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Results in Journal Advances in Enzyme Research: 72

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Xiangyang Xu, Zaiwei Song, Yunchao Yin, Faguo Zhong, Junying Song, Jiachao Huang, Wangli Ye, Peng Wang
Advances in Enzyme Research, Volume 8, pp 49-57; doi:10.4236/aer.2020.84005

Chitosanases EAG1 is a classical glycoside hydrolase from Bacillus ehimensis. The previous researches showed that this Chitosanases can not only hydrolyze the b1,4-glycosidic bonds of chitosan to COS in different sizes but also keep a high catalytic activity in organic, which was useful for producing chitooligosaccharides and GlcN for use in the food and pharmacological industries. While it is instable in the liquid state. This shortcoming seriously restricts its industrial application. Here we used the modeled structure of EAG1 and the molecular modeling software package to screen the free chemical database ZINC. Moreover, the strategies including “initial filter” and consensus scoring were applied to accelerate the process and improve the success rate of virtual screening. Finally, five compounds were screened and they were purchased or synthetized to test their binding affinity against EAG1. The test results showed that one of them could inhibit the enzyme with an apparent Ki of 1.5 μM. The result may take the foundation for further inhibitor screening and design against EAG1 and the screened compound may also help to improve the liquid stability of EAG1 and expand its industrial application.
Chanjugaa Uthayakumar, Santhosh Rupert
Advances in Enzyme Research, Volume 8, pp 1-18; doi:10.4236/aer.2020.81001

The medical herb Phyllanthus amarus play a crucial role in indigenous medicine. Therapeutically, these plants’ extract acts as potential players that inhibits several digestive enzymes that are relevant to the management of Peptic ulcers and Diabetes Mellitus, which occur due to the overproduction of such enzymes. Evaluation of inhibitory effect of this extract was carried out against Pepsin, α-amylase, Trypsin enzymes along with the effect of thermal stability and ammonium sulphate precipitation on these inhibitory assays. P. amarus leave’s extract with different concentration gradients were used in this research analysis. Results obtained along with the literature analysis revealed photochemical compounds such as polyphenols causes inhibitory nature in the extract. Maximal percentage of inhibition of amylase, pepsin and trypsin were found to be 71% (0.32 mg/ml), 85% (0.08 mg/ml) and 87% (1.28 mg/ml) respectively. In thermal stability assay the maximum percentage of inhibition for amylase, pepsin and trypsin was observed at 30% (80°C), 68% (4°C) and 5% (37°C). Enzymes inhibitory assays on ammonium sulphate precipitation elicited maximum percentage of inhibition for amylase, pepsin and trypsin as 42% (at 45% of (NH4)2SO4), 58% (at 15% of (NH4)2SO4) and 40% (at 30% of (NH4)2SO4) respectively. This research concluded that Phyllanthus amarus leave extracts are potential inhibitors of α-amylase, pepsin and trypsin enzymes. Ammonium sulphate precipitation was helpful to purify the polyphenols the active compounds to a good extend. Also, thermal stability was helpful to check the stability of these active photochemical compounds present in the extract. Thus, P. amarus is an effective inhibitor to be used as supplements in the disease management.
Afaf O. B. Shart, Elhadi A. I. Elkhalil
Advances in Enzyme Research, Volume 8, pp 39-48; doi:10.4236/aer.2020.84004

The aim of the present work was to isolate Bacillus spp. With high lipase activity; to characterize the isolates using both biochemical and molecular methods; to produce lipase using Bacillus isolates and to study the biochemical and biophysical characteristics of the produced lipase. Sixty five Bacillus isolates were isolated from soil 20 isolates from guar field soil (G), 15 isolates from Abusabein field soil (B), 15 isolates from sun flower field soil (S) and 15 isolates from oil effluent (O). Lipase producing isolates were screened; a Chromogenic plate’s method was used. Enzyme activity was quantitatively assayed. Lipase production under submerged fermentation (SMF) conditions using a production medium that contained metal salts, Tween-20 and olive oil as substrate at different period 24, 48, 72 and 96 h, the optimum pH, temperature for lipase activity was determinated and kinetics as well. The isolates showed the highest lipase activity which was identified as Bacillus sp. The optimum pH, temperature, thermostability and kinetic of the produced enzymes were found in three isolates G14, O1 and B10 with the highest enzyme activity and best stability. The isolates G14, O1 and B10 revealed the highest lipase activity of 63.4, 41.2 and 28.3 U/ml, respectively. The results showed optimum pH of the lipase activity from isolates G14, O1 and B10 8.0, 6.0 and 6.0 and the optimum temperature 40, 60 and 75˚C, respectively. Lipase enzymes from isolates O1 and B10 were found to be more thermostable after incubation time for 120 min at 90˚C. The Vmax and Km values of lipase for isolates G14, OI and B10 were 17.6, 135 and 24.4 μmole∙min𕒵 and 1.3, 1.6 and 0.681 mM, respectively. According to these results Bacillus spp. with high lipase activity and thermostability can be used to promote food, pharmaceuticals, paper, detergents agrochemicals industries and pollution control in Sudan.
Yukio Morimoto, Koichi Takamiya
Advances in Enzyme Research, Volume 8, pp 19-26; doi:10.4236/aer.2020.82002

Organomercury lyase (MerB) overexpressed in Escherichia coli captured and decomposed organomercury compounds, and it has been detected by radioactive analysis with neutron irradiation. Genetically modified E. coli captures a lot of mercury from a cultivation solution with about 80% recovery, when the bacteria are growing during 24 to 72 hours. Since the modified E. coli has no additive gene for mercury metabolism, the bacteria could hold mercury tightly by the MerB enzyme in their cell and do not release them into medium. In the later, 72 hours after, bacteria have less recovery ratio; it may be affected by undecompsed mercury compounds in bacteria growth. The recovery ability of the bacteria would not be changed by addition of the MerB producing reagent (IPTG). A quantitative value of mercury atom is estimated by an emission of γ-ray by reactor neutron from a dried cell or solution on a filter paper, which is available for nondestructive testing of bacteria holding mercury atoms. In this method an efficient recovery system of toxic mercury from a polluted solution has been archived without destruction of samples, so called in-cell analysis.
Dominic W. S. Wong, Sarah Batt, William Orts
Advances in Enzyme Research, Volume 8, pp 27-37; doi:10.4236/aer.2020.83003

Combinatorial chemistry involves the chemical or biological synthesis of diverse variation of the structures of a target molecule and the library is then screened for variants of desirable target properties. The approach has been a focus of research activity in drug discovery and biotechnology. This report is to demonstrate the application of enzyme technology using the concept of combinatorial chemistry as a novel approach for the bioconversion of plant fibers. Wheat insoluble fiber was subjected to combinatorial enzyme digestion to create structural variants of feruloyl oligosaccharides (FOS). Fractionation and screening resulted in the isolation of a fraction of bioactive FOS species showing antimicrobial activity. These results demonstrate the feasibility and usefulness of the combinatorial enzyme technique in the transformation of plant biomass to value-added products.
Michael O. Okpara, Olufemi S. Bamidele, Joshua O. Ajele
Advances in Enzyme Research, Volume 7, pp 45-56; doi:10.4236/aer.2019.74004

Proteases are important industrial enzymes that account for about 60% of the total enzyme market globally due to their large application in food, feed, textile and pharmaceutical industries. The effect of salt stress on protease production was evaluated on Aspergillus flavus and Aspergillus niger. The enzyme production was enhanced by stepwise optimization of the culture parameters, notably, carbon source, nitrogen source, pH, and temperature of the submerged fermentation process while using a minimal salt media and casein as substrate for the protease activity. The fungi species were found to be good producers of both acid and alkaline proteases under 4% salt stress condition. The optimum culture conditions for alkaline protease production by Aspergillus flavus were sucrose 4%, peptone 1%, pH 8 at 40°C with maximum enzymatic activities of 8.85 mM/min/mg protein, 5.22 mM/min/mg protein, 3.75 mM/min/mg protein, and 1.64 mM/min/mg protein, respectively. Lactose 4%, peptone 1%, pH 6 at 50°C were the optimum culture conditions for acid protease production by Aspergillus flavus with maximum enzymatic activities of 4.59 mM/min/mg protein, 2.06 mM/min/mg protein, 1.24 mM/min/mg protein, and 1.23 mM/min/mg protein, respectively. For Aspergillus niger, the optimum culture conditions for alkaline protease production were corn starch 4%, yeast extract 1%, pH 6 at 40°C with maximum enzymatic activities of 5.99 mM/min/mg protein, 3.85 mM/min/mg protein, 6.18 mM/min/mg protein, and 3.72 mM/min/mg protein, respectively. While lactose 4%, yeast extract 1%, pH 6 at 50°C were the best culture conditions for acid protease production by Aspergillus niger with maximum enzymatic activities of 4.81 mM/min/mg protein, 0.93 mM/min/mg protein, 5.71 mM/min/mg protein, and 3.34 mM/min/mg protein, respectively.
Kohji Ishihara, Natsumi Adachi, Takumu Mishima, Chiharu Kuboki, Ayaka Shuto, Kazuya Okamoto, Manami Inoue, Hiroki Hamada, Daisuke Uesugi, Noriyoshi Masuoka, et al.
Advances in Enzyme Research, Volume 7, pp 15-25; doi:10.4236/aer.2019.72002

We screened 15 Agromyces strains from the Microbacteriaceae family and 16 Gordonia strains from the Gordoniaceae family to investigate their biocatalytic ability to reduce carbonyl compounds. Two Agromyces strains (A. soli NBRC109063 and A. humatus NBRC109085) and two Gordonia strains (G. hydrophobica NBRC16057 and G. malaquae NBRC108250) grew well in 230 medium. The stereoselective reduction of various carbonyl compounds using these four strains was investigated. We discovered that these strains can reduce aliphatic and aromatic α-keto esters and an aromatic α-keto amide. On the basis of the conversion rate and stereoselectivity of the alcohols produced, G. hydrophobica NBRC16057 is a potential biocatalyst for the stereoselective reduction of α-keto esters and an aromatic α-keto amide to the corresponding chiral alcohols. Our results also suggest that the reduction of ethyl 2-methylacetoacetate by wet G. hydrophobica NBRC16057 cells in the presence of L-glutamate is useful for the production of chiral ethyl 3-hydroxy-2-methylbutanoate.
, Zachary N. Senwo
Advances in Enzyme Research, Volume 7, pp 1-13; doi:10.4236/aer.2019.71001

Yueming Li, Hongqing Xu, Jianchun Xu, Ruilian Pang, Bingzheng Xu
Advances in Enzyme Research, Volume 7, pp 57-65; doi:10.4236/aer.2019.74005

Phytase is a kind of enzyme that hydrolyzes phytic acid and its salts to produce inositol and phosphoric acid. As a new feed additive, phytase has great potential in animal nutrition and environmental protection. Because of its good stability, large-scale production and high activity, microbial phytase has become a hot spot in industrial application. Here, we reported the predicted structure and enzymatic properties of a phytase from Bacillus subtilis, which was named as phyS. It was clear that the optimal temperature is 35°C, and the optimal pH is 8. Meanwhile, the enzyme activity was kept at above 90% in the range of pH 8 - 9, this result demonstrated that phyS is an alkaline phytase. This study lays a foundation for the extensive application of phyS.
Sergei V. Stovbun, Kirill V. Ermakov, Alexander A. Bukhvostov, Alexander S. Vedenkin, Dmitry A. Kuznetsov, V. Stovbun Sergei, V. Ermakov Kirill, A. Bukhvostov Alexander, S. Vedenkin Alexander, A. Kuznetsov Dmitry
Advances in Enzyme Research, Volume 7, pp 27-44; doi:10.4236/aer.2019.73003

A conventionally synthesized thio- and cyano-modified single-stranded poly(dNTP) sequences of different molecular sizes (20n - 200n) and the same lengths routine poly(dNTP) and poly(NTP) species were obtained through the good services provided by the Russian Federal Bioorganic Products Group and by the ThermoFischer, Inc., and then tested for their impact on catalytic activities of β-like DNA polymerases from chromatin of HL-60, WERI-1A and Y-79 cells as well as for the affinity patterns in DNApolβ-poly(dNTP)/ (NTP) pairs, respectively. An essential link between the lengths of ultrashort (50n - 100n) single-stranded poly(dNTP) sequences of different structures and their inhibitory effects towards the cancer-specific DNA polymerases β has been found. A possible significance of this phenomenon for both DNA repair suppression in tumors and a consequent anti-cancer activity of the DNA repair related short poly(dNTP) fragments has been for the first time emphasized with a respect to their pharmacophore revealing potential. Thus, this work presents an experimental attempt to upgrade a contemporary attitude towards the DNA derived products applied for anti-cancer agenda, particularly, for acute myeloid leukemia and retinoblastoma cell DNA repair machinery breakdown. In this study, tumor specific DNA polymerases β were found of being the targets for attack promoted with the primer-like single-stranded DNA fragments followed by consequent cytostatic phenomena. A novel concept of the DNA related anti-cancer medicines is under discussion.
R. A. Abdulrauf, F. A. Dawud, N. S. Emmanuel, H. D. Muhammad, A. S. Dange, B. A. David, A. E. Ogweje, A. U. Alexander, M. Yahuza
Advances in Enzyme Research, Volume 6, pp 21-28; doi:10.4236/aer.2018.63003

This study was designed to assess the effect to apple cider vinegar (ACV) on oxidative stress biomarkers in male and female Wistar rats exposed to chronic restraint stress. Severe and persistent stress elevates reactive oxygen species (ROS) production by metabolic and physiological processes; causing cellular damage. Thirty (30) Adult Wistar rats of both sexes weighing about 150 - 200 g were divided into 3 groups each consisting of a male and female subgroup and given the following treatments once a day for 21 days: Normal control group received 0.5 ml distilled water orally, the restraint stress (RS) group was exposed to chronic restraint stress 6 hours daily while the Apple cider vinegar (ACV)-treated group received 4 ml/kg of apple cider vinegar orally in addition to chronic restraint stress 6 hours daily. The rats were sacrificed after the experimental period and blood was collected via cardiac puncture for assessing oxidative stress biomarkers. ACV (4 ml/kg) treatment decreased lipid peroxidation (MDA) and serum catalase (CAT) activity while upregulating endogenous superoxide dismutase (SOD) activity. The findings of this study show that the female Wistar rats are more predisposed to the antioxidant effect of ACV than the males.
Dominic W. S. Wong, Sarah Batt Doris Feng, William Orts
Advances in Enzyme Research, Volume 6, pp 11-20; doi:10.4236/aer.2018.62002

Yan Chen, Gaozhong Pu, Bo Lian, Xiuxia Pei, Guifang Huang, QiFeng Wang, Yanna Lv
Advances in Enzyme Research, Volume 6, pp 1-9; doi:10.4236/aer.2018.61001

, Paul M. Johnson
Advances in Enzyme Research, Volume 6, pp 29-52; doi:10.4236/aer.2018.63004

Glutamate dehydrogenase (GDH)-synthesized RNA, a nongenetic code-based RNA is suitable for unraveling the structural constraints imposed on the regulation (transcription, translation, siRNA etc.) of metabolism by genetic code. GDH-synthesized RNAs have been induced in whole plants to knock out target mRNA populations thereby producing plant phenotypes that are allergen-free; enriched in fatty acids, essential amino acids, shikimic acid, resveratrol etc. Methods applied hereunder for investigating the structural properties of GDH-synthesized RNA included purification of GDH isoenzymes, synthesis of RNA by the isoenzymes, reverse transcription of the RNA to cDNA, sequencing of the cDNA, computation of the G+C-contents, profiling the stability through PCR amplification compared with genetic code-based DNA; and biochemical characterization of the RNAs synthesized by individual hexameric isoenzymes of GDH. Single product bands resulted from the PCR amplification of the cDNAs of GDH-synthesized RNA, whereas several bands resulted from the amplification of genetic code-based DNA. The cDNAs have wide G+C-contents (35% to 59%), whereas genetic code-based DNA has narrower G+C-contents (50% to 60%). The GDH β6 homo-hexameric isoenzyme synthesized the A+U-rich RNAs, whereas the a6, and α6 homo-hexameric isoenzymes synthesized the G+C-rich RNAs. Therefore, the RNA synthesized by GDH is different from genetic code-based RNAs. In vitro chemical reactions revealed that GDH-synthesized RNA degraded total RNA to lower molecular weight products. Therefore, GDH-synthesized RNA is RNA enzyme. Dismantling of the structural constraints imposed on RNA by genetic code liberated RNA to become an enzyme with specificity to degrade unwanted transcripts. The RNA enzyme activity of GDH-synthesized RNA is ubiquitous in cells; it is readily induced by treatment of plants with mineral nutrients etc. and may simplify experimental approaches in plant enzymology and molecular biology research projects.
Kashini Andrew, Nachamada Solomon Emmanuel, Deborah Chioma Ejiogu, Chinonso Nicodemus Chima, Fatima Mohammed Yahuza, Emmanuel Chukwuemeka Umeh, Philemon Paul Mshelia
Advances in Enzyme Research, Volume 5, pp 33-44; doi:10.4236/aer.2017.53004

Isoniazid induced hepatotoxicity is a major concern in patients taking anti tuberculosis treatment and prophylaxis. It can result in elevated serum liver enzymes and hepatic failure. The aim of the study was to evaluate the phytochemicals and ameliorative effects of aqueous extracts of Brysocarpus coccineus on serum liver enzymes in isoniazid (INH) induced hepatotoxicity in adult male Wistar rats. Thirty six (36) adult male Wistar rats were divided into six groups of six rats each and were treated orally for 30 days as follows: Group I: 1 ml/kg of distilled water; group II: Isoniazid (27 mg/kg); group III: Isoniazid (27 mg/kg) + Livolin forte (20 mg/kg); group IV: Isoniazid (27 mg/kg) + B. coccineus (200 mg/kg); group V: Isoniazid (27 mg/kg) + B. coccineus (400 mg/kg); group VI: Isoniazid (27 mg/kg) + B. coccineus (800 mg/kg). At the end of the experiments, the Wistar rats were sacrificed and sera obtained for liver enzymes assay, whereas the liver tissue was also harvested and used for histological studies. Tanins, saponins, alkaloids and flavonoids were quantitatively present at 2.29%, 18.05%, 23.24% and 18.99%, respectively. There was an increase in the serum AST and ALT in the isoniazid treated group, which was reversed by livolin forte and the aqueous extracts at a dose of 200 mg/kg, however the extracts increased the serum levels of AST and ALT at higher doses, which was however not significant (p > 0.05) when compared to the controls. There was evidence of a reduction in hepatocytes damage in the extract treated groups when compared to the Isoniazid untreated group. In conclusion, aqueous extracts of B. coccineus shows hepatoprotective effects at 200 mg/kg in isoniazid hepatotoxicity in adult male Wistar rats.
Takafumi Tezuka, Atsunori Higashino, Mitsuo Akiba, Takashi Nakamura
Advances in Enzyme Research, Volume 5, pp 13-23; doi:10.4236/aer.2017.52002

Assays of stress enzymes related to active oxygen species were performed by using an in vitro preparation from the liver of a monkey (Japanese Macaque). Ge-132, an organic germanium compound, viz. poly-trans-[(2-carboxyethyl) germasesquioxane] [(GeCH2CH2COOH)2O3]n, suppressed the activities of NADH-dependent oxidase and NADPH-dependent oxidase [NAD(P)H-OD] and xanthine oxidase (XOD) as superoxide-forming enzymes, while promoting the activities of superoxide dismutase (SOD) as a superoxide-scavenging enzyme and catalase (CAT) as an enzyme responsible for degradation of hydrogen peroxide (H2O2). The evidence suggests that the levels of active oxygen species such as and H2O2 would be reduced by Ge-132. The possible connection between Ge-132 and activities of stress enzymes is discussed on the basis of these results.
Paulo Alexandre A. De Almeida Neves, Eliane Novato Silva, Paulo S. L. Beirão
Advances in Enzyme Research, Volume 5, pp 1-12; doi:10.4236/aer.2017.51001

Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalysis, thiocholine, readily generates a light absorbing product upon reaction with Elman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB). The hydrolysis of acetylcholine cannot be assayed with this method. The isothermal titration calorimetry can assay the hydrolysis of both substrates, without requiring additional reagents other than the enzyme and the substrate. To compare kinetic values obtained in the hydrolysis of acetylcholine (ACh) and acetylthiocholine (ATCh), with carbaryl acting as inhibitor, a calorimetric technique was used to evaluate kinetic properties of the two reactions. This method can show the hydrolysis of both substrates by the heat exchange that occurs during catalysis. In addition, it allowed the assessment of the AChE inhibition by carbaryl, a common insecticide. The results show a similarity between values obtained with both substrates, which are slightly higher for acetylcholine, the enzyme natural substrate. Enzymatic parameters values from ATCh and ACh were similar to each other and inhibitory constants using carbaryl were also similar, displaying that any approach to ACh is feasible using ATCh. The results obtained from ITC show the precision achieved by the calorimetric method.
I. S. Malgwi, , M. U. Kawu, E. D. Eze, H. A. Salami, N. S. Emmanuel, A. Mohammed
Advances in Enzyme Research, Volume 5, pp 24-31; doi:10.4236/aer.2017.52003

In mammals, lactation is the most energetically demanding period of a female’s reproductive life. This study was designed to evaluate the effect of fermented Soya bean and Vitamin C supplement on lipid peroxidation and antioxidant enzymes in lactating albino rats. Thirty five (35) adult female rats were used for this study. At parturition, the animals were randomly divided into five groups of five (5) rats each. Except group four (4) that was subdivided into three (3) sub groups of five animals each (n = 5). Treatment was carried out as follows: Group I: (Normal control) was given normal feed and distilled water, orally (1 ml/kg), Group II: metoclopramide (5 mg/kg), Group III: 100 mg/kg of Vitamin C. The three (3) sub groups under group four (4) received 10%, 20% and 40% soya bean, respectively, Group V: was co-admin- istered with 20% soya bean supplement and Vitamin C (100 mg/kg). Treatment was done for the period of ten (10) days at 06:00 h daily. Although there was an increase in serum MDA concentrations in all the treated groups compared to the control, lipid peroxidation was however significantly higher (P < 0.05) in the metoclopramide group relative to the soya bean supplemented groups. This study has shown that supplementation with soya bean induces a mild antioxidant effect by increasing serum level of superoxide dismutase. There was however a significant decrease in serum SOD in the 10% SB group compared to the control. There was a significant difference in serum catalase activity in the group treated with METCL (46.20 ± 1.53), SB 10% (44.00 ± 1.14) and SB 20% (45.20 ± 1.28) compared to the control (52.00 ± 0.71) (P < 0.05). Serum level of glutathione peroxidase GPx showed a significant difference in the group treated with VIT C, SB 10% and SB 20% compared to the control (P < 0.05).
Xueke Zhou, Tingting Wang, Renqiang Li
Advances in Enzyme Research, Volume 4, pp 1-6; doi:10.4236/aer.2016.41001

SDS-PAGE was applied to determine trypsin activity and inhibition. After the hydrolysis by trypsin to substrate bovine serum albulnin (BSA) under different temperatures and pH, the hydrolysis degree of BSA was conducted using SDS-PAGE. From the quantitative analysis to the electrophoresis bands of BSA and its hydrolysis products in SDS-PAGE pattern, the change of trypsin activity was determined, and then the optimum temperature at 40°C and the optimum pH at pH 8.5 - 8.7 for trypsin activity were obtained. All the target bonds in BSA molecule could be hydrolyzed at the same time by trypsin. The inhibition was due to the binding of inhibitor to trypsin, which made it impossible for trypsin to touch the substrate protein. SDS-PAGE was demonstrated to be also an effect method for assaying the characteristics of trypsin activity and its inhibition.
Muhammad Imran, , , Muhammad Javaid Asad, Hassan Ashfaq
Advances in Enzyme Research, Volume 4, pp 44-55; doi:10.4236/aer.2016.42005

In energy deficient world, cellulases play a major role for the production of alternative energy resources utilizing lignocellulosic waste materials for bioethanol and biogas production. This study highlights fungal and bacterial strains for the production of cellulases and its industrial applications. Solid State Fermentation (SSF) is more suitable process for cellulase production as compared to submerge fermentation techniques. Fungal cellulosomes system for the production of cellulases is more desirable and resistant to harsh environmental conditions. Trichoderma species are considered as most suitable candidate for cellulase production and utilization in industry as compared to Aspergillus and Humicola species. However, genetically modified strains of Aspergillus have capability to produce cellulase in relatively higher amount. Bacterial cellulase are more resistant to alkaline and thermophile conditions and good candidate in laundries. Cellulases are used in variety of industries such as textile, detergents and laundries, food industry, paper and pulp industry and biofuel production. Thermally stable modified strains of fungi and bacteria are good future prospect for cellulase production.
Nadezhda Andreeva, Ludmila Trilisenko, Mikhail Eldarov, Tatiana Kulakovskaya
Advances in Enzyme Research, Volume 4, pp 144-151; doi:10.4236/aer.2016.44013

The Saccharomyces cerevisiae polyphosphatase PPN1 (uniprot/Q04119) degrades inorganic polyphosphates both by cleaving Pi from the chain end and by fragmenting long-chain polymers into shorter ones. In this study, we have found a new activity of this protein: it releases phosphate from dATP. The dATP phosphohydrolase activity of pure PPN1 was ~7-fold lower compared to the exopolyphosphatase activity. This activity was strongly stimulated by Co2+ ions, as well as by ammonium ions, and inhibited by heparin and pyrophosphate similar to the exopolyphosphatase activity of PPN1. The Km value for dATP was 0.88 ± 0.14 mM. The dATP phosphohydrolase activity in the cells of PPN1-overexpressing yeast strain was several-fold higher than that in the parent strain. The other exopolyphosphatase of S. cerevisiae, PPX1, did not split Pi from dATP.
Harold W. Gardner
Advances in Enzyme Research, Volume 4, pp 56-61; doi:10.4236/aer.2016.42006

It is demonstrated that (3Z)-nonenal (NON) and (3Z)-hexenal (HEX) are oxidized in a cascade by lipoxygenase (LOX) and hydroperoxide peroxygenase (HP peroxygenase) into (2E)-4-hydroxy-2- nonenal (HNE) and (2E)-4-hydroxy-2-hexenal (HHE), respectively. In turn, HNE inactivates LOX terminating the cascade. The hydroxy-alkenals produced serve to inhibit plant pathogens, which initiated the cascade. In addition to LOX, other unknown oxygenases may be involved in the cascade.
Paulo R. Adalberto, Camilla C. Golfeto, Ariele C. Moreira, Fernando G. Almeida, Douglas Ferreira, , Dulce H. F. Souza
Advances in Enzyme Research, Volume 4, pp 7-19; doi:10.4236/aer.2016.41002

The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two chromatographic steps. PGaseLG was identified with MS analysis and molecular exclusion chromatography revealed the monomeric nature of a protein with an estimated molecular weight of about 39 kDa. PGaseLg has an optimum temperature of 60°C and optimum pH activity at 5.0. Using polygalacturonate as a substrate, the calculations of KM, Vmax and kcat were 0.65 mg·mL-1, 1800 μmol·min-1·mg-1 and 35.97 s-1, respectively. The enzyme was stable for more than 3 h at 50°C at pH 5.0; otherwise, at lower or higher pH values, the PGaseLg was less stable. The influence of several metals, EDTA and β-mercaptoethanol on enzyme activity was also determined. Thin layer chromatography (TLC) analyses indicated that PGaseLg is an exopolygalacturonase.
, Trine Aakvik Strand, Tone Haugen, Geir Klinkenberg, Hans Kristian Kotlar, , ,
Advances in Enzyme Research, Volume 4, pp 68-86; doi:10.4236/aer.2016.42008

With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90°C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.
Osmar Soares Da Silva, Rodrigo Lira De Oliveira, Cristina Maria Souza-Motta,
Advances in Enzyme Research, Volume 4, pp 125-143; doi:10.4236/aer.2016.44012

This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited KM = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 - 11.0) during 24 h. The optimum temperature was 40°C. The proteolytic activity was inhibited by Cu2+ (33.98%) and Hg2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.
Rishikesh K. Tiwari, Shikha Singh, ,
Advances in Enzyme Research, Volume 4, pp 113-124; doi:10.4236/aer.2016.44011

The importance of the earthworms in the agricultural practices is well known. The increasing applications of pesticides and chemicals in the agricultural farms have adversely influenced the flora and fauna of the soil. Earthworms which immensely contribute in increasing the quality and fertility of agricultural soil are reported be worst hit organisms under such conditions. Recent reports have indicated growing interests among researchers to explore biochemical and molecular markers as indicators of accumulation of pollutants in the soil in general and pesticides in particular. The varying levels of several biomolecules in different parts of the earthworm have been reported which are indicative of sensitivity of the organisms to different xenobiotics. However, the existing information lacks the literature displaying stock of information regarding the impact of pesticides on the levels of some key enzymes regulating many crucial functions in the earthworm at one place. Keeping in view this issue, it was envisaged to bring out a mini review which illustrates updated information available on the impact of pesticides on the activities of certain key enzymes reported to be responsible for catalysing metabolic pathways concerning the neurotransmission system, energy metabolism, oxidative stress and amino acids metabolism in different body parts of the earthworms, a prospective bioindicators of pesticides contamination in the soil.
Mirza Baasit Ali, Muhammad Irshad, Zahid Anwar, Mudassar Zafar, Muhammad Imran
Advances in Enzyme Research, Volume 4, pp 20-33; doi:10.4236/aer.2016.41003

Xylanases are mostly produced through submerged fermentation; nonetheless solid-state fermentation has increased profound attention and consideration of scholars having high conversion level biomass to energy conservation. This study depicted the purification of xylanases and their possible utilization in industry. The present study was carried out to examine the culture influence of fungal strain Fomes fomentarius (F. fomentarius) using different agro-industrial residues (wheat straw, rice husk, sugarcane bagasse and siris pods). F. fomentarius showed maximum enzyme production after 72 h of fermentation, when grown on wheat straw in solid state fermentation process while maximum activity showed on pH 6.0 at 30°C. The other parameters optimized by statistical design (RSM) showed maximum xylanase activity (146 ± 8 IU/mL) at 65% moisture content, 4 mL inoculums size, 175 mg Ammonium sulphate, 200 mg Calcium carbonate and 1.4 grams of glucose. Xylanase was salted out at 60% ammonium sulphate concentration and enzyme was further purified by Sephadex G-100 gel filtration chromatography with 2.2 fold increase in activity. The purified xylanase from F. fomentarius had optimum pH 6.0 and 40°C. Xylanase showed higher specificity for oat spelt xylan with kinetic constants Km 1.25 mg/mL and Vmax 54 mM/min. Xylanases have an industrial important enzyme used extensively in food, feed and paper industry.
Sung-Tae Kang,
Advances in Enzyme Research, Volume 4, pp 152-157; doi:10.4236/aer.2016.44014

Castor oil is the source of numerous products and is the only commercial source of the fatty acid ricinoleate, 12-hydroxy-oleate. Hydrogenated castor oil is similarly useful as the source of 12-hydroxy-stearic acid, best known as a component of lithium grease. Mono- and diacylglycerols are derived from castor oil and are useful in development of lubricants and emulsifiers for cosmetics, pharmaceutical and food use. Acylglycerols derived from hydrogenated castor oil may be similarly useful, albeit with different physical and chemical properties. We have evaluated the use of immobilized lipases to generate acylglycerols, using organic solvents to modulate the action of lipase to produce mono- and diacylglycerols, using tri-(12-hydroxy stearoyl)-glycerol as a model for hydrogenated castor oil. The presence of an alkylated oxygen in the solvent appears to be an important factor in supporting lipase activity, with diisopropyl ether providing the best yield of di-(12-hydroxy stearoyl)-glycerol.
Lluvia Sánchez-Pérez, Silvia Rodríguez-Navarro, Victor Hugo Marín-Cruz, Miguel Ángel Ramos-López, Alejandro Palma Ramos,
Advances in Enzyme Research, Volume 4, pp 98-112; doi:10.4236/aer.2016.43010

The application of enzymatic extracts and conidia of Beauveria bassiana in Metamasius spinolae and Cyclocephala lunulata was evaluated. The variables were mortality and time of death. In M. spinolae, mortality with extracts 29%, conidia 27% and the combination of both 31%, all had a time of death of four days. Although with different symptoms, used enzymatic extracts: contraction and softening of the joints; by conidia: mycelium in the joints; in the combination of conidia and enzymatic extracts: abundant aerial mycelium. In C. lunulata, 100% mortality in all treatments; Time of death: enzymatic extracts and extracts with conidia 1.2 days; conidia 2.8 days. Symptoms were different, enzymatic extracts: melanization and internal tissue lysis; enzymatic extract and conidia: mycelium emerged and melanization; conidia: mycelium emerged. Enzymatic extracts showed insecticidal activity in M. spinolae and C. lunulata. These results suggest the potential of enzymatic extracts as biocontrol agents to improve the use of entomopathogenic fungi.
Shamim Mushtaq, Meraj Zehra, Ayesha Khan, Mehwish Ahmed, Rubina Ghani
Advances in Enzyme Research, Volume 4, pp 87-97; doi:10.4236/aer.2016.43009

Aim: To evaluate the functional relationship between the nitric oxide synthase (NOS) and superoxide dismutase (SOD) enzymes in the pathogenesis of human senile cataract lenses of non-diabetic patients. Methods: Total solubilized proteins from human cataract lens were compared with normal lens (control) by 2-Dimenstional gel electrophoresis (2-DE). Proteins with different abundances were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot analysis was used to verify the changes in expression of NOS3 and SOD2. A further functional association of NOS3 with SOD2 and other proteins was seen by STRING 8.3 databases. Results: In the 2-DE maps, the cataract and normal lens proteins migrated in the region of pH 3 - 10 with a relative molecular weight of 20 - 130 kDa. Approximately two protein spots with differential intensity were detected as NOS3 and SOD2 using MALDI-TOF-MS. Western blot analysis showed high expression of NOS3 in cataract and SOD2 in normal lens samples. String interaction network revealed strong interactions between NOS3 and SOD2 at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor in the pathogenesis of cataract. Conclusion: This study will offer new avenues for mechanistic evaluation and future prevention of cataractogensis. However, large scale studies will be required to evaluate the effect of this interaction on the clinical outcome in human cataract.
Zhanara Suleimenova, Nurlan Akhmetsadykov, Aigul Kalieva, Kairat Mustafin, Zhazira Saduyeva
Advances in Enzyme Research, Volume 4, pp 62-67; doi:10.4236/aer.2016.42007

In agriculture, phytase is one of the most important monogastric animal sources of nutrient components because it effectively catalyzes the release of phosphate from phytate and phosphorylated compounds. In present work, Aspergillus niger strain (own collection) was used. Various physical and chemical factors have been known to affect the growth and the production of phytase. The effect of carbon and nitrogen sources, temperature and pH for extra cellular phytase production was investigated. Maximal phytase activity of Aspergillus niger was detected in media with 1.0% sucrose as a carbon source. Among the inorganic and organic nitrogen sources, ammonium nitrate in concentration of 0.5% was found to be a favorable nitrogen source for phytase production in Aspergillus niger. Optimum temperature and pH for phytase production by Aspergillus niger were 30°C and 5.5.
Mezajoug Kenfack Laurette Blandine, Ngangoum Eric Serge, Tchiégang Clergé
Advances in Enzyme Research, Volume 4, pp 35-43; doi:10.4236/aer.2016.42004

Crude enzyme extracts were prepared from leaves and stems of Linn. (Fabaceae) from Cameroon under optimized conditions. Proteolytic enzymes were precipitated with ammonium sulfate at 35% (w/v) saturation and assayed for enzyme activity. The effects of temperature, pH, incubation time and substrate specificity were studied. SDS-PAGE was used to determine molecular weight of precipitated protease. Results indicated that proteolytic activity of crude extract was 35.20 U/ml compared to 51.03 U/ml of partial purified extract. The optimum enzyme activity was found to be at 40°C, while 50% of activity was maintained at 60°C after 60 min incubation. Partial purified crude extract exhibited two optimum pH (2.75 and 9.0). The highest enzyme activity towards Bovine Serum Albumine (25.9 U/ml) was noted. SDS-PAGE gels exhibited molecular weight between 40 - 60 KDa. This result confirms that partial purified extract of A. precatorius contains proteases and could be a promising source for proteolytic enzyme extraction.
Published: 10 February 2015
Advances in Enzyme Research, Volume 1, pp 77-78

Valon Ejupi, Shpend Dragusha, Tsutomu Kabashima, , Ahmed F. M. El-Mahdy, Sheng Yin, Takayuki Shibata, Masaaki Kai
Advances in Enzyme Research, Volume 3, pp 19-29; doi:10.4236/aer.2015.31003

A selective, sensitive, and convenient assay for human collagenase is required because of its implication in diseases such as rheumatoid arthritis, osteoarthritis, and tumors. Here, a novel assay for human collagenase activity is described in which enzymatic degradation of native collagen or acetyl peptide is determined by using a fluorogenic reaction for oligopeptides. The oligopeptides are quantified spectrofluorometrically with either 3,4-dihydroxyphenylacetic acid or 1,2-dihy- droxybenzen reaction in the presence of sodium periodate and sodium borate (pH 7 - 8). These reactions can selectively convert N-terminal Gly-containing oligopeptides and N-terminal Ile-con- taining oligopeptides to fluorescence (FL) compounds, respectively, but not proteins, acetyl peptides or amino acids. Under optimized conditions using 1.65 μM collagen IV or 1.5 mM Ac-GPQGI- AGQ as substrates, this assay exhibits a proportional relationship between FL intensities and human collagenase-3 (MMP-13) concentrations. It can assay endogenous collagenase activities in several biological samples, such as cultured human cells and cheek tissue.
K. N. Unni, Panichikkal Abdul Faisal, , , Sreedharan Sreedevi, E. S. Hareesh, Trikariyoor Asokan Nidheesh Roy,
Advances in Enzyme Research, Volume 3, pp 31-38; doi:10.4236/aer.2015.32004

This study explored the utility of flours of rubber seed, coconut and groundnut kernels, and de-oiled cakes of coconut and groundnut as solid substrate for the production of lipase by Pseudomonas aeruginosa strain BUP2 (MTCC No. 5924), a novel bacterium reported from the rumen of Malabari goat. Various proportions (10%, 20%, 30%, 40% or 50%) of flours or cakes were prepared (w/v) with BUP medium (pH 4, 5, 6, 7 or 8), and incubated at different temperature (25°C, 28°C, 30°C or 32°C) for 24 to 96 h. The samples were assayed for lipase activity at 24 h intervals. The rubber seed flour (20%)-BUP medium supported the maximum lipase production (871 U/gds) at 48h incubation (pH 6, 28°C), followed by ground nut flour (398 U/gds), while ground nut cake supported the least lipase production (244 U/gds). From this, it is evident that the cheaply available rubber seed is an efficient substrate for the production of lipase, irrespective of its known demerit that it contains the limarin, a toxin; in fact, we could not detect limarin in the fermented matter. Thus, the utility of rubber seed for the production of a costly enzyme is reported from a novel rumen bacterium, which would be advantageous for rubber farmers.
, Victor V. Bratus
Advances in Enzyme Research, Volume 3, pp 1-8; doi:10.4236/aer.2015.31001

Chronic, low-level inflammation may be an independent marker of Metabolic Syndrome (MetS). Systemic Enzyme Therapy (SET), the oral administration of proteolytic enzymes, is safe and effective in the management of inflammation. Therefore, the effects of SET, as Wobenzym®, on the prevention and treatment of inflammation and other metabolic risk factors were assessed in a rabbit model of diet-induced MetS. Animals were fed a lipid-enriched diet for 8 weeks during which they were administered a vehicle control (control group) or Wobenzym either throughout the study period (prevention group) or beginning at the 5th week, after the development of biomarkers of MetS (treatment group). At the 8th week, both prevention and treatment groups demonstrated improved insulin sensitivity relative to the control group and reduced serum C-reactive protein (CRP) and glycosylated hemoglobin (HbA1c, P < 0.001). At 8 weeks, the prevention group, but not the treatment group, exhibited reduced total cholesterol and oxidative stress, measured as serum malondialdehyde (P < 0.001). Triglycerides and free fatty acids were reduced in both the treatment (P < 0.01) and prevention groups (P < 0.001) relative to the control group at the 8th week. Body weight and blood glucose were not affected. Enzyme therapy may have a positive effect on inflammation, insulin sensitivity, and other metabolic risk factors of MetS.
, Margarita N. Karpova, Lada V. Kuznetsova, Natalya Y. Klishina
Advances in Enzyme Research, Volume 3, pp 9-18; doi:10.4236/aer.2015.31002

This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - ATPase complex of the neuronal membrane and this enzyme is a Cl--pump that is coupled to GABAA receptors. The greatest (44%) activating effect on the enzyme is found with HCO3- (20 - 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM HCO3- /~5mM Cl-. Blockers of the GABAA receptor, namely bicuculline (10 - 50 μM) and picrotoxin (50 - 100 μM), inhibit this anion activation, whereas the HCO3- -ATPase activity is not sensitive to these ligands. Autoradiographic analysis of the spectrum of the partially purified enzyme phosphorylated with [γ-32P]ATP allowed us to distinguish three major 32P-labeled protein whose molecular weight are about 57, 53, and 48 kDa. In the presence of 5 mM Cl-/25mM HCO3- and 100 μM picrotoxin, the intensity of the phosphorylation of bands significantly decreased, thereby confirming the assumption about coupled of binding sites for anions and GABAA-ergic ligands. It was suggested scheme of Cl--transport through the plasma membrane by utilizing neuronal Cl-/ -HCO3- ATPase in the low (5 mM) Cl- and high (25 mM) HCO3- concentrations. The data demonstrated for the first time that the GABAA-coupled Cl-/ HCO3- -ATPase from rat brain neuronal membranes is maximally activated at a Cl-/HCO3- ratio of 1:5 and it remains stable at high concentrations of substrate and buffer.
Sabrina Moro Villela Pacheco, Américo Cruz Júnior, Ayres Ferreira Morgado, Agenor Furigo Júnior, Onyetugo Chioma Amadi, José Manuel Guisán, Benevides C C Pessela
Advances in Enzyme Research, Volume 3, pp 101-114; doi:10.4236/aer.2015.34011

Nineteen fungal strains were isolated from a chicken slaughterhouse effluent and within those, only one showed high values of lipolytic activity in submerged cultures. This fungus was identified as Trametes hirsuta. The crude extract was immobilized in chitosan/clay beads, with an immobilization yield of 80.9%. The analyses of the crude extract and the immobilized derivative at different temperatures, pH (s), solvents, metallic ions and storage showed that the immobilization process increased the enzyme life span. Ethyl esters were obtained in solvent free systems using chicken viscera oil and the enzyme crude extract. For effective comparison, a reaction using viscera oil and commercial lipase Novozym 435 was carried out. The result revealed 35% and 28% esters conversion in the reactions containing chicken viscera oil, using Novozym 435 and the crude extract respectively. The extract was also used in a reaction with soybean oil, traditionally used as starting substrate for biodiesel production.
Yukiko Ozawa, Yasufumi Umena, Takeo Imai,
Advances in Enzyme Research, Volume 3, pp 75-80; doi:10.4236/aer.2015.33008

2-ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of aliphatic amino acid-derived 2-keto acids. The enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (αβγδ)2 consisting of four different subunits, α = 45 kDa, β = 31 kDa, γ = 22 kDa and δ = 13 kDa, respectively, and it has three [4Fe-4S] clusters per αβγδ-protomer, similar to other ferredoxin oxidoreductases. In the present study, the native enzyme was purified from this strain and crystallized to give rod-like crystals that were suitable for X-ray diffraction experiments. The crystals belonged to space group P41212, with unit-cell parameters a = b = 136.20 Å, c = 221.07 Å. Diffraction images were processed to a resolution of 3.0 Å. The data collected so far indicate the approximate molecular boundaries and a partial main-chain trace of the enzyme.
, Jia-Kun Zeng, Pi-Han Wang, Wen-Chin Chen
Advances in Enzyme Research, Volume 3, pp 53-65; doi:10.4236/aer.2015.33006

Isolating cellulase-secreting microbes followed-by screening their cellulolytic activities has been an essential approach to discover novel and potential cellulases for cellulolytic industrial applications. This study was aimed to explore competitive exoglucanases by screening avicelase activities for 92 fungal strains isolated from environmental airborne-fungal-spore samples. Results showed that an isolated fungal strain numbered 58 exhibited the best avicelase activity of 0.209 U/mL when cultured for six days at pH 5.0 - 5.3 and 25℃ - 27℃, and was lately identified as a yeast strain of Meyerozyma sp. (96% ITS fragment similar with Meyerozyma caribbica, HG970748). Based on amino acid sequences revealed from LC/MS/MS, the target exoglucanase was identical to 1,4-beta-D-glucan cellobiohydrolases and was named Mc-CBHI which had optimal avicelase reaction conditions of pH 5 and 70℃ and could remain fairly stable after 4hr incubation at acid conditions (pH 3 - 5) or wide temperature ranges (30℃ - 80℃). Additionally, the Mc-CBHI (~70 kDa and ~3.6% of crude enzyme) had specific FPase and avicelase activities of 0.179 U/mg and 0.126 U/mg, respectively (which were about 40% - 50% activities of a commercial cellulase Accellerase-1000). These results demonstrated that the newly-found Mc-CBHI could become one of potential exoglucanase resources for related cellulolytic industrial applications.
Lidiya Lichko, Tatiana Kulakovskaya
Advances in Enzyme Research, Volume 3, pp 93-100; doi:10.4236/aer.2015.34010

The recombinant exopolyphosphatase PPX1 with a specific activity of ~300 U/mg protein was purified from the strain of Saccharomyces cerevisiae with the inactivated PPN1 gene transformed by the expression vector carrying the yeast PPX1 gene. The recombinant PPX1 was similar to the PPX1 of wild strains in its substrate specificity and requirement for cations. PPX1 had the high substrate specificity to polyphosphates. The preparation was suitable for polyphosphate assay in the presence of orthophosphate and nucleoside phosphates not hydrolyzed by PPX1. The yield of the enzyme preparation was 250 assays per 1 g of the biomass. The recombinant PPX1 was successfully used in polyphosphate assay in different yeast species and some foodstuffs.
Folasade M. Olajuyigbe, Kevin I. Ehiosun, Kikelomo F. Jaiyesimi
Advances in Enzyme Research, Volume 3, pp 66-74; doi:10.4236/aer.2015.33007

Peroxidases (POXs) are the key extracellular enzymes produced by crude oil degrading microbes. Knowledge of optimum conditions for POXs activity is crucial for providing effective environment for bioremediation. In this study, physicochemical properties of POXs produced by Actinomyces israelii and Actinomyces viscosus during growth on crude oil were studied. The POXs exhibited similarities in activity and stability with striking differences in response to two divalent metal ions. The POXs from both species had optimum pH of 7.0 and were very stable over a narrow pH range (6.0 - 8.0). The POXs demonstrated similar thermostability exhibiting relative residual activity of 62% at 50°C after 30 min incubation and 45% residual activity at the same temperature after 60 min despite the fact that POXs from A. viscosus and A. israelii had optimum temperatures of 50°C and 40°C, respectively. The POXs from A. viscosus and A. israelii were greatly activated by Fe2+ at 5.0 and 10.0 mM. The enzymes were both strongly inhibited by Cu2+, Mg2+ and Hg2+. Surprisingly, these congeneric POXs demonstrated striking differences in their response to Ca2+ and Mn2+. POX from A. viscosus was activated by Ca2+ and Mn2+ exhibiting relative activity of 136% and 106% at 5 mM, respectively. In contrast, POX from A. israelii was strongly inhibited by Ca2+ and Mn2+ exhibiting 62.5% relative activity in the presence of 5 mM of each metal ion. Increasing the concentration of Ca2+ and Mn2+ led to further activation of POX from A. viscosus and inhibition of POX from A. israelii. Results provide deeper insights into functional properties of studied POXs from closely related microbes. The physicochemical properties are very similar; however, notable differences provide a strong basis for structural characterization of these congeneric enzymes.
Thiago Pajeú Nascimento, Amanda Emmanuelle Sales, Camila Souza Porto, Romero Marcos Pedrosa Brandão, Galba Maria Campos Takaki, Jose Antônio Couto Teixeira, Tatiana Souza Porto, Ana Lúcia Figueiredo Porto
Advances in Enzyme Research, Volume 3, pp 81-91; doi:10.4236/aer.2015.33009

Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25°C for 72 hours. The optimum temperature for the produced enzyme was 45°C and most of its original activity was retained after being subjected to 80°C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+; but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy.
Advances in Enzyme Research, Volume 3, pp 39-51; doi:10.4236/aer.2015.32005

Phosphatases are diverse groups of enzymes that deserve special attention because of their significant roles in organic phosphorus (OP) mineralization to inorganic available forms (Pi). This work 1) compared the catalytic potentials of commercially acid phosphatase from wheat germ, sweet potato, and potato, and alkaline phosphatase from E. coli; 2) demonstrated that the rate of hydrolysis, catalytic efficiency, thermal stability, and optimal pH of these enzymes depended on enzyme sources and the stereochemical or stereoisomeric structures of the substrates; 3) revealed that both acid and alkaline phosphatases exhibited broad range of substrate hydrolysis with high affinity for p-nitrophenyl phosphate bis (cyclohexylammonium) than the widely used p-nitrophenyl phosphate disodium hexahydrate for phosphatase assay. Sweet potato had relatively higher reaction kinetics (Vmax, Km, Kcat, Kcat/Km) values with most substrates tested. The order of catalytic activity was in the order: sweet potato > wheat germ > potato, while the order of substrate hydrolyzed was: PNPBC > PNP > PNP2A2E > DG6P2Na > DG6PNa > Bis-PNP > phytate. The optimum pH for the acid phosphatase was observed to be 5.0. Generally, the activity of alkaline phosphatase was similar to that of acid phosphatase with optimal pH between 10 and 13, depending on the substrates. Knowledge derived from this work would be helpful in enzyme catalysis in soils and water environments.
Advances in Enzyme Research, Volume 2, pp 134-149; doi:10.4236/aer.2014.24014

Glucose-6-phosphate dehydrogenase has been purified from pigeon pea (Cajanus cajan) seeds and subjected to characterization. The enzyme was purified 123.69 fold with a yield of 21.37% by ammonium sulphate fractionation, PEG-4000 precipitation, CM cellulose column chromatography and DEAE cellulose column chromatography. The catalytically active enzyme is a dimer of 113 KDa with a subunit molecular weight of 55 KDa. Thermal inactivation of enzyme follows first order kinetics at 30°C and 40°C with half life of 6 and 1.5 min respectively. Km value for glucose-6-phosphate and NADP+ was found to be 2.68 mM and 0.75 mM respectively whereas Vmax value was found to be 0.11 U/mL and 0.13 U/mL respectively. The enzyme shows more affinity towards NADP+ than glucose-6-phosphate. The pKa value was found to be 10.41 indicating that the amino acid residue at active site might be lysine. The enzyme exhibited maximum catalytic activity at pH 8.2. The enzyme was found to be highly thermosensitive with gradual loss of activity above 30°C temperature.
Eriko Osumi, Chihiro Kondo, Mitsumasa Mizuno, Takahiro Suzuki, Mamoru Matsubara, Kazuo Shimozato,
Advances in Enzyme Research, Volume 2, pp 100-112; doi:10.4236/aer.2014.22011

Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephosphorylation of destrin. It is suggested that destrin dephosphorylation is involved in cortical F-actin disruption observed in parallel with β-agonist-induced amylase secretion. At present, the phosphorylation/dephosphorylation mechanism of destrin in parotid tissue is not known. We previously detected, in a crude rat parotid extract, a constitutively active protein kinase catalyzing phosphorylation of destrin; however, its identification has been hampered by difficulty in its enrichment. The purpose of this study was to explore a simple purification method(s) for this enzyme. To this end, we first developed a high-throughput dot-blot assay for the kinase with an anti-phosphodestrin antibody and then studied its purification by column chromatography on several media. We found that the kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite columns. In each chromatography, however, the kinase could be eluted, at the cost of resolution, only by sharp increases in the elution power of the eluent; gradual increases in the elution power resulted in unacceptably poor recovery. We confirmed that enzymatic properties of the kinase were not basically altered during the purification. Further purification of the kinase was achieved by native polyacrylamide gel electrophoresis (PAGE), which resolved the kinase activity into two bands and separated the activity from most proteins (the kinase activity after PAGE was detected with destrin-coated polyvinylidene difluoride membranes and the anti-phosphodestrin antibody). The two bands seem to constitute the major destrin-phosphorylating activity in the resting rat parotid gland. We here report its partial purification and characterization together with the detection methods.
Rushyannah Killens-Cade, Rachel Turner, Christine MacInnes, Amy Grunden
Advances in Enzyme Research, Volume 2, pp 1-13; doi:10.4236/aer.2014.21001

Lipid-producing microalgae are emerging as the leading platform for producing alternative biofuels in response to diminishing petroleum reserves. Optimization of fatty acid production is required for efficient conversion of microalgal fatty acids into usable transportation fuels. Microbial lipases/esterases can be used to enhance fatty acid production because of their efficacy in catalyzing hydrolysis of esters into alcohols and fatty acids while minimizing the potential poisoning of catalysts needed in the biofuel production process. Although studies have extensively focused on lipases/esterases produced by mesophilic organisms, an understanding of lipases/esterases produced by thermophilic, acidic tolerant microbes, such as Metallosphaera sedula, is limited. In this work, the carboxylesterase from Metallosphaera sedula DSM5348 encoded by Msed_1072 was recombinantly expressed in Escherichia coli strain BL21 (λDE3). The purified enzyme either with a hexahistidine (His6)-tag (Msed_1072Nt and Msed_1072Ct) or without the hexahistidine (His6)-tag (Msed_1072) was biochemically characterized using a variety of substrates over a range of temperatures and pH and in the presence of metal ions, organic solvents, and detergents. In this study, the fusion of the protein with a hexahistidine (His6)-tag did not result in a change in substrate specificity, but the findings provide information on which enzyme variant can hydrolyze fatty acid esters in the presence of various chemicals, and this has important implication for their use in industrial processes. It also demonstrates that Metallosphaera sedula Msed_1072 can have application in microalgae-based biofuel production systems.
Sudarshan R. Nelatury,
Advances in Enzyme Research, Volume 2, pp 14-26; doi:10.4236/aer.2014.21002

Enzyme kinetic parameters have been estimated using MATLAB software via the Wilkinson non-linear regression technique. The MATLAB script file written to implement this technique is short and very straightforward. Several software tools are commercially available for this purpose, with many graphical user interface (GUI) features. A routine use of these packages might offer immediate satisfaction of interactive hands-on experience; but in some cases the researcher might wish to write his/her own code and compare the results for further confirmation. Today MATLAB is in use in almost all the schools and laboratories as a standard software tool. So this paper is aimed at helping enzyme researchers to make use of this powerful software for estimation of parameters. It enables the incorporation of the analytical steps behind parameter estimation in an easy-to-follow manner and furnishes better visualization.
Robert Louis Bertrand,
Advances in Enzyme Research, Volume 2, pp 77-81; doi:10.4236/aer.2014.22008

The reduction of nitroblue tetrazolium by superoxide radicals generated from photo-reactive riboflavin has been in use for more than four decades to detect superoxide dismutase (SOD) on nondenaturing polyacrylamide gels. SOD research in medicine and biochemistry has warranted the development of multiple assay variants to overcome specific experimental constraints or to combine the SOD assay with other enzyme assays. Fine-tuning reagent concentrations to effectively visualize bands continue to be a major research obstacle in assay development. Herein we describe a straightforward technique to reliably adjust the background color of polyacrylamide gels without compromising assay efficacy. Low micromolar to low millimolar concentrations of yellow riboflavin can be mixed with the blue of reduced nitroblue tetrazolium to controllably produce blue, purple, yellow-brown, or yellow gel backgrounds. The advantage of this technique is that the assay is not modified by the introduction of new reagents. Quantitative reliability of these alternative stains was assessed by plotting determined band intensity values against known enzyme loads. The correlation (R2) values of trial averages were compared against the average correlation of the standard 0.028 mM riboflavin solution using pooled standard deviation and Student’s T-test at 95% confidence. Assay sensitivity was assessed by comparing lowest possible visible enzyme load of the experimental stains with the 0.028 mM riboflavin standard. No difference in the quantitative reliability was found in any riboflavin concentration. The minimum reliable sensitivity of the assay was found to be 10 ng for each concentration of riboflavin. This technique has already been employed to analyze SOD protein expression levels in extracts of Escherichia coli (Bertrand et al., Med Hypotheses 2012; 78:130-133, 2012; Bertrand & Eze, Adv. Enz. Res., 1: 132-141, 2013).
Panichikkal Abdul Faisal, Erandapurthukadumana Sreedharan Hareesh, , Kizhakkepowathial Nair Unni, , , Moolakkariyil Sarath Josh,
Advances in Enzyme Research, Volume 2, pp 125-133; doi:10.4236/aer.2014.24013

Solid-state fermentation (SSF) holds tremendous potentials for the production of industrially significant enzymes. The present study describes the production of lipase by a novel rumen bacterium, Pseudomonas sp. strain BUP6 on agro-industrial residues. Pseudomonas sp. strain BUP6 showed higher lipase production when grown in Basal salt medium (BSM) supplemented with oil cakes. Initially, five different oil cakes (obtained after extracting oil from coconut, groundnut, cotton seed, gingelly or soybean) were screened to find out the most suitable substrate-cum-inducer for the production of lipase. Among them, groundnut cake supported the maximum production of lipase (107.44 U/gds). Box-Behnken Design (BBD), followed by response surface methodology (RSM) was employed to optimize the culture parameters for maximizing the production of lipase. Using the software Minitab 14, four different parameters like temperature, pH, moisture content and incubation time were selected for the statistical optimization, which resulted in 0.7 fold increase (i.e., 180.75 U/gds) in production of lipase under the optimum culture conditions (temperature 28°C, pH 5.9, moisture 33% and incubation 2 d). Thus, this study signifies the importance of SSF for the production of industrially-significant lipase using agro-industrial residues as solid support.
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