PROPERTIES OF CYSTEINE PROTEINASE INHIBITORS FROM BLACK GRAM AND RICE BEAN

Cysteine proteinase inhibitors (CPI) were purified to 59 and 54 fold from black gram (Vignaraungo (L.) Hepper) and rice bean (Vignaumbellata Thunb.), respectively, by using heal treatment, followed by chromatography on a carboxymethyl (CM)‐papain‐Sepharose affinity column. The purified inhibitors were highly inhibitory to papain and Pacific whiting cathepsin L in a concentration dependent manner. They were detected as a dark band on tricine‐SDS‐PAGE gel stained for inhibitory activity. The apparent molecular weights of purified CPI from black gram and rice bean seeds were estimated to be 12, 000 daltons. The purified inhibitors were thermostable up to 90C and active in the neutral and alkaline pH ranges.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A PROTEINASE INHIBITOR FROM TEPARY BEAN (PHASEOLUS ACUTIFOLIUS) SEEDS

In a qualitative screening of 36 accessions of tepary beans seeds, all the accessions inhibit the activity of bovine trypsin and trypsin-like proteinases from the insect P. truncatus, and the majority of them inhibit α-amylase activity of several important insect pests. A protein proteinase inhibitor was purified from accession L-242-45, using fractional precipitation, gel filtration, ion exchange chromatography and reverse-phase HPLC. The protein showed an apparent molecular weight of 7,100 by PAGE. However, contrary to other inhibitors previously reported, the inhibitory activity was only present in the trimeric form. The protein was characterized as a serine-proteinase inhibitor that recognized trypsin, chymotrypsin and trypsin-like proteinases, but it also recognized aspartic acid proteinases from different insects. It contained no carbohydrate residues and showed a high stability at 96C at low pH.     

USE OF CYSTEINE PROTEINASE INHIBITORS FROM INJURED TOMATO LEAVES IN WHITING SURIMI

Cooking surimi paste from Pacific whiting results in a gel with poor texture due mainly to myosin degradation caused by a cysteine proteinase. Cysteine and serine proteinase inhibitors were isolated from injured and methyl jasmonate treated tomato leaves. Tomato cysteine proteinase inhibitor was stable at 60C but inactivated at 90C, making it suitable for use in surimi. Tomato proteinase inhibitors (TPI), having 7.9 papain inhibitor units, inhibited autolysis about 95% in 10 g of Pacific whiting surimi. Gel strength of Pacific whiting surimi was improved by adding only 0.0 27% of TPI to the surimi formulation. Addition of TPI did not affect the color of whiting surimi gel, while egg white needed to prevent gel weakening caused the gels to have more yellow hue (P<0.05). SDS-PAGE showed that myofibrillar protein degradation was prevented during cooking when 0.027% of TPI was included in the surimi. TPI extracted from tomato plants has potential for use as food grade additive in Pacific whiting surimi.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

MODULATING THE CONDITIONING OF MEAT BY THE USE OF ORYZACYSTATIN, A CYSTEINE PROTEINASE INHIBITOR OF RICE SEED ORIGIN

A new process was developed to regulate the papain-induced overtenderization of meat by using oryzacystatin, a cysteine proteinase inhibitor of rice seed origin. Meat cubes (2 × 2 × 2 cm each), aged 3 days, were treated with a 0.1 fold weight of papain solution and incubated at 8°C. The most favorable degree of tenderization resulted when a 2% papain solution was used for 2 h at 8°C. When the incubation was made for 24 h or longer, overtenderization resulted. The incubation of a meat cube with papain for the first 2 h and then with an adequate amount of added oryzacystatin for the subsequent 22 h was effective in regulating overtenderization. It was also confirmed by SDS-polyacrylamide gel electro-phoresis that the use of oryzacystatin under similar incubation conditions regulated the papain-aided hydrolysis of myofibrillar proteins.     

PURIFICATION AND CHARACTERIZATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF SILVER CARP

Recently, a myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S‐200, High Q ion‐exchange and affinity column of Arginine Sepharose‐4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l ‐3‐carboxy‐trans‐2, 3‐epoxypropionyl‐l ‐leucine‐4‐guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin‐like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.     

CHARACTERIZATION OF PROTEINASE RECOVERED FROM PACIFIC WHITING SURIMI WASH WATER

Pacific whiting surimi wash water (SWW) proteinase was recovered by ohmic heating, ultrafiltration, and freeze-drying. By these processes, 5.9-fold purification was achieved. The most efficient step was ohmic heating, which concentrated the proteinase by 4.8 fold. Specific activity of the recovered SWW proteinase on casein and Z-Phe-Arg-NMec was 28.2 and 0.17 U/mg protein, respectively. The SWW proteinase showed good hydrolytic activity towards casein, acid-denatured hemoglobin and myofibrils. Acidification increased specific activity on all substrates tested but reduced thermal stability. β-Mercaptoethanol, dithiothreitol and urea enhanced activity against Z-Phe-Arg-NMec. Proteinase activity on Z-Phe-Arg-NMec showed an optimum pH of 4.0. The recovered proteinase showed 18.5% residual activity after 7 week storage at 4C.     

CYSTEINE AS AN INHIBITOR OF POLYPHENOL OXIDASE

The effect of L-cysteine on mushroom polyphenol oxidase (PPO) activity was investigated using three spectrophotometric assays. The formation of pigment (melanin) , o- phenylquinone and cysteine-quinone adduct from catechol were each assayed under similar conditions. Cysteine had two effects; first, a lag phase was seen when melanin formation was measured, and secondly, the rate of browning was decreased after the lag phase. The lag phase was not observed when the formation of cysteine-quinone adduct rather than melanin formation was measured. This suggests that the lag phase observed in melanin formation is due to adduct formation and not quinone reduction. The velocity of adduct formation was similar to the velocity of o- phenylquinone and melanin formation without cysteine suggesting that reduction of o- phenylquinone to catechol was not significant. The structure of the adduct formed between cysteine and o- phenylquinone was synthesized and unequivocally determined by NMR spectroscopy to be S- (2,3-dihydroxyphenyl)cysteine.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF TROPOMYOSIN-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF YELLOW CROAKER (PSEUDOSCIAENA CROCEA)

A myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of yellow croaker (Pseudosciaena crocea) was isolated from myofibril by heat treatment and chromatographies on Sephacryl S‐200, fast performance liquid chromatography (FPLC) on Mono Q column and high performance liquid chromatography (HPLC) using a Bio‐Sil SEC‐125 column. A single protein band with a molecular weight of 34 kDa was observed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Optimum temperature and pH of the purified protein was 55C and 8.0. Inhibitor susceptibility analysis indicated that the enzymatic activity was effectively suppressed by serine proteinase inhibitors such as Pefabloc 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride and aprotinin, while inhibitors for other proteinases (namely cysteine, asparatic and metallo) did not show any inhibitory effect. At the last purification stage, the electrophoretic study revealed that the proteinase associated with tropomyosin, as the N‐terminal amino acid sequence of the 34 kDa main band protein, exhibited high sequence homology (90%) to α‐tropomyosin from white croaker, human and rat. This result suggested that yellow croaker MBSP is an α‐tropomyosin‐binding proteinase.     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM MAISE (ZEA MAIZE)

α-Amylase inhibitor is presented in maize seeds. It is a protein as indicated by precipitation with ammonium sulfate and trichloroacetic acid, denaturation by heat, digestion with proteases and by dye-staining. It was purified to homogeneity by ammonium sulfate precipitation and Sephadex G-75 gel filtration. It had an apparent molecular weight of 29,600 and did not contain any carbohydrate. Its properties differed from those of previously reported α-amylase inhibitors, since it was active against α-amylase of maize, produced during germination as well as against Bacillus subtilis α-amylase. It was also active against α-amylase from the insects Tribolium castaneum, Sitophilus zeamais and Rhyzopertha dominica, but it was inactive against α-amylase from human saliva, hog pancreas, Aspergillus oryzae, wheat, rye, barley, triticale, and sorghum. It was stable for 5 min at 96°C at pH 7. Maximal inhibition required at least 10 min of preincubation with the enzyme at pH 6.8 and 257deg;C. Polyacrylamide gel electrophoresis gave three protein bands, but only one was obtained in S.D.S. and mercaptoethanol.     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM RYE (SECALE CEREALE) FLOUR

An α-amylase inhibitor from rye (Secale cereale) flour has been purified to homogeneity by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE- and CM-cellulose. The isoelectric point was pH 5.8, and the molecular weight 28,000 by polyacrylamide gel electrophoresis with different gel concentrations and 27,000 by sedimentation equilibrium centrifugation. Under denaturating conditions the molecular weight was about 14,000, indicating two subunits identical in size. The inhibitor was active towards human salivary and hog pancreatic α-amylases but inactive towards Bacillus subtilis and Aspergillus oryzae α-amylases. The pH optimum for the reaction between the rye inhibitor and human salivary α-amylase was 6.0. The inhibitor did not change activity when exposed to pH 2 (0.01M HCl), but prolonged digestion by trypsin destroyed the inhibitor. The rye α-amylase inhibitor lost about 80% of its activity after 10 min at 100°C.     

PURIFICATION AND CHARACTERIZATION OF ESCULENTAMIN, A PROTEINACEOUS ALPHA-AMYLASE INHIBITOR FROM THE TARO ROOT, COLOCASIA ESCULENTA

A proteinaceous alpha-amylase inhibitor from the cormel of taro (Colocasia esculenta) was purified by extraction, heat treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-100, BioGel P-30, and DE-53 cellulose. This protein (aptly named esculentamin) was shown to be an acidic (pI = 4.4) glycoprotein with a carbohydrate content of 3.64% and a molecular weight of 11,800 daltons. Esculentamin activity was essentially stable to boiling for 3 h (pH 7.0), to a pH range of 2.0 to 12.0 (at 4°C), and to 6 M guanidine hydrochloride and 8 M Urea. Amino acid analysis indicated a predominance of acidic over basic amino acids, while side-by-side neutral and alkaline absorption spectra strongly suggested a lack of tryptophan. “Bifunctional” inhibitory activity towards both α-amylase and proteases was not observed with esculentamin (i. e., it did not inhibit the several proteases tested, viz. trypsin and subtilisin).     

ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITOR FROM THE WATER FERN, AZOLLA PINNATA R.Br.

Trypsin inhibitor from water fern (Azolla pinnata R. Br.) was isolated and characterized. Extraction of Azolla leaf meal in NaCl (0.15 M) rendered a higher recovery of trypsin inhibitor compared to other solvents tested (P < 0.05). The extraction time affected the inhibitor recovery significantly (P < 0.05). The extraction time of 3 h was optimum for the recovery of trypsin inhibitor. Based on inhibitor activity of a zone separated by electrophoresis, the molecular mass of the inhibitor from Azolla leaf was 21 kDa. The partially purified inhibitor was heat stable up to 10 min at 90C, at pH 7.0. High activity was also retained over a wide pH range (4–8) at 37C.     

FRACTIONATION OF SMALL PEPTIDES FROM BEER

Small peptides (MW<1000) were isolated from beer by hollow fibre ultrafiltration followed by column chromatography using the gel matrix Sephadex LH20. This simple procedure enabled the isolation of small peptides relatively free of other low molecular weight nitrogenous compounds. Analysis of the composition of subfractions from the small peptide fraction from pilot brewery beer and a commercial beer using reverse phase HPLC showed superior resolution of individual components compared to previously reported analytical techniques and demonstrated the presence of many peptide components. Acid hydrolysis and amino acid analysis of small peptide subfractions showed that the principal amino acid in each subfraction was glutamic acid/glutamine consistent with the release of peptides by proteolytic degradation of barley and malt proteins.