Applied Biochemistry and Biotechnology (v.180, #1)

From the New Editor-in-Chief by Jonathan N. Sachs (1-2).

Activity Enhancement Based on the Chemical Equilibrium of Multiple-Subunit Nitrile Hydratase from Bordetella petrii by Yi Liu; Ping Liu; Lu Lin; Yueqin Zhao; Wenjuan Zhong; Lunjie Wu; Zhemin Zhou; Weifeng Sun (3-9).
The maturation mechanism of nitrile hydratase (NHase) of Pseudomonas putida NRRL-18668 was discovered and named as “self-subunit swapping.” Since the NHase of Bordetella petrii DSM 12804 is similar to that of P. putida, the NHase maturation of B. petrii is proposed to be the same as that of P. putida. However, there is no further information on the application of NHase according to these findings. We successfully rapidly purified NHase and its activator through affinity his tag, and found that the cell extracts of NHase possessed multiple types of protein ingredients including α, β, α2β2, and α(P14K)2 who were in a state of chemical equilibrium. Furthermore, the activity was significantly enhanced through adding extra α(P14K)2 to the cell extracts of NHase according to the chemical equilibrium. Our findings are useful for the activity enhancement of multiple-subunit enzyme and for the first time significantly increased the NHase activity according to the chemical equilibrium.
Keywords: NHase; Bordetella petrii ; Self-subunit swapping; Chemical equilibrium; Activity

Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach by Tzuu-Wang Chang; Pavithra Janardhanan; Charlene M. Mello; Bal Ram Singh; Shuowei Cai (10-25).
Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2′-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2′-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance.
Keywords: RNA; Aptamer; 2′-fluorine-pyrimidine; Surface plasmon resonance; Botulinum neurotoxin; Antidote; Endopeptidase

Reduced seed germination is among the most important factors adversely affecting crop stand and subsequent plant growth. Fennel (Foeniculum vulgare Mill) is an important medicinal plant with poor seed germination rate, occasionally. It is accordingly pertinent to find methods which can enhance fennel seed germination and remove the barriers of dormancy breaking. The present experiments studied the effects of two different priming (cold moist stratification and osmopriming) and 14 dormancy breaking techniques (hormonal, osmopriming, biopriming, chemical priming, and hydropriming) on the seed germination and seedling growth of two different fennel genotypes under growth chamber conditions. In the first and second experiment, the priming techniques including the time lengths of cold moist stratification (0, 15, 30, and 45 days) and the concentrations of polyethylene glycol 6000 (PEG6000, osmopriming at −0.99, −1.35, and −2.33 MPa) were used as the main plots. However, in both experiments, the dormancy breaking techniques and fennel genotypes were factorially combined and used as the subplots. Different seed- and seedling-related parameters including germination (%), plumule, radicle and seedling length, average germination time, rate and homogeneity of germination, and seed vigor index were determined. Both priming techniques were efficient on the enhancement of seed germination and seedling growth. Among the dormancy breaking techniques, Aminol Forte (biopriming), kadostim (biopriming), benzyl adenine + kinetin (biopriming), distilled water (hydropriming), gibberellin + kinetin (hormonal priming), and benzyl adenine + kinetin + gibberellin (biopriming) were the most effective ones. The related concentrations were equal to 100 mg/l, 10−5 M, and 0.4 %. The fennel genotypes reacted significantly different under priming conditions. It is possible to enhance seed germination and seedling growth of fennel using priming and dormancy breaking techniques, which is useful for the increased production of fennel under different conditions. The results indicate that bio and hydropriming techniques were among the most effective ones, which significantly increased seed germination and seedling growth, and removed the seed dormancy barriers.
Keywords: Priming techniques; Fennel (Foeniculum vulgare Mill); Seed germination; Seedling growth

Heavy metal pollution in water emerges as a severe socio-environmental problem originating primarily from the discharge of industrial wastewater. In view of the toxic, non-biodegradable, and persistent nature of most of the heavy metal ions, remediation of such components becomes an absolute necessity. Biosorption is an emerging tool for bioremediation that has gained momentum for employing low-cost biological materials with effective metal binding capacities. Even though biological materials possess excellent metal adsorption abilities, they show poor mechanical strength and low rigidity. Other disadvantages include solid–liquid separation problems, possible biomass swelling, lower efficiency for regeneration or reuse, and frequent development of high pressure drop in the column mode that limits its applications under real conditions. To improve the biosorption efficiency, biomasses need to be modified with a simple technique for selective/multi-metal adsorption. This review is intended to cover discussion on biomass modification for enhanced biosorption efficiency, mechanism studies using various instrumental/analytical techniques, and future direction for research and development including the fate of spent biosorbent. In most of the previously published researches, difficulty of the process in scaling up has not been addressed. The current article outlines the application potential of biosorbents in the development of hybrid technology integrated with membrane processes for water and wastewater treatment in industrial scale.
Keywords: Surface modification; Biosorption mechanism; Heavy metals; Hybrid process; Spent biosorbent

Rhizomes of Dioscorea species are traditionally used for relieving menopausal syndromes in Chinese medicine. The estrogen-stimulating bioactive principles have been demonstrated in our previous study. In this study, the estrogen-stimulating effects of proteins isolated from four Dioscorea species [D. alata L. (DA), D. zingiberensis C.H. Wright (DH), D. collettii var. hypoglauca (Palib.) S.J. Pei & C.T. Ting (DH), and D. oppositifolia L. (DO)] have been investigated and compared. Microscopic authentication of four Dioscorea species was performed by using paraffin and powder sections of the rhizomes. The potential bioactive proteins of four Dioscorea species have been rapidly isolated by using a DOI-antibody affinity column chromatography on immobilized antibodies against on estradiol-stimulating protein from DO (DOI), and their bioactivity has been rapidly confirmed and compared by phenotypic (i.e., estradiol-stimulating effect) and target-based (i.e., STAR, aromatase, estrogen receptors) screening approaches. The estrogen-stimulating activity of bioactive proteins from DO is the highest. In addition, bioactive proteins from DO upregulated the estradiol-metabolizing enzymes (aromatase and steroidogenic acute regulatory protein). Meanwhile, bioactive proteins from DA, DH and DO upregulated estrogen receptor β (ERβ). All bioactive proteins did not change the expression of estrogen receptor β (ERα). The estrogen-stimulating bioactive proteins isolated from DO increased biosynthesis of estradiol and upregulated the protein expression of aromatase, steroidogenic acute regulatory protein, and ERβ. The results scientifically support the traditional use of DO in Chinese medicine for relieving menopausal syndrome. Besides, proteins from DA and DZ could also upregulate the translational levels of ERβ, and potentially reducing the risk of ovarian cancer, which also support the clinical use of them for treating female aging disorder. Graphical Abstract Comparative Analysis of DOI-like Proteins with Stimulating Activity on Ovarian Estradiol Biosynthesis from Four Different Dioscorea Species in vitro
Keywords: Dioscorea protein; Aromatase; Steroidogenic acute regulatory protein; Estrogen receptor; Menopause; Estrogen; Estrogen-stimulating protein

Steam explosion was studied as a means to improve the enzymatic digestibility of carbohydrates in Brewer’s spent grain, a protein and lipid-rich lignocellulosic by-product of the brewing industry. Having temperature, treatment time and the presence of acid catalyst as variables, a treatment at 200 °C for 10 min without an acid catalyst was found to be the most efficient, dissolving 12.1 % of the dry matter. Mainly oligomeric non-cellulosic glucan and arabinoxylan were dissolved, and the remaining insoluble carbohydrates could be efficiently hydrolysed by an enzyme cocktail (75 % hydrolysis yield). The process also caused partial protein degradation and dissolved over a third of the total nitrogen. Meanwhile, the insoluble protein appeared to become more strongly associated with acid-insoluble lignin. Compositional changes observed in the proteins and carbohydrates were supported by the results of epifluorescence microscopy. The process yielded three chemically different fractions which could serve as biorefinery products or intermediates.
Keywords: Brewer’s spent grain; Pretreatment; Enzymatic hydrolysis; Microscopy; Biorefinery

This investigation utilized sugarcane bagasse aqueous extract (SBAE), a nontoxic, cost-effective medium to boost triacylglycerol (TAG) accumulation in novel fresh water microalgal isolate Scenedesmus sp. IITRIND2. Maximum lipid productivity of 112 ± 5.2 mg/L/day was recorded in microalgae grown in SBAE compared to modified BBM (26 ± 3 %). Carotenoid to chlorophyll ratio was 12.5 ± 2 % higher than in photoautotrophic control, indicating an increase in photosystem II activity, thereby increasing growth rate. Fatty acid methyl ester (FAME) profile revealed presence of C14:0 (2.29 %), C16:0 (15.99 %), C16:2 (4.05 %), C18:0 (3.41 %), C18:1 (41.55 %), C18:2 (12.41), and C20:0 (1.21 %) as the major fatty acids. Cetane number (64.03), cold filter plugging property (−1.05 °C), and oxidative stability (12.03 h) indicated quality biodiesel abiding by ASTM D6751 and EN 14214 fuel standards. Results consolidate the candidature of novel freshwater microalgal isolate Scenedesmus sp. IITRIND2 cultivated in SBAE, aqueous extract made from copious, agricultural waste sugarcane bagasse to increase the lipid productivity, and could further be utilized for cost-effective biodiesel production.
Keywords: Scenedesmus sp. IITRIND2; Sugarcane bagasse; Triacylglycerol; Biodiesel; Lipid productivity

Loops Adjacent to Catalytic Region and Molecular Stability of Man1312 by Haiyan Zhou; Jie Yong; Han Gao; Zhihui Yuan; Wenjiao Yang; Yun Tian; Yongyao Wu (122-135).
Hemicelluloses are the second major polysaccharides in nature and can be converted to ethanol product by a variety of enzymes including mannanases. Mannanase is an important enzyme that hydrolyses mannose-containing polysaccharides which are abundant in plants. An optimized mannanase could help to improve conversion process and make the technology efficiently and competitively. In this work, the effects of loops adjacent to active region on enzymic properties of Man1312 were investigated. Loop 6 and 10 are two loops neighboring to Man1312 catalytic region, and deletion mutagenesis and residue substitution were performed on both loops. Deletion on sites S145, Q148, N244, and S255 and substitution on sites N146, S147, S156, and T157 gave significant increased stability to enzyme. The quadruplet mutant ManD4I4 combined all the mutations and had higher optimal temperature and T m value by 5 and 4 °C than Man1312, respectively. From our data, we are able to conclude the loops of enzymes are important to design mutagenesis and obtain improved properties, especially the loops neighboring to catalytic region from tertiary structure. In our experiment, residue deletion and substitution on loops neighboring to catalytic region made significant improvement on enzyme properties.
Keywords: Mannanase; Rational design; Loop; Molecular stability

The costs of amylase represent ca. 24 % of the expenditures in the starch industry and an increase in amylase production and/or activity will greatly cut down on production costs. In the present study, we obtained a high amylase-producing strain of bacteria, WangLB, and identified it as a member of the Bacillus genus based on 16S rDNA analysis. The fermentation conditions for amylase production in the strain were optimized, and the maximum amylase activity we obtained was 26,670 ± 1390 U/mL, under the optimized conditions of 48-h incubation in liquid starch medium, 35 °C, pH 10, 1 % v/v inoculum concentration, 20 g/L starch concentration, and 0.1 % w/v peptone. The influences of 16 small organic inducers on amylase production were tested, and the results showed that 20 mmol/L alanine greatly enhanced amylase production to 290 % of the baseline level. We also conducted an amylase enzymology analysis. The molecular weight of the amylase was 55 kD, determined by SDS-PAGE. The optimum temperature and pH for the amylase were 55 °C and pH 9, respectively. The enzyme also showed high activity over a wide range of temperatures (50–85 °C) and pH values (3–10), and the activity of the amylase was Ca2+ independent. The kinetic parameters K m and V max were 0.37 ± 0.02 mg/mL and 233 U/mg, respectively. Finally, the amylase was applied to the hydrolysis of five different brands of starch. It was found that the hydrolyzability of the substrate by amylase increased along with starch solubility.
Keywords: Bacillus sp; Amylase; Fermentation parameters; Enzymology

Comparative Analyses of the Relative Effects of Various Mutations in Major Histocompatibility Complex I—a Way to Predict Protein-Protein Interactions by Ananya Ali; Ria Biswas; Sanchari Bhattacharjee; Prabahan Nath; Sumanjit Pan; Angshuman Bagchi (152-164).
Protein-protein interactions (PPIs) play pivotal roles in most of the biological processes. PPI dysfunctions are therefore associated with disease situations. Mutations often lead to PPI dysfunctions, but there are certain other types of mutations which do not cause any appreciable abnormalities. This second type of mutations is called polymorphic mutations. So far, there are many studies that deal with the identification of PPI sites, but clear-cut analyses of the involvements of mutations in PPI dysfunctions are few and far between. We therefore made an attempt to link the appearances of mutations and PPI disruptions. We used major histocompatibility complex as our reference protein complex. We analyzed the mutations leading to the disease amyloidosis and also the other mutations that do not lead to disease conditions. We computed various biophysical parameters like relative solvent accessibility to discriminate between the two different types of mutations. Our analyses for the first time came up with a plausible explanation for the effects of different types of mutations in disease development. Our future plans are to build tools to detect the effects of mutations in disease developments by disrupting the PPIs.
Keywords: PPI; MHC I; Amyloidosis; Relative solvent accessibility; Mutation; Poisson distribution

Refolded Recombinant Human Paraoxonase 1 Variant Exhibits Prophylactic Activity Against Organophosphate Poisoning by Priyanka Bajaj; Rajan K. Tripathy; Geetika Aggarwal; Ashok K. Datusalia; Shyam S. Sharma; Abhay H. Pande (165-176).
Organophosphate (OP) compounds are neurotoxic chemicals, and current treatments available for OP-poisoning are considered as unsatisfactory and inadequate. There is an urgent need for the development of more effective treatment(s) for OP-poisoning. Human paraoxonase 1 (h-PON1) is known to hydrolyze a variety of OP-compounds and is a leading candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. Non-availability of effective system(s) for the production of recombinant h-PON1 (rh-PON1) makes it hard to produce improved variant(s) of this enzyme and analyze their in vivo efficacy in animal models. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop variant(s) of h-PON1. Recently, we have developed a procedure to produce active rh-PON1 enzymes by using E. coli expression system. In this study, we have characterized the OP-hydrolyzing properties of refolded rh-PON1(wt) and rh-PON1(H115W;R192K) variant. Our results show that refolded rh-PON1(H115W;R192K) variant exhibit enhanced OP-hydrolyzing activity in in vitro and ex vivo assays and exhibited prophylactic activity in mouse model of OP-poisoning, suggesting that refolded rh-PON1 can be developed as a therapeutic candidate.
Keywords: Acetylcholinesterase; Chlorpyrifosoxon; Organophosphate; Paraoxonase 1; Prophylactic

Preparation and Biological Activity of New Collagen Composites, Part I: Collagen/Zinc Titanate Nanocomposites by Madalina G. Albu; Todorka G. Vladkova; Iliana A. Ivanova; Ahmed S. A. Shalaby; Veselina S. Moskova-Doumanova; Anna D. Staneva; Yanko B. Dimitriev; Anelya S. Kostadinova; Tanya I. Topouzova-Hristova (177-193).
The aim of this investigation was to develop new antimicrobial collagen/zinc titanate (ZnTiO3) biomaterials using a sol–gel cryogenic draying technology in keeping the native collagen activity. Broad-spectrum antimicrobial activity was demonstrated against Firmicutes (Staphylococcus epidermidis, Bacillus cereus, and Candida lusitaniae) and Gracilicutes (Escherichia coli, Salmonella enterica, and Pseudomonas putida) microorganisms. The antimicrobial activity as well as the cytotoxicity were specific for the different test microorganisms (Gram-positive and Gram-negative bacteria and fungi) and model eukaryotic cells (osteosarcoma, fibroblast, and keratinocyte cells), respectively, and both were depending on the ZnTiO3 concentration. Three mechanisms of the antimicrobial action were supposed, including (i) mechanical demolition of the cell wall and membrane by the crystal nanoparticles of the ZnTiO3 entrapped in the collagen matrix, (ii) chelation of its metal ions, and (iii) formation of free oxygen radicals due to the interaction between the microbial cells and antimicrobial agent. It was concluded that the optimal balance between antimicrobial activity and cytotoxicity could be achieved by a variation of the ZnTiO3 concentration. The antifungal and broad-spectrum antibacterial activity of the studied collagen/ZnTiO3 nanocomposites, combined with a low cytotoxicity, makes them a promising anti-infection biomaterial.
Keywords: Collagen/ZnTiO3 nanocomposites; Broad-spectrum antibacterial and antifungal activity; Cytotoxicity (osteosarcoma, fibroblast, and keratinocyte cells)