Ginger Protease Research Articles

Efficient Absorption of X-Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease.

Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

Partial characterization of an enzymatic extract from Bentong ginger (Zingiber officinale var. Bentong).

Extraction of protease from a local ginger rhizome (Zingiber officinale var. Bentong) was carried out. The effect of extraction pH (6.4, 6.8, 7.0, 7.2, 7.6, 8.0, 8.4, and 8.8) and stabilizers (0.2% ascorbic acid, 0.2% ascorbic acid and 5 mM EDTA, or 10 mM cysteine and 5 mM EDTA) on protease activity during extraction was examined. pH 7.0 potassium phosphate buffer and 10 mM cysteine in combination with 5 mM EDTA as stabilizer were found to be the most effective conditions. The extraction procedure yielded 0.73% of Bentong ginger protease (BGP) with a specific activity of 24.8 ± 0.2 U/mg protein. Inhibitory tests with some protease inhibitors classified the enzyme as a cysteine protease. The protease showed optimum activity at 60 °C and pH 6–8, respectively. The enzyme was completely inhibited by heavy metal cations such as Cu2+, and Hg2+. SDS stimulated the activity of enzyme, while emulsifiers (Tween 80 and Tween 20) slightly reduced its activity. The kinetic analysis showed that the protease has Km and Vmax values of 0.21 mg mL−1 and 34.48 mg mL−1 min−1, respectively. The dried enzyme retained its activity for 22 months when stored at −20 °C.

Zingipain, a ginger protease with acetylcholinesterase inhibitory activity.

In order to search for new acetylcholinesterase inhibitors (AChEIs), 15 Zingiberaceae plants were tested for AChEI activity in rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Zingiber officinale contained a significant AChEI activity. Eighty percent saturation ammonium sulfate precipitation and diethylaminoethyl cellulose ion exchange chromatography (unbound fraction) enriched the protein to a single band on nondenaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (approximately 33.5 kDa). Gelatin-degrading zymography showed that the AChEI-containing band also contained cysteine protease activity. The AChEI activity was largely stable between -20 and 60 °C (at least over 120 min) and over a broad pH range (2-12). The AChEI activity was stimulated strongly by Mn(2+) and Cu(2+) at 1-10 mM and weakly by Ca(2+), Fe(2+), Mg(2+), and Zn(2+) at 1 mM, but was inhibited at 10 mM. In contrast, Hg(2+) and ethylenediaminetetraacetic acid were very and moderately strongly inhibitory, respectively. In-gel tryptic digestion with liquid chromatography-tandem mass spectroscopy resolution revealed two heterogeneous peptides, a 16-amino-acid-long fragment with 100 % similarity to zingipain-1, which is a cysteine protease from Z. officinale, and a 9-amino-acid-long fragment that was 100 % identical to actinidin Act 2a, suggesting that the preparation was heterogeneous. AChEI exhibited noncompetitive inhibition of AChE for the hydrolysis of acetylthiocholine iodide with a K(i) value of 9.31 mg/ml.

Purification, characterization, and milk coagulating properties of ginger proteases

Ginger proteases are used as milk coagulants in making a Chinese traditional milk product (Jiangzhinai or Jiangzhuangnai), suggesting their potential as a source of rennet substitute that might be applicable in the modern dairy industry. In this study, ginger proteases were extracted from fresh ginger rhizome by using phosphate buffer and subsequently purified by ion exchange chromatography. Ginger proteases, all with a molecular weight around 31kDa, were found to exist in 3 forms with isoelectric point values around 5.58, 5.40, and 5.22, respectively. These enzymes had very similar biochemical behavior, exhibiting optimal proteolytic activity from 40 to 60°C and maximum milk clotting activity at 70°C. They were capable of hydrolyzing isolated αS1-, β-, and κ-casein, of which αS1-casein was most susceptible to the enzyme; κ-casein was hydrolyzed with a higher specificity than αS1- and β-casein. In addition, the ginger proteases exhibited a similar affinity for κ-casein and higher specificity with increasing temperature. Gel electrophoresis and mass spectra indicated that Ala90-Glu91 and His102-Leu103 of κ-casein were the preferred target bonds of ginger proteases. The milk clotting activity, affinity, and specificity toward κ-casein showed that ginger protease is a promising rennet-like protease that could be used in manufacturing cheese and oriental-style dairy foods.

Ginger protease used as coagulant enhances the proteolysis and sensory quality of Peshawari cheese compared to calf rennet

The worldwide increase in cheese consumption combined with a scarcity of rennet as well as ethical concerns have resulted in a global interest for natural milk coagulants from plant sources. In this study, the influence of ginger protease in comparison to calf rennet on the physicochemical, microbiological, and sensory characteristics of Peshawari cheese manufactured from cow’s milk was examined. For most of the physicochemical parameters (fat, protein, lactose, acidity, pH), and the main groups of microorganisms (total viable, enterobacteria, Lactobacilli, and molds and yeasts) investigated, no significant (P > 0.05) differences were observed between the two cheeses made by using different coagulants. However, significantly lower (P < 0.05) levels of moisture and higher levels of soluble nitrogen were observed in the cheese produced by ginger protease compared to that made using calf rennet. The main sensory attributes (appearance, body texture, and flavor) were significantly enhanced (P < 0.05) in Peshawari cheese prepared with ginger protease. Importantly, no bitterness was noted by the sensory panel in the Peshawari cheese made with ginger protease. The results reveal that the ginger protease may have potential application for the manufacture of Peshawari cheese.

Ginger rhizome as a potential source of milk coagulating cysteine protease

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS–PAGE, and exhibited a p I of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg 2+ and Cu 2+. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α s-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, p I, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.

Effects of Ginger Proteases on Meat Collagen

Effects of Ginger Proteases on Meat Collagen

Purification of ginger protease using affinity chromatography and chromatofocusing

Purification of ginger protease using affinity chromatography and chromatofocusing

Characterization of ginger proteases and their potential as a rennin replacement

AbstractBACKGROUND: Ginger rhizome (Zingiber officinale Roscoe) contains ginger proteases and has proteolytic activity. Ginger proteases have been used for tenderizing meat but rarely for milk clotting. The purpose of this study was to purify ginger proteases and to research their biochemical characteristics.RESULTS: The milk clotting activity (MCA) and proteolytic activity (PA) of the proteases was stable after storage at 4 °C for 24 h. The MCA and PA of fresh ginger juice with 0.2% L‐ascorbic acid remained stable for 6 days at 4 °C. When under storage at −80 °C for 2 months, the MCA and PA of the fresh ginger juice and acetone precipitate were still high. Two peaks with protease activity were purified from a DEAE FF ion‐exchange column; the specific activity (units mg−1 protein) of the MCA (MCSA) and PA (PSA) for the first peak was significantly higher than the second peak (P &lt; 0.05). The protease activity of the ginger proteases was significantly inhibited by E‐64, leupeptin, and iodoacetic acid. Zymography results showed that two protease fractions purified from ginger juice with 62 and 82 kDa had a higher PA against α‐ and β‐casein than against κ‐casein.CONCLUSION: The ascorbic acid addition significantly stabilized the MCA and PA of ginger proteases. The protease inhibition test suggested that ginger proteases belonged to the cysteine type. The biochemical characteristics of ginger protease described in this paper can provide useful information for making new milk curd products. Copyright © 2009 Society of Chemical Industry

Plant collagenase: Unique collagenolytic activity of cysteine proteases from ginger

Two cysteine proteases, GP2 and GP3, have been isolated from ginger rhizomes ( Zingiber officinale). GP2 is virtually identical to a previously identified ginger protease GPII [K.H. Choi, and R.A. Laursen, Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale, Eur. J. Biochem. 267 (2000) 1516–1526.], and cleaves native type I collagen at multiple discrete sites, which are in the interior of the triple helical region of this molecule. In reaction with proline-containing peptides GP2 shows preference for Pro in the P2 position, and at least 10-fold higher efficiency of hydrolysis than papain. Comparison of models of GP2 and GP3 with the crystal structure of papain shows that the three enzymes have different S2 pocket structures. The S2 pocket in GP2 and GP3 is half the size of that of papain. GP2 is the only reported plant cysteine protease with a demonstrated ability to hydrolyse native collagen. The results support a role for ginger proteases as an alternative to papain, in commercial applications such as meat tenderization, where collagen is the target substrate.

Stabilization and Partial Purification of a Protease from Ginger Rhizome (Zingiber offinale Roscoe)

ABSTRACT: Ginger protease (GP) or zingibain is of interest as a meat tenderizing agent. The objective of this research was to investigate food-compatible methods for stabilizing GP during storage or enzyme fractionation. Crude GP extracted from fresh ginger had a half-life (t1/2) of 2.1 (±0.16) d at 5°C decreasing to 20 min at 30°C. Addition of ascorbate (0.2% w/v) increased the t1/2 for GP from 2 to 20 d at 5°C. Dithiothreitol or Ethylenediaminetetraacetic acid (EDTA) had no effect on GP stability. Acetone powder preparations from ginger yielded GP with t1/2 of 18 mo at 5°C. Crude GP extracted from acetone powder was sufficiently stabilized to allow fractionation by ion exchange chromatography without the addition of toxic or expensive additives. GP was partially purified 252-fold with a recovery of 61%. The nomimal molecular weight of GP was 34.8 kDa compared with 25.1 kDa for papain. This work shows that the stability of GP can be greatly improved, increasing its attractiveness as a commercial product. Some possible routes of GP deactivation and stabilization are discussed.

Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale.

The ginger proteases (GP-I and GP-II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P2 position. The complete amino-acid sequence of GP-II, a glycoprotein containing 221 amino acids, and about 98% that of GP-I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine-histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is approximately 50%. GP-II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision-induced dissociation MS showed that both sites were occupied by the glycans (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)3(Xyl)1(Fuc)1(GlcNAc)3, in a ratio of approximately 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man1-->6(Man1-->3) (Xyl1-->2)Man1-->4GlcNAc1-->4(Fuc1-->3)GlcNAc for the major form, with an additional N-acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.

Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing

Ginger proteases in ginger rhizome (Zingiber officinale roscoe) were extrected from the ginger acetone powder and purified on DEAE-Sepharose and Sephadex G-75 columns. Before the purification, excess p-chloromercuribenzoate was added to the enzymes to prevent their autodigestion. The mercuribenzoate-proteases were further purified and fractionated by isoelectric focusing in Ampholine of pH 3–10 or pH 4–6. The proteases were fractionated into three components by the isoelectric focusing, having p I value of 4.5, 4.6 and 4.8 respectively. All these proteases had a molecular mass of 29 000 as measured by SDS- polyacrylamide gel electrophoresis and by TSK G2000SW XL gel chromatography. The Ampholine in the purified enzymes can quickly be removed by the gel chromatography of TSK G2000SW. Some divalent metal ions, such as Hg 2+, Cu 2+, Cd 2+ and Zn 2+, strongly inhibited these purified enzymes.

GINGER RHIZOME: A NEW SOURCE OF PROTEOLYTIC ENZYME

The proteolytic activity of ginger rhizome was studied with bovine serum albumin (BSA), collagen and actomyosin as substrates. A semipurified, powdered enzyme preparation was prepared by buffer extraction of an acetone powder of ginger rhizome and subsequent acetone precipitation of the proteolytic principle from the buffer extract. With 3% BSA as substrate, a relatively high proteolytic activity occurred over a pH range of 4.5–6.0, with an optimum pH of 5.0. The optimum temperature for proteolysis of BSA was 60°C during a 10 min reaction time, with rapid denaturation of the enzyme occurring at 70° C. NaCl in cone up to 10% produced about a 20 and 50% reduction in proteolysis of collagen and BSA, respectively. The ginger protease was protected by dithiothreitol during extraction and reaction, indicating the involvement of −SH groups at the active site. The analyses of soluble peptide amino acids or terminal amino acids suggest that the proteolysis of collagen is many fold greater than that of actomyosin. The combined proteolysis of these two muscle protein fractions by the ginger protease resulted in significantly more tender meat. According to conventional nomenclature, “Zingibain” is the proper name for this proteolytically active principle in Zingiber officinale roscoe or ginger rhizome which is commonly referred to as gingerroot. For meat applications, a possible advantage of zingibain over papain and ficin is the greater proteolysis of collagen in comparison to actomyosin. When compared to reported values for bromelain, zingibain has a higher optimum activity temperature, which is desirable in some applications