and II (Pin 2) [[2], [3], [4]]. Protease inhibitors primarily act to regulate protease activity, and they have been found to occur abundantly in plants; for instance, they constitute up to 10% of total protein in seeds where they provide a defensive mechanism against insects and microorganisms [5]. There is ample evidence that trypsin inhibitors also have special physiological functions, such as anti-cancer and anti-inflammatory activities [6,7]. Thus, the discovery of novel and more potent trypsin inhibitors would have potential benefits and market prospects in the fields of agriculture (e.g., biological control), food technology (e.g., food preservation), and in clinical practice (e.g., therapeutics and antiretroviral agents) [8].

There are two types of soybean trypsin inhibitors (SBTI), viz., the soybean Kunitz trypsin inhibitor (SKTI) and the soybean Bowman-Birk inhibitor (SBBI) [9]. SKTI has been much more studied and applied compared to SBBI. SBBI protein consists of 71 amino acid residues, and contains seven disulfide bridges which confers configurational stability to the active molecule [10]. Furthermore, Bowman-Birk inhibitors have two tandem homologous domains, which enables inhibition of two protease molecules simultaneously and independently [11]. SKTI protein is composed of 170–200 amino acid residues, a molecular weight of about 21.5 kDa, and an isoelectric point at pH 4.5. SKTI also has 2 disulfide bridges in its structure, and is a non-glycosylated, monomeric, globulin type protein. It has 12 criss-crossed antiparallel β−strands, hence it has extensive hydrogen bonding that contribute to the stabilization of its special structure [12]. The presence of both the 12 β−strands and 2 disulfides stabilize the active configuration of SKTI. It is resistant to thermal and acid treatments, as well as proteolysis. It has even been reported to survive enzymatic denaturation in simulated gastric and intestinal environment in vitro [13]. Soybean trypsin inhibitors were considered for many years to be one kind of antinutritional factor, and research about SBTI has mainly focused on strategies for their inactivation [14]. Because of its stability, SKTI is used as an external loading control for Western blot analysis [15], additive to protect protein against proteolysis in food systems [16], and as good model for studying the interaction of proteases and inhibitors [17].

Buckwheat trypsin inhibitor (BTI), is a peptide obtained from buckwheat (Fargopyrum esculentum) seeds, and belongs to the potato type-I family of inhibitors, and was first purified in 1995 [18]. Recombinant buckwheat trypsin inhibitor (rBTI) was successfully expressed in Escherichia coli by our group [[19], [20], [21]] and shown to be composed of 69 amino acids with a molecular weight of 7.9 kDa. It shares high sequence homology with other potato type-I inhibitors derived from other plant sources such as Amaranthus hypochondriacus [22], Cucurbita maxima [23], Momordica charantia [24,25]. The crystal structures of rBTI and rBTI complexed with bovine trypsin have also been reported [26]. Three conserved domains exist in rBTI, α−sheet, an α−helix and a reactive site. The P₈’ residue plays important role for its activity. The rBTI exhibited high tumor suppressor activity in vitro and could also induce apoptosis in several cancer lines including Hep G2, Hela and EC9706 [20,21]. It was further demonstrated that rBTI enters Hep G2 by clathrin-dependent endocytosis and has the potential to deliver anticancer drugs, proteins and therapeutic genes [27]. rBTI could also extend life expectancy by mimicking calorie restriction in C. elegans [28,29]. In addition, rBTI has good water solubility and heat resistance, and retains its high inhibitor activity during preparation and processing. Thus, it is speculated that with these advantages, rBTI has prospects for broad applications in biomedical, food and related fields. However, rBTI has not attained commercialization status as yet. With its smaller molecular weight, rBTI may exhibit important differences from SBTI, the only commercially available plant-derived trypsin inhibitor to make it better suited for applications where size is important. Thus, by comparing the physical and chemical properties of rBTI and SBTI, this work seeks to highlight its relative advantages and to provide the requisite information needed to rationalize potential applications.