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Insights into the stereospecificity of the d-specific dehalogenase from Rhizobium sp. RC1 toward d- and l-2-chloropropionate

Sudi IY, Hamid AA, Shamsir MS, Jamaluddin H, Wahab RA, Huyop F

Biotechnology, biotechnological equipment, 2014 Jul 04;28(4):608-615.
PMID: 26740767

Halogenated compounds are recalcitrant environmental pollutants prevalent in agricultural fields, waste waters and industrial by-products, but they can be degraded by dehalogenase-containing microbes. Notably, 2-haloalkanoic acid dehalogenases are employed to resolve optically active chloropropionates, as exemplified by the d-specific dehalogenase from Rhizobium sp. RCI (DehD), which acts on d-2-chloropropionate but not on its l-enantiomer. The catalytic residues of this dehalogenase responsible for its affinity toward d-2-chloropropionate have not been experimentally determined, although its three-dimensional crystal structure has been solved. For this study, we performed in silico docking and molecular dynamic simulations of complexes formed by this dehalogenase and d- or l-2-chloropropionate. Arg134 of the enzyme plays the key role in the stereospecific binding and Arg16 is in a position that would allow it to activate a water molecule for hydrolytic attack on the d-2-chloropropionate chiral carbon for release of the halide ion to yield l-2-hydroxypropionate. We propose that within the DehD active site, the NH group of Arg134 can form a hydrogen bond with the carboxylate of d-2-chloropropionate with a strength of ∼4 kcal/mol that may act as an acid-base catalyst, whereas, when l-2-chloropropionate is present, this bond cannot be formed. The significance of the present work is vital for rational design of this dehalogenase in order to confirm the involvement of Arg16 and Arg134 residues implicated in hydrolysis and binding of d-2-chloropropionate in the active site of d-specific dehalogenase from Rhizobium sp. RC1.

Matched MeSH terms: Hydrolases
Fulltext Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

Abusham RA, Rahman RN, Salleh AB, Basri M

Microb Cell Fact, 2009 Apr 09;8:20.
PMID: 19356254 DOI: 10.1186/1475-2859-8-20

BACKGROUND: Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia.
RESULTS: A thermostable organic solvent-tolerant protease producer had been identified as Bacillus subtilis strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological characteristics and biochemical properties. The production of the thermostable organic solvent-tolerant protease was optimized by varying various physical culture conditions. Inoculation with 5.0% (v/v) of (AB600 = 0.5) inoculum size, in a culture medium (pH 7.0) and incubated for 24 h at 37 degrees C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth and production of protease (444.7 U/ml; 4042.4 U/mg). The Rand protease was not only stable in the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and 80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60 degrees C, respectively.
CONCLUSION: Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic solvent. This unique property makes it attractive and useful to be used in industrial applications.

Matched MeSH terms: Peptide Hydrolases
Fulltext NS3 protease from flavivirus as a target for designing antiviral inhibitors against dengue virus

Natarajan S

Genet Mol Biol, 2010 Apr;33(2):214-9.
PMID: 21637471 DOI: 10.1590/S1415-47572010000200002

The development of novel therapeutic agents is essential for combating the increasing number of cases of dengue fever in endemic countries and among a large number of travelers from non-endemic countries. The dengue virus has three structural proteins and seven non-structural (NS) proteins. NS3 is a multifunctional protein with an N-terminal protease domain (NS3pro) that is responsible for proteolytic processing of the viral polyprotein, and a C-terminal region that contains an RNA triphosphatase, RNA helicase and RNA-stimulated NTPase domain that are essential for RNA replication. The serine protease domain of NS3 plays a central role in the replicative cycle of dengue virus. This review discusses the recent structural and biological studies on the NS2B-NS3 protease-helicase and considers the prospects for the development of small molecules as antiviral drugs to target this fascinating, multifunctional protein.

Matched MeSH terms: Acid Anhydride Hydrolases
Seeking key microorganisms for enhancing methane production in anaerobic digestion of waste sewage sludge

Mustapha NA, Hu A, Yu CP, Sharuddin SS, Ramli N, Shirai Y, et al.

Appl Microbiol Biotechnol, 2018 Jun;102(12):5323-5334.
PMID: 29696331 DOI: 10.1007/s00253-018-9003-8

Efficient approaches for the utilization of waste sewage sludge have been widely studied. One of them is to use it for the bioenergy production, specifically methane gas which is well-known to be driven by complex bacterial interactions during the anaerobic digestion process. Therefore, it is important to understand not only microorganisms for producing methane but also those for controlling or regulating the process. In this study, azithromycin analogs belonging to macrolide, ketolide, and lincosamide groups were applied to investigate the mechanisms and dynamics of bacterial community in waste sewage sludge for methane production. The stages of anaerobic digestion process were evaluated by measuring the production of intermediate substrates, such as protease activity, organic acids, the quantification of bacteria and archaea, and its community dynamics. All azithromycin analogs used in this study achieved a high methane production compared to the control sample without any antibiotic due to the efficient hydrolysis process and the presence of important fermentative bacteria and archaea responsible in the methanogenesis stage. The key microorganisms contributing to the methane production may be Clostridia, Cladilinea, Planctomycetes, and Alphaproteobacteria as an accelerator whereas Nitrosomonadaceae and Nitrospiraceae may be suppressors for methane production. In conclusion, the utilization of antibiotic analogs of macrolide, ketolide, and lincosamide groups has a promising ability in finding the essential microorganisms and improving the methane production using waste sewage sludge.

Matched MeSH terms: Peptide Hydrolases
Fulltext Effective production of keratinase by gellan gum-immobilised Alcaligenes sp. AQ05-001 using heavy metal-free and polluted feather wastes as substrates

Yusuf I, Ahmad SA, Phang LY, Yasid NA, Shukor MY

3 Biotech, 2019 Jan;9(1):32.
PMID: 30622870 DOI: 10.1007/s13205-018-1555-x

The ability of gellan gum-immobilised cells of the heavy metal-tolerant bacterium Alcaligenes sp. AQ05-001 to utilise both heavy metal-free and heavy metal-polluted feathers (HMPFs) as substrates to produce keratinase enzyme was studied. Optimisation of the media pH, incubation temperature and immobilisation parameters (bead size, bead number, gellan gum concentration) was determined for the best possible production of keratinase using the one-factor-at-a-time technique. The results showed that the immobilised cells could tolerate a broader range of heavy metal concentrations and produced higher keratinase activity at a gellan gum concentration of 0.8% (w/v), a bead size of 3 mm, bead number of 250, pH of 8 and temperature of 30 °C. The entrapped bacterium was used repeatedly for ten cycles to produce keratinase using feathers polluted with 25 ppm of Co, Cu and Ag as substrates without the need for desorption. However, its inability to tolerate/utilise feathers polluted with Hg, Pb, and Zn above 5 ppm, and Ag and Cd above 10 ppm resulted in a considerable decrease in keratinase production. Furthermore, the immobilised cells could retain approximately 95% of their keratinase production capacity when 5 ppm of Co, Cu, and Ag, and 10 ppm of As and Cd were used to pollute feathers. When the feathers containing a mixture of Ag, Co, and Cu at 25 ppm each and Hg, Ni, Pb, and Zn at 5 ppm each were used as substrates, the immobilised cells maintained their operational stability and biological activity (keratinase production) at the end of 3rd and 4th cycles, respectively. The study indicates that HMPF can be effectively utilised as a substrate by the immobilised-cell system of Alcaligenes sp. AQ05-001 for the semi-continuous production of keratinase enzyme.

Matched MeSH terms: Peptide Hydrolases
Fulltext Reassessment of the catalytic activity and substrate specificity of fkbp35 from plasmodium knowlesi using protease-free assay

Cahyo Budiman, Carlmond Goh Kah Wun, Lee, Ping Chin, Rafida Razali, Thean, Chor Leow

Borneo International Journal of Biotechnology, 2020;1(1):125-143.
MyJurnal

FK506-binding protein35 of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) and is considered as a promising avenue of antimalarial drug target development. This protein is organized into the N-terminal domain responsible for PPIase catalytic activity followed and the tetratricopeptide repeat domain for its dimerization. The protease-coupling and protease-free assays are known to be the common methods for investigating the catalytic properties of PPIase. Earlier, the protease-coupling assay was used to confirm the catalytic activity of Pk-FKBP35 in accelerating cis-trans isomerization of the peptide substrate. This report is aimed to re-assess the catalytic and substrate specificity of Pk-FKBP35 using an alternative method of a protease-free assay. The result indicated that while Pk-FKBP35 theoretically contained many possible cleavage sites of chymotrypsin, experimentally, the catalytic domain was relatively stable from chymotrypsin. Furthermore, under protease-free assay, Pk-FKBP35 also demonstrated remarkable PPIase catalytic activity with kcat/KM of 4.5 + 0.13 × 105 M−1 s−1, while the kcat/KM of active site mutant of D55A is 0.81 + 0.05 × 105 M−1 s−1. These values were considered comparable to kcat/KM obtained from the protease-coupling assay. Interestingly, the substrate specificities of Pk-FKBP35 obtained from both methods are also similar, with the preference of Pk-FKBP35 towards Xaa at P1 position was Leu>Phe>Lys>Trp>Val>Ile>His>Asp>Ala>Gln>Glu. Altogether, we proposed that protease-free and protease-coupling assays arereliable for Pk-FKBP35.

Matched MeSH terms: Peptide Hydrolases
Fulltext Optimizing the acceleration of Cheddar cheese ripening using response surface methodology by microbial protease without altering its quality features

Alhelli AM, Mohammed NK, Khalil ES, Hussin ASM

AMB Express, 2021 Mar 22;11(1):45.
PMID: 33751265 DOI: 10.1186/s13568-021-01205-9

Cheddar cheese proteolysis were accelerated employing Penicillium candidum PCA1/TT031 protease into cheese curd. In the present study, several of the significant factors such as protease purification factor (PF), protease concentration and ripening time were optimized via the response surface methodology (RSM). The ideal accelerated Cheddar cheese environment consisted of 3.12 PF, 0.01% (v/v) protease concentration and 0.6/3 months ripening time at 10 °C. The RSM models was verified to be the most proper methodology for the maintain of chosen Cheddar cheese. Under this experimental environment, the pH, acid degree value (ADV), moisture, water activity (aw), soluble nitrogen (SN)%, fat and overall acceptability were found to be 5.4, 6.6, 35%, 0.9348, 18.8%, 34% and 13.6, respectively of ideal Cheddar cheese. Furthermore, the predicted and experimental results were in significant agreement, which confirmed the validity and reliability of the suggested method. In spite of the difference between the ideal and commercial Cheddar cheese in the concentration of some of amino acids and free fatty acids, the sensory evaluation did not show any significant difference in aroma profile between them.

Matched MeSH terms: Peptide Hydrolases
Fulltext Separation and identification of bromelain-generated antibacterial peptides from Actinopyga lecanora

Ghanbari R, Ebrahimpour A

Food Sci Biotechnol, 2018 Apr;27(2):591-598.
PMID: 30263784 DOI: 10.1007/s10068-017-0267-z

Actinopyga lecanora, as a rich protein source was hydrolysed to generate antibacterial bioactive peptides using different proteolytic enzymes. Bromelain hydrolysate, after 1 h hydrolysis, exhibited the highestantibacterial activities against Pseudomonas aeruginosa, Pseudomonas sp., Escherichia coli and Staphylococcus aureus. Two dimensional fractionation strategies, using a semi-preparative RP-HPLC and an isoelectric-focusing electrophoresis, were applied for peptide profiling. Furthermore, UPLC-QTOF-MS was used for peptides identification; 12 peptide sequences were successfully identified. The antibacterial activity of purified peptides from A. lecanora on P. aeruginosa, Pseudomonas sp., E. coli and S. aureus was investigated. These identified peptides exhibited growth inhibition against P. aeruginosa, Pseudomonas sp., E. coli and S. aureus with values ranging from 18.80 to 75.30%. These results revealed that the A. lecanora would be used as an economical protein source for the production of high value antibacterial bioactive peptides.

Matched MeSH terms: Peptide Hydrolases
Fulltext Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOG(R)1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens

Izawati AM, Masani MY, Ismanizan I, Parveez GK

Front Plant Sci, 2015;6:727.
PMID: 26442041 DOI: 10.3389/fpls.2015.00727

DOG(R)1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOG(R)1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l(-1) 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOG(R)1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm.

Matched MeSH terms: Phosphoric Monoester Hydrolases
Fulltext Enhancing food waste biodegradation rate in a food waste biodigester with the synergistic action of hydrolase-producing Bacillus paralicheniformis GRA2 and Bacillus velezensis TAP5 co-culture inoculation

Roslan MAM, Jefri NQUA, Ramlee N, Rahman NAA, Chong NHH, Bunawan H, et al.

Saudi J Biol Sci, 2021 May;28(5):3001-3012.
PMID: 34012331 DOI: 10.1016/j.sjbs.2021.02.041

Food waste (FW) minimization at the source by using food waste biodigester (FWBs) has a vast potential to lower down the impact of increasing organic fraction in municipal solid waste generation. To this end, this research sought to check the performance of locally isolated hydrolase-producing bacteria (HPB) to improve food waste biodegradation rate. Two under-explored HPB identified as Bacillus paralicheniformis GRA2 and Bacillus velezensis TAP5 were able to produce maximum amylase, cellulase, protease and lipase activities, and demonstrated a significant hydrolase synergy in co-culture fermentation. In vitro biodegradation analysis of both autoclaved and non-autoclaved FW revealed that the HPB inoculation was effective to degrade total solids (>62%), protein (>19%), total fat (>51), total sugar (>86%), reducing sugar (>38%) and starch (>50%) after 8-day incubation. All co-culture treatments were recorded superior to the respective monocultures and the uninoculated control. The results of FW biodegradation using batch-biodigester trial indicated that the 1500 mL and 1000 mL inoculum size of HPB inoculant reached a plateau on the 4th day, with gross biodegradation percentage (GBP) of >85% as compared to control (66.4%). The 1000 mL inoculum was sufficient to achieve the maximum GBP (>90%) of FW after an 8-day biodigestion in a FWB.

Matched MeSH terms: Peptide Hydrolases
Global Transcriptome Analysis of Gracilaria changii (Rhodophyta) in Response to Agarolytic Enzyme and Bacterium

Lim EL, Siow RS, Abdul Rahim R, Ho CL

Mar Biotechnol (NY), 2016 Apr;18(2):189-200.
PMID: 26631182 DOI: 10.1007/s10126-015-9680-6

Many bacterial epiphytes of agar-producing seaweeds secrete agarase that degrade algal cell wall matrix into oligoagars which elicit defense-related responses in the hosts. The molecular defense responses of red seaweeds are largely unknown. In this study, we surveyed the defense-related transcripts of an agarophyte, Gracilaria changii, treated with β-agarase through next generation sequencing (NGS). We also compared the defense responses of seaweed elicited by agarase with those elicited by an agarolytic bacterium isolated from seaweed, by profiling the expression of defense-related genes using quantitative reverse transcription real-time PCR (qRT-PCR). NGS detected a total of 391 differentially expressed genes (DEGs) with a higher abundance (>2-fold change with a p value <0.001) in the agarase-treated transcriptome compared to that of the non-treated G. changii. Among these DEGs were genes related to signaling, bromoperoxidation, heme peroxidation, production of aromatic amino acids, chorismate, and jasmonic acid. On the other hand, the genes encoding a superoxide-generating NADPH oxidase and related to photosynthesis were downregulated. The expression of these DEGs was further corroborated by qRT-PCR results which showed more than 90 % accuracy. A comprehensive analysis of their gene expression profiles between 1 and 24 h post treatments (hpt) revealed that most of the genes analyzed were consistently upregulated or downregulated by both agarase and agarolytic bacterial treatments, indicating that the defense responses induced by both treatments are highly similar except for genes encoding vanadium bromoperoxidase and animal heme peroxidase. Our study has provided the first glimpse of the molecular defense responses of G. changii to agarase and agarolytic bacterial treatments.

Matched MeSH terms: Glycoside Hydrolases
Fulltext Crustacean Proteases and Their Application in Debridement

Perera E, Rodriguez-Viera L, Montero-Alejo V, Perdomo-Morales R

Trop Life Sci Res, 2020 Jul;31(2):187-209.
PMID: 32922675 DOI: 10.21315/tlsr2020.31.2.10

Digestive proteases from marine organisms have been poorly applied to biomedicine. Exceptions are trypsin and other digestive proteases from a few cold-adapted or temperate fish and crustacean species. These enzymes are more efficient than enzymes from microorganism and higher vertebrates that have been used traditionally. However, the biomedical potential of digestive proteases from warm environment species has received less research attention. This review aims to provide an overview of this unrealised biomedical potential, using the debridement application as a paradigm. Debridement is intended to remove nonviable, necrotic and contaminated tissue, as well as fibrin clots, and is a key step in wound treatment. We discuss the physiological role of enzymes in wound healing, the use of exogenous enzymes in debridement, and the limitations of cold-adapted enzymes such as their poor thermal stability. We show that digestive proteases from tropical crustaceans may have advantages over their cold-adapted counterparts for this and similar uses. Differences in thermal stability, auto-proteolytic stability, and susceptibility to proteinase inhibitors are discussed. Furthermore, it is proposed that the feeding behaviour of the source organism may direct the evaluation of enzymes for particular applications, as digestive proteases have evolved to fill a wide variety of feeding habitats, natural substrates, and environmental conditions. We encourage more research on the biomedical application of digestive enzymes from tropical marine crustaceans.

Matched MeSH terms: Peptide Hydrolases
Natural DENV-2 NS2B/NS3 protease inhibitors from Myristica cinnamomea King

Sivasothy Y, Liew SY, Othman MA, Abdul Wahab SM, Hariono M, Mohd Nawi MS, et al.

Trop Biomed, 2021 Jun 01;38(2):79-84.
PMID: 33973577 DOI: 10.47665/tb.38.2.044

The NS2B/NS3 protease is crucial for the pathogenesis of the DENV. Therefore, the inhibition of this protease is considered to be the key strategy for the development of new antiviral drugs. In the present study, malabaricones C (3) and E (4), acylphenols from the fruits of Myristica cinnamomea King, have been respectively identified as moderate (27.33 ± 5.45 μM) and potent (7.55 ± 1.64 μM) DENV-2 NS2B/NS3 protease inhibitors, thus making this the first report on the DENV-2 NS2B/NS3 protease inhibitory activity of acylphenols. Based on the molecular docking studies, compounds 3 and 4 both have π-π interactions with Tyr161. While compound 3 has hydrogen bonding interactions with Gly151, Gly153 and Tyr161, compound 4 however, forms hydrogen bonds with Ser135, Asp129, Phe130 and Ile86 instead. The results from the present study suggests that malabaricones C (3) and E (4) could be employed as lead compounds for the development of new dengue antivirals from natural origin.

Matched MeSH terms: Peptide Hydrolases
Fulltext Development of MTH1-Binding Nucleotide Analogs Based on 7,8-Dihalogenated 7-Deaza-dG Derivatives

Shi H, Ishikawa R, Heh CH, Sasaki S, Taniguchi Y

Int J Mol Sci, 2021 Jan 28;22(3).
PMID: 33525366 DOI: 10.3390/ijms22031274

MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds.

Matched MeSH terms: Phosphoric Monoester Hydrolases/antagonists & inhibitors; Phosphoric Monoester Hydrolases/metabolism; Phosphoric Monoester Hydrolases/chemistry*
Fulltext Efficacy of selected purification techniques for bromelain

Nadzirah, K.Z., Zainal, S., Noriham, A., Normah, I.

International Food Research Journal, 2013;20(1):43-46.
MyJurnal

Bromelain is one of the vegetal proteases found in pineapple plant. It has numerous applications in food and pharmaceuticals. This review discussed different bromelain purification techniques which will assist in determining the effect of processing conditions on the purification efficacy. There are four purification techniques to be discussed, namely; reverse micellar system, aqueous two phase extraction, cation exchange chromatography and ammonium sulphate precipitation. Of the four techniques, cation exchange chromatography had shown the best bromelain purification technique with purification fold of 10.0 followed by reverse micellar system containing CTAB/ isooctane/ hexanol/ butanol, ATPE containing PEG polymer, ammonium sulphate precipitation and ATPE containing PEO-PPO-PEO with purification fold of 5.2, 4.0, 2.81 and 1.25, respectively.

Matched MeSH terms: Peptide Hydrolases
Fulltext Characterization and purification of protease extracted from two maturity stages of ‘noni’ (Morinda citrifolia L.) fruit

Normah Ismail, Nurulain Abd Razak

Scientific Research Journal, 2014;11(2):0-0.
MyJurnal

Protease was extracted from two maturity stages of noni fruits (Morinda citrifolia L.), unripe (stage 1) and ripe (stage 5). The crude extract was partially purified by acetone precipitation method followed by dialysis, gel filtration chromatography and freeze drying. Protein concentrations, proteolytic activity, molecular weight distribution, pH stability, temperature stability and storage efficiency of the resulting protease were evaluated. The unripe and ripe noni fruit contains 0.65 and 0.35% protein, respectively. Molecular weight of the proteases from both stages ranged approximately between 3 to 28 kDa based on the SDS-PAGE results. The optimum activity were at pH 7s and 6, temperatures of 40 and 50°C, respectively for proteases obtained from the unripe and ripe fruit. Analysis from the freeze dried protease indicated that protease from ripe noni fruits had higher protein concentration and specific activity compared to those from unripe fruit. However, it is more sensitive to pH and temperature and less stable during storage as it shows lower proteolytic activity compared to protease from unripe fruit. Based on its high proteolytic activity reaching up to 70.31 U/mg and storage stability (30% lost of activity), noni fruit could be an alternative source of plant protease.

Matched MeSH terms: Peptide Hydrolases
Fulltext In silico design of potentially functional artificial metallo-haloalkane dehalogenase containing catalytic zinc

Ang TF, Salleh AB, Normi YM, Leow TC

3 Biotech, 2018 Jul;8(7):314.
PMID: 30023146 DOI: 10.1007/s13205-018-1333-9

Artificial metalloenzymes are unique as they combine the good features of homogeneous and enzymatic catalysts, and they can potentially improve some difficult catalytic assays. This study reports a method that can be used to create an artificial metal-binding site prior to proving it to be functional in a wet lab. Haloalkane dehalogenase was grafted into a metal-binding site to form an artificial metallo-haloalkane dehalogenase and was studied for its potential functionalities in silico. Computational protocols regarding dynamic metal docking were studied using native metalloenzymes and functional artificial metalloenzymes. Using YASARA Structure, a simulation box covering template structure was created to be filled with water molecules followed by one mutated water molecule closest to the metal-binding site to metal ion. A simple energy minimization step was subsequently run using an AMBER force field to allow the metal ion to interact with the metal-binding residues. Long molecular dynamic simulation using YASARA Structure was performed to analyze the stability of the metal-binding site and the distance between metal-binding residues. Metal ions fluctuating around 2.0 Å across a 20 ns simulation indicated a stable metal-binding site. Metal-binding energies were predicted using FoldX, with a native metalloenzyme (carbonic anhydrase) scoring 18.0 kcal/mol and the best mutant model (C1a) scoring 16.4 kcal/mol. Analysis of the metal-binding site geometry was performed using CheckMyMetal, and all scores for the metalloenzymes and mutant models were in an acceptable range. Like native metalloenzymes, the metal-binding site of C1a was supported by residues in the second coordination shell to maintain a more coordinated metal-binding site. Short-chain multihalogenated alkanes (1,2-dibromoethane and 1,2,3-trichloropropane) were able to dock in the active site of C1a. The halides of the substrate were in contact with both the metal and halide-stabilizing residues, thus indicating a better stabilization of the substrate. The simple catalytic mechanism proposed is that the metal ion interacted with halogen and polarized the carbon-halogen bond, thus making the alpha carbon susceptible to attack by nucleophilic hydroxide. The interaction between halogen in the metal ion and halide-stabilizing residues may help to improve the stabilization of the substrate-enzyme complex and reduce the activation energy. This study reports a modified dynamic metal-docking protocol and validation tests to verify the metal-binding site. These approaches can be applied to design different kinds of artificial metalloenzymes or metal-binding sites.

Matched MeSH terms: Hydrolases
Insertion of single-chain variable fragment (scFv) peptide linker improves surface display of influenza hemagglutinin (HA1) on non-recombinant Lactococcus lactis

Jee PF, Chen FS, Shu MH, Wong WF, Abdul Rahim R, AbuBakar S, et al.

Biotechnol Prog, 2017 Jan;33(1):154-162.
PMID: 27802566 DOI: 10.1002/btpr.2400

Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N-acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non-recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA, and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single-chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51.7 ± 1.4% and 3,594.0 ± 675.9, respectively) in comparison to the HA1/AcmA without the scFv peptide linker (41.1 ± 1.5% and 1,652.0 ± 34.1, respectively). Higher amount of HA1/L/AcmA (∼2.9 × 10(4) molecules per cell) was displayed on L. lactis when compared to HA1/AcmA (∼1.1 × 10(4) molecules per cell) in the immunoblotting analysis. The HA1/L/AcmA completely agglutinated RBCs at comparable amount of protein to that of HA1/AcmA and HA1. Computational modeling of protein structures suggested that scFv peptide linker in HA1/L/AcmA kept the HA1 and the AcmA domain separated at a much longer distance in comparison to HA1/AcmA. These findings suggest that insertion of the scFv peptide linker between HA1 and AcmA improved binding of recombinant proteins to L. lactis. Hence, insertion of scFv peptide linker can be further investigated as a potential approach for improvement of heterologous proteins displayed on the surface of L. lactis using the AcmA binding domain. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:154-162, 2017.

Matched MeSH terms: Glycoside Hydrolases
Fulltext Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater

Obayashi Y, Wei Bong C, Suzuki S

Front Microbiol, 2017;8:1952.
PMID: 29067013 DOI: 10.3389/fmicb.2017.01952

Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems; especially, low protein binding materials should be chosen to use at overall processes of the measurement.

Matched MeSH terms: Peptide Hydrolases
Fulltext Genome Sequence ofSerratia marcescenssubsp.sakuensisStrain K27, a Marine Bacterium Isolated from Sponge (Haliclona amboinensis)

Cheng TH, Saidin J, Danish-Daniel M, Gan HM, Mat Isa MN, Abu Bakar MF, et al.

Genome Announc, 2018 Feb 08;6(6).
PMID: 29439033 DOI: 10.1128/genomeA.00022-18

Serratia marcescens
subsp.sakuensisstrain K27 was isolated from sponge (Haliclona amboinensis). The genome of this strain consists of 5,325,727 bp, with 5,140 open reading frames (ORFs), 3 rRNAs, and 67 tRNAs. It contains genes for the production of amylases, lipases, and proteases. Gene clusters for the biosynthesis of nonribosomal peptides and thiopeptide were also identified.

Matched MeSH terms: Peptide Hydrolases

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