Fanghua Wang

Biochemical Properties of Recombinant Leucine Aminopeptidase II from Bacillus stearothermophilus and Potential Applications in the Hydrolysis of Chinese Anchovy (Engraulis japonicus) Proteins

The effects of various factors on the activity and conformation of recombinant leucine aminopeptidase II (rLAP II) from Bacillus stearothermophilus and its potential utilization in the hydrolysis of anchovy proteins were investigated. The optimal temperature and pH of rLAP II were 55 °C and 8.0 in phosphate buffer, and its activity was strongly stimulated by Co(2+). Conformational studies indicated that maintaining the α-helical structure had a critical effect on rLAP II activity. rLAP II was used to hydrolyze anchovy proteins, and it exhibited high specificity for peptides with molecular weight between 6000 and 1000 Da and positive coordination with endogenous enzymes and commercial Flavourzyme. Its use will enhance protein hydrolysis in species of aquatic animals. rLAP II could potentially be used to remove bitterness in the protein hydrolysis industry.

Efficient purification of native recombinant proteins using proteases immobilized on cellulose

Cellulose binding domain (CBD) fusion protease was employed to digest the CBD fusion protein. After cleavage, the tag free target proteins can be separated from the CBD tag and CBD fusion protease which still adsorbed to the cellulose by centrifugation. The green fluorescent protein was efficiently purified using this method.

Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223

Experiments were carried out to investigate the effects of various factors on the activity and conformation of recombinant leucine aminopeptidase of Bacillus kaustophilus CCRC 11223 (BkLAP) and potential utilization of BkLAP in the hydrolysis of anchovy protein. Optimal temperature and pH of BkLAP were 70 °C and 8.0 in potassium-phosphate buffer, respectively, and the activity was strongly stimulated by Ni2+, followed by Mn2+ and Co2+. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of BkLAP to different extents and further induce the changes in enzymatic activity. The secondary structure of BkLAP was slightly modified by Ni2+ at the concentration of 1×10−4 M, however, significant changes on the secondary structures of the enzyme were observed when Hg2+ was added to the concentration of 1×10−4 M. The potential application of BkLAP was evaluated through combination with the commercial or endogenous enzyme to hydrolysis the anchovy protein. Results showed that combining the BkLAP with other enzymes could significantly increase the degree of hydrolysis and amino acid component of hydrolysate. In this regard, BkLAP is a potential enzyme that can be used in the protein hydrolysate industry.

Recombinant Lipase from Gibberella zeae Exhibits Broad Substrate Specificity: A Comparative Study on Emulsified and Monomolecular Substrate

Using the classical emulsified system and the monomolecular film technique, the substrate specificity of recombinant Gibberella zeae lipase (rGZEL) that originates from Gibberella zeae was characterized in detail. Under the emulsified reaction system, both phospholipase and glycolipid hydrolytic activities were observed, except for the predominant lipase activity. The optimum conditions for different activity exhibition were also determined. Compared with its lipase activity, a little higher ratio of glycolipid hydrolytic activity (0.06) than phospholipase activity (0.02) was found. rGZEL preferred medium chain-length triglycerides, while lower activity was found for the longer-chain triglyceride. Using the monomolecular film technique, we found that the preference order of rGZEL to different phospholipids was 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS) > 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (PG) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) > l-α-phosphatidylinositol (PI) > cardiolipin (CL) > 3-sn-phosphatidic acid sodium salt (PA) > l-α-phosphatidylethanolamine (PE), while no hydrolytic activity was detected for sphingomyelin (SM). Moreover, rGZEL showed higher galactolipase activity on 1,2-distearoyimonoglactosylglyceride (MGDG). A kinetic study on the stereo- and regioselectivity of rGZEL was also performed by using three pairs of pseudodiglyceride enantiomers (DDGs). rGZEL presented higher preference for distal DDG enantiomers than adjacent ester groups, however, no hydrolytic activity to the sn-2 position of diglyceride analogs was found. Furthermore, rGZEL preferred the R configuration of DDG enantiomers. Molecular docking results were in concordance with in vitro tests.

Preparation of anchovy (Engraulis japonicus) protein hydrolysates with high free radical-scavenging activity using endogenous and commercial enzymes

Anchovy protein hydrolysates with high free radical-scavenging activity were prepared by endogenous and commercial enzymes. Various hydrolytic factors (commercial protease composition, protease concentration, temperature, and reaction time) were optimized. Using a single-factor experiment, three commercial proteases (Protamex, Flavourzyme 500 MG, and Alcalase 2.4 L) were selected for further optimization using a simplex lattice design. The optimum composition of Protamex:Flavourzyme 500 MG:Alcalase 2.4 L was found to be 1.1:1.0:0.9. The hydrolytic conditions (commercial protease concentration, temperature, and reaction time) for the optimum protease composition were optimized using a Box-Behnken design. The optimum hydrolytic conditions were as follows: total commercial protease concentration of 3.27%, pH of 7.5, temperature of 55.4℃, and reaction time of 2.7 h. Under these conditions, hydrolysate with a 1, 1-diphenyl-2-picryhydrazyl scavenging activity of 84.7% was obtained. Meanwhile, a degree of hydrolysis of 33.2% and high protein nitrogen recovery of 87.5% were achieved. The amino acid composition of the hydrolysates demonstrated that they have high nutritional value, thereby suggesting that the hydrolysates have potential to be used as raw material for functional food.

Expression and Characterization of a Novel Glycerophosphodiester Phosphodiesterase from Pyrococcus furiosus DSM 3638 That Possesses Lysophospholipase D Activity

Glycerophosphodiester phosphodiesterases (GDPD) are enzymes which degrade various glycerophosphodiesters to produce glycerol-3-phosphate and the corresponding alcohol moiety. Apart from this, a very interesting finding is that this enzyme could be used in the degradation of toxic organophosphorus esters, which has resulted in much attention on the biochemical and application research of GDPDs. In the present study, a novel GDPD from Pyrococcus furiosus DSM 3638 (pfGDPD) was successfully expressed in Escherichia coli and biochemically characterized. This enzyme hydrolyzed bis(p-nitrophenyl) phosphate, one substrate analogue of organophosphorus diester, with an optimal reaction temperature 55 °C and pH 8.5. The activity of pfGDPD was strongly dependent on existing of bivalent cations. It was strongly stimulated by Mn2+ ions, next was Co2+ and Ni2+ ions. Further investigations were conducted on its substrate selectivity towards different phospholipids. The results indicated that except of glycerophosphorylcholine (GPC), this enzyme also possessed lysophospholipase D activity toward both sn1-lysophosphatidylcholine (1-LPC) and sn2-lysophosphatidylcholine (2-LPC). Higher activity was found for 1-LPC than 2-LPC; however, no hydrolytic activity was found for phosphatidylcholine (PC). Molecular docking based on the 3D-modeled structure of pfGDPD was conducted in order to provide a structural foundation for the substrate selectivity.

Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus

The effects of N-terminal (1–34 amino acids) and C-terminal (434–487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor “a” indicated that the loop structure (1–25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26–34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434–487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434–469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.

Function of C-terminal peptides on enzymatic and interfacial adsorption properties of lipase from Gibberella zeae

BACKGROUND The crystal structure of lipase from Gibberella zeae (GZEL) indicates that its C-terminal extension is composed of a loop and a α-helix. This structure is unique, possibly providing novel evidence on lipase mechanisms. METHODS Two C-terminally truncated mutants (GZEL-Δ(α-helix) and GZEL-Δ(α-helix+loop)) were constructed. The role of these secondary structure segments on enzymatic activities and interfacial binding properties of GZEL was investigated by using conventional pH-stat method and monomolecular film techniques. In addition, inactive variants (Ser144Ala) of wild-type GZEL and two truncated mutants were constructed and produced specifically for interfacial binding experiments. RESULTS Compared to the wild-type GZEL, lipase and phospholipase activities were significantly decreased in the two mutants. Deletion of the α-helix had great influence on the lipase activity of GZEL, resulting in residual 7.3% activity; the additional deletion of the loop led to 8.1% lipase activity. As for the phospholipase function, residual activities of 63.0% and 35.4% were maintained for GZEL-Δ(α-helix) and GZEL-Δ(α-helix+loop), respectively. Findings obtained with monomolecular film experiments further indicated that the reduction in phospholipase activity occurred with the anionic phospholipid as substrate, but was not seen with zwitterionic phospholipid. Results of the maximum insertion pressure, synergy factor and binding kinetic parameters documented that the α-helix structure of GZEL strongly influence the binding and insertion of enzyme to the phospholipid monolayer. Moreover, the interfacial binding function of α-helix was partly conformed by connecting to the C-terminal of Aspergillus oryzae lipase. GENERAL SIGNIFICANCE Our results provide important information on the understanding of the structure-function relationship of GZEL.