Trypsin/a-amylase

Among the EST database of ragi sequences, there are two groups of bifunctional proteinase inhibitor trypsin a-amylase from seeds of ragi sequences. The upper clade was further subdivided (Fig. 6.10). Wang et al. (2008) concluded that there was great diversity in the sequence of different Bowman-Birk inhibitors in emmer wheat both within and between populations. 

Shivraj, B., Rao, H. N., and Pattiraman, T. N. (1982). Natural plant inhibitors. Isolation of a trypsin/a-amylase inhibitor and a chymotrypsin inhibitor from ragi (Eleusine coracana) grains by affinity chromatography and study of their properties. J. Sci. Food Agric. 33,1080-1091. 

Some of the pancreatic enzymes in the lumen include pancreatic amylase, pancreatic lipase, elastase, trypsin, a-chymotrypsin, and carboxypeptidase A. For example, the aspirin derivatives aspirin phenylalanine ethyl ester, aspirin phenyllactic ethyl ester, and aspirin phenylalanine amide have been studied as substrates for carboxypeptidase A [67,68], with the phenylalanine ethyl ester derivative proving to be the best substrate. This study indicated that the carboxypeptidase A may serve as a reconversion site for many drug derivatives. 

As indicated in Table 2.1, most of the promoters used in plant tissue culture have been based on the constitutive cauliflower mosaic virus (CaMV) 35S promoter. In contrast, inducible promoters have the advantage of allowing foreign proteins to be expressed at a time that is most conducive to protein accumulation and stability. Although a considerable number of inducible promoters has been developed and used in plant culture applications, e.g. [32-37], the only one to be applied thus far for the production of biopharmaceutical proteins is the rice a-amylase promoter. This promoter controls the production of an a-amylase isozyme that is one of the most abundant proteins secreted from cultured rice cells after sucrose starvation. The rice a-amylase promoter has been used for expression of hGM-CSF [10], aranti-trypsin [12, 29, 38, 39] and human lysozyme [30]. 

Contacts with the catalytic residues, in combination with hydrophobic interactions, are also observed in the complex of an insect a-amylase with the Ragi bifunctional a-amylase/trypsin inhibitor (RBI) [174]. Conversely, the mechanism of inhibition of barley a-amylase by the barley a-amylase/subtilisin inhibitor (BASI) did not involve direct contact between inhibitor residues and the catalytic site [175]. The inhibitor sterically blocks the catalytic site, but does not extend into it. A cavity is created, which is occupied by a calcium ion coordinated by water-mediated interactions with the catalytic residues. 

Hafkenscheid, J.C.M., Hessels, M., and van der Hoek, E.W. 1983. Determination of a-amylase, trypsin and lipase in duodenal fluid Comparison of methods. J. Clin. Chem. Clin. Biochem. 21 167-174. 

Alam et al. (2001) studied the inhibitory action of CNBR fragments of the RATI on amylase 2 purified from porcine pancreas and reported that the fragment comprising the first 10 amino acids of the N-terminal segment inhibited amylase competitively. NH2-Ser-Val-Gly-Thr-Ser-Cys-Ile-Pro-Gly-OH was most effective. A serine to alanine transformation diminished the inhibitory effect. Blocking the N-terminus abolished the a-amylase inhibitory activity. It may be presumed that the abolition of the a-amylase inhibitory activity with 2,4,6-trinitrobenzenesulfonic acid as observed by Shivraj and Pattabhiraman (1981) may have been due to its interaction with cysteine. Figure 6.9 shows a representation of this ragi protein with its ability to bind both a-amylase and trypsin with its two arms. 

FIGURE 6.9 Depiction of the two heads of the ragi double headed a-amylase and trypsin inhibitor. (Gourinath et al., 2000. Reproduced with permission from the lUCr). 

Alam, N., Gourinath, S., Dey, S., Srinivasan, A., and Singh, T. P. (2001). Substrate-inhibitor interactions in the kinetics of a-amylase inhibition by ragi a-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments. Biochemistry 40, 4229M233. 

Campos, F. A. P. and Richardson, M. (1983). The complete amino acid sequence of the bifunctional a-amylase/trypsin inhibitor from seeds of ragi (Indian finger millet, Eleusine coracana Gaertn.). FEBS Lett. 152, 300-304. 

Barwell, C. J., Blunde, G., and Manandhar, P.D., Isolation and characterization of brown algal polyphenols as inhibitors of a-amylase, lipase and trypsin, J. Appl. Phycol., 1, 319, 1989. 

Relevant quality control should not be restricted to the usual triad of activities of pancreas lipase, a-amylase, and trypsin, but should be extended to the content of colipasc, the activities of the two other lipolytic enzymes present in pancreatine (phospholipase Aj and carboxylester lipase), and the dissolution characteristics of enteric-coated preparations as a function of time and pH (Fig. 16). The availability of such information will certainly contribute to a better tailoring of flic management of maldigestion in the individual patient and to a more appropriate correction of the obligate nonphysio logical route of delivery of these enzyme supplements. 

Cereal dual function a-amylase/trypsin inhibitor proteins... 

The cereal dual function a-amylase/trypsin inhibitor proteins are cysteine-rich, disulphide-rich, double-headed, 13-16 kDa, dual function inhibitor proteins that inhibit both of the digestion enzymes a-amylase and trypsin [290-325] (Table 11). Thus the Zea (com) member of this family, com Hageman factor inhibitor (CHFI), is a double-headed 14 kDa protein that inhibits a-amylase and the serine proteases trypsin and blood clotting Factor Xlla [323-324] (Table 11). The structures of the bifunctional a-amylase/trypsin inhibitor proteins from Eleusine (ragi) (RBI) [292-295] and Zea (com) (CHFI) [325] have been determined. These proteins are structurally similar to the lipid transfer proteins, being composed of a bundle of 4 a-helices together with a short [3-sheet element connected by loops, the a-amylase- and protease-inhibitory domains being separately located [325]. 

Eleusine coracana (ragi, Indian finger millet) (Poaceae) [seed] RBI (Ragi bifunctional I) = RATI (Ragi a-Amylase and Trypsin Inhibitor) (122 aa 13 kDa lOCys) Trypsin (R34-L35) [1 nM], (a-Amylase inhibitor, K 11 nM) [290- 297]... 

Hordeum vulgare (barley) (Poaceae) [seed] Homologous Barley chloroform-methanol soluble proteins a-e = Barley CMa-e = BTAI-CMa-e, CMx (-16 kDa 10 Cys) Trypsin (CMc CMe) [CMa (= BTAI-CMa) inhibits a-Amylase] [298- 303]... 

Oryza sativa (Poaceae) [seed] RA5 (14 kDa 10 Cys), RAM (15 kDa 10 Cys) Cereal a-Amylase /Trypsin inhibitor homologue [306]... 

Triticum aestivum (wheat) (Poaceae) Chloroform-methanol soluble proteins CM , CM2, CM3, CM16.CM17 (13-16 kDa 10 Cvs heterotetramers) Cereal a-Amylase /Trypsin inhibitors [308- 311]... 

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