scholarly journals N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

2015 ◽  
Vol 82 (4) ◽  
pp. 1004-1014 ◽  
Author(s):  
Canfang Niu ◽  
Huiying Luo ◽  
Pengjun Shi ◽  
Huoqing Huang ◽  
Yaru Wang ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Biochemical Characterization ◽  
Animal Feed ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Acidic Ph ◽  
Cleavage Sites ◽  
Ph Optimum ◽  
Content Type ◽  
Histidine Acid Phosphatase

ABSTRACTN-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases fromYersinia kristensenii(YkAPPA) andYersinia rohdei(YrAPPA), each having anN-glycosylation motif, and one pepsin-sensitive HAP phytase fromYersinia enterocolitica(YeAPPA) that lacked anN-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering theN-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed inPichia pastorisfor biochemical characterization. Compared with those of theN-glycosylation site deletion mutants andN-deglycosylated enzymes, allN-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of theN-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of theN-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced inEscherichia colibut had no effect on the pepsin resistance ofN-glycosylated enzymes produced inP. pastoris. Thus,N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation ofN-glycosylation, for improvement of phytase properties for use in animal feed.

scholarly journals Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Canfang Niu ◽  
Peilong Yang ◽  
Huiying Luo ◽  
Huoqing Huang ◽  
Yaru Wang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Side Chains ◽  
Cleavage Sites ◽  
Corn Meal ◽  
Feed Enzymes ◽  
The Stability ◽  
Phytate Content

Abstract Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3–2.4-fold, improved their stability at 60 °C and pH 1.0–2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content.

scholarly journals Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas

1989 ◽  
Vol 261 (2) ◽  
pp. 601-609 ◽  
Author(s):  
A R Hayman ◽  
M J Warburton ◽  
J A S Pringle ◽  
B Coles ◽  
T J Chambers
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Tartrate Resistant Acid Phosphatase ◽  
Bovine Bone ◽  
Ph Optimum ◽  
Original Tumour

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.

scholarly journals Comparative Biochemical Characterization of Three Exolytic Oligoalginate Lyases from Vibrio splendidus Reveals Complementary Substrate Scope, Temperature, and pH Adaptations

2014 ◽  
Vol 80 (14) ◽  
pp. 4207-4214 ◽  
Author(s):  
Sujit Sadashiv Jagtap ◽  
Jan-Hendrik Hehemann ◽  
Martin F. Polz ◽  
Jung-Kul Lee ◽  
Huimin Zhao
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Vibrio Splendidus ◽  
Marine Microbes ◽  
Ph Optimum ◽  
Brown Seaweeds ◽  
Content Type ◽  
Alginate Lyases

ABSTRACTMarine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exo-type oligoalginate lyases (OalA, OalB, and OalC) fromVibrio splendidus12B01 (12B01), a marine bacterioplankton species. OalA was most active at 16°C, had a pH optimum of 6.5, and displayed activities toward poly-β-d-mannuronate [poly(M)] and poly-α-l-guluronate [poly(G)], indicating that it is a bifunctional enzyme. OalB and OalC were most active at 30 and 35°C, had pH optima of 7.0 and 7.5, and degraded poly(M·G) and poly(M), respectively. Detailed kinetic analyses of oligoalginate lyases with poly(G), poly(M), and poly(M·G) and sodium alginate as substrates demonstrated that OalA and OalC preferred poly(M), whereas OalB preferred poly(M·G). The catalytic efficiency (kcat/Km) of OalA against poly(M) increased with decreasing size of the substrate. OalA showedkcat/Kmfrom 2,130 mg−1ml s−1for the trisaccharide to 224 mg−1ml s−1for larger oligomers of ∼50 residues, and 50.5 mg−1ml s−1for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.

scholarly journals Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

2013 ◽  
Vol 79 (13) ◽  
pp. 4072-4077 ◽  
Author(s):  
Xuguo Duan ◽  
Jian Chen ◽  
Jing Wu
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Optimum ◽  
Widespread Application ◽  
Content Type ◽  
Major Factors

ABSTRACTPullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase fromBacillus deramificanswere selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that theKmvalues for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and theKcat/Kmvalues increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

Immunological and biochemical evidence for identity of tartrate-resistant isoenzymes of acid phosphatases from human serum and tissues.

1980 ◽  
Vol 26 (3) ◽  
pp. 420-422 ◽  
Author(s):  
K W Lam ◽  
P Lee ◽  
C Y Li ◽  
L T Yam
Keyword(s):  
Acid Phosphatase ◽  
Substrate Specificity ◽  
Human Serum ◽  
Giant Cell ◽  
Bone Tumor ◽  
Biochemical Characterization ◽  
Acid Phosphatases ◽  
Ph Optimum ◽  
Human Spleen ◽  
Biochemical Evidence

Abstract We purified acid phosphatase isoenzyme 5b from a human spleen affected by leukemic reticuloendotheliosis and used it to produce a specific antiserum. The antiserum was used to show complete immunological identity among isoenzymes 5a and 5b in human serum, and 5b isolated from a giant-cell bone tumor and from the spleen of a case of Hodgkin's disease. Acid phosphatase 5b in a giant-cell bone tumor was isolated for biochemical characterization. Its pH optimum and substrate specificity were very similar to those of isoenzyme 5b from human spleen.

Immunological and biochemical evidence for identity of tartrate-resistant isoenzymes of acid phosphatases from human serum and tissues.

1980 ◽  
Vol 26 (3) ◽  
pp. 420-422 ◽  
Author(s):  
K W Lam ◽  
P Lee ◽  
C Y Li ◽  
L T Yam
Keyword(s):  
Acid Phosphatase ◽  
Substrate Specificity ◽  
Human Serum ◽  
Giant Cell ◽  
Bone Tumor ◽  
Biochemical Characterization ◽  
Acid Phosphatases ◽  
Ph Optimum ◽  
Human Spleen ◽  
Biochemical Evidence

Abstract We purified acid phosphatase isoenzyme 5b from a human spleen affected by leukemic reticuloendotheliosis and used it to produce a specific antiserum. The antiserum was used to show complete immunological identity among isoenzymes 5a and 5b in human serum, and 5b isolated from a giant-cell bone tumor and from the spleen of a case of Hodgkin's disease. Acid phosphatase 5b in a giant-cell bone tumor was isolated for biochemical characterization. Its pH optimum and substrate specificity were very similar to those of isoenzyme 5b from human spleen.

scholarly journals Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-d-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-d-Rhamnosyltransferase WbpZ

2015 ◽  
Vol 197 (12) ◽  
pp. 2012-2019 ◽  
Author(s):  
Shuo Wang ◽  
Youai Hao ◽  
Joseph S. Lam ◽  
Jason Z. Vlahakis ◽  
Walter A. Szarek ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Metal Ion ◽  
Specific Antigen ◽  
Site Directed Mutagenesis ◽  
Significant Activity ◽  
Polysaccharide Antigen ◽  
Ph Optimum ◽  
Content Type ◽  
Enzymatic Function ◽  
The Common

ABSTRACTThe opportunistic pathogenPseudomonas aeruginosaproduces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer ofd-rhamnose (d-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes,wbpX,wbpY, andwbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of thewbpZgene product, we chemically synthesized the donor substrate GDP-d-Rha and enzymatically synthesized GDP-d-[3H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred oned-Rha residue from GDP-d-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferringd-mannose (d-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-d-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-rhamnosyltransferase that has significant activity of GDP-d-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs ofwbpZ(D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZknockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of ad-Rha-transferase and a critical step in the development of CPA synthesis inhibitors.IMPORTANCEThis is the first characterization of ad-rhamnosyltransferase and shows that it is essential inPseudomonas aeruginosafor the synthesis of the common polysaccharide antigen.

scholarly journals Modulatory effect of divalent metal cations on the phosphotyrosine activity of the frog liver acid phosphatase.

1999 ◽  
Vol 46 (1) ◽  
pp. 217-221
Author(s):  
A Szalewicz ◽  
B Strzelczyk ◽  
A Kubicz
Keyword(s):  
Acid Phosphatase ◽  
Metal Cations ◽  
Divalent Metal ◽  
Acidic Ph ◽  
Ph Optimum ◽  
Divalent Metal Cations

Frog liver acid phosphatase hydrolyzes phosphotyrosine at acidic pH optimum. Mn2+, Ca2+ and Mg2+ (but not Zn2+) ions modulate this activity by shifting its pH optimum to physiological pH. This effect is not observed when p-nitrophenylphosphate is used as a substrate. Phosphoserine and phosphothreonine are not hydrolyzed under the same conditions.

scholarly journals Engineering of Phytase for Improved Activity at Low pH

2002 ◽  
Vol 68 (4) ◽  
pp. 1907-1913 ◽  
Author(s):  
Andrea Tomschy ◽  
Roland Brugger ◽  
Martin Lehmann ◽  
Allan Svendsen ◽  
Kurt Vogel ◽  
...  
Keyword(s):  
Animal Feed ◽  
Specific Activity ◽  
Three Dimensional ◽  
Industrial Applications ◽  
Low Ph ◽  
Site Directed Mutagenesis ◽  
Single Mutation ◽  
Sequence Alignments ◽  
Ph Optimum ◽  
Double Mutation

ABSTRACT For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.

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