Site saturation mutagenesis: Methods and applications in protein engineering

Rodrigo M.P. Siloto ,Randall J. Weselake^,

                                                                      

Abstract

Mutagenesis strategies have been applied to fine-tune different enzyme properties including substrate specificity, thermostability, enantioselectivity or simply to increase activity. The mechanistic understanding of how a particular enzyme operates often dictates suitable mutagenesis strategies. Site saturation mutagenesis is used to substitute targeted residues to any other naturally occurring amino acid. Here, we review several aspects of this mutagenesis approach, comparing different molecular techniques to produce libraries of single-residue substitutions. A discussion of experimental design and combinatorial mutagenesis in light of screening capabilities is provided. Several examples of this mutagenesis strategy applied to directed evolution and structure–function studies are also discussed.

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Keywords

• Mutagenesis;Directed evolution;Structure–function;Codon-degeneracy;Biocatalysis;Protein engineering

Antibacterial activity of 7,10-dihydroxy-8(E)-octadecenoic acid against food-borne pathogenic bacteria

 

Hye-Ran Sohn^a,Ka-Yeon Baek^a,Ching T. Hou^b,Hak-Ryul Kim^a,

                                                                                                     

Abstract

A bacterial strain Pseudomonas aeruginosa (PR3) has been intensively studied to produce a novel 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from oleic acid or natural vegetable oils containing oleic acid. However, biological properties of DOD remained unknown so far. In this study, as a trial to determine the biological properties of DOD molecule, antibacterial activities of DOD against food-borne pathogenic bacteria were determined quantitatively and qualitatively. DOD presented strong antibacterial activities against all the bacterial strains tested with an MIC value being in the range of 125–500 μg/ml and there was no activity preference between gram-positive and gram-negative strains.

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Keywords

• Antibacterial activity;Bioconversion;7,10-dihydroxy-8(E)-octadecenoic acid;Oleic acid;Pseudomonas aeruginosa;Vegetable oil

Utilization of enzymatically interesterified cottonseed oil and palm stearin-based structured lipid in the production of trans-free margarine

 

Garima Pande,Casimir C. Akoh^, ,Robert L. Shewfelt

 

                                                                                                                     

Abstract

trans-Free margarine fat was enzymatically synthesized from palm stearin (PS) and regular cottonseed oil (CO). Response surface methodology (RSM) was used for optimization. The independent variables were substrate molar ratio (PS:CO, 2–5), temperature (50–65 °C), time (6–22 h), and lipases (Lipozyme^® TLIM and Novozym^® 435). Incorporation of stearic acid (mol%) was the dependent variable. Desirable products composition 5.2 mol% stearic acid were achieved at 57 °C, 14 h, 4:1, using Lipozyme TLIM, and at 56 °C, 6 h, 4:1, using Novozym 435 (5.9 mol% stearic acid). Using optimal conditions, structured lipids (SLs) were synthesized in a 1 L stir-batch reactor. Solid fat contents at 25 °C were lower for SLs (24.8–30.8%) than the corresponding physical blends (34.7–39.3%). Novozym 435-catalyzed SL product had desirable fatty acid profile, physical properties, β′ polymorph, and was further used to formulate margarine. Compared to commercial margarine (19.1 mol% trans fatty acids (TFA), the SL containing margarine had no trans fat. However, it was harder and less spreadable than commercial margarine but no difference was observed in their flavor. In this study, we were able to formulate trans-free margarine suitable for possible use as hard/industrial margarine.

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Keywords

• Enzymatic synthesis;Trans-free;Margarine;Response surface methodology

Substitution of yeast extract by ammonium sulfate for mannitol production in cashew apple juice

Cláudia P.M.L. Fontes^a,Mariana Santiago Silveira^b,Alexandre A. Guilherme^b,Fabiano A.N. Fernandes^b, Sueli Rodrigues^a,

                                                                                                                        

Abstract

The use of agriculture excess as substrate in industrial fermentations became an interesting alternative to reduce production costs and to reduce negative environmental impact caused by the disposal of these products. In this work, the use of cashew apple juice supplemented with ammonium sulfate as substrate for mannitol production by two Leuconostoc strains was studied. The carbohydrates of cashew apple juice are glucose and fructose. Process pH was not significant on fructose conversion into mannitol by Leuconostoc citreum B-742. However, for L. mesenteroides B-512F the process pH showed a significant effect on fructose conversion into mannitol. The use of cashew apple juice supplemented with ammonium sulfate in replacement of yeast extract enhanced the yield L. mesenteroides B-512F was the best mannitol producer. The optimal pH for mannitol production from fructose by this strain in pH-controlled fermentation was at pH 5.0 with mannitol yield of 95% and productivity of 1.6 g/L h. Lactic acid bacteria are known as nutritional exigent microorganisms. Usually, they are cultivated in yeast extract and other complex nitrogen source. Herein, the combination of an inorganic exogenous nitrogen source and the amino acids from the cashew apple juice was enough to support LAB growth in a low cost substrate.

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Keywords

• Mannitol production;pH-controlled fermentation;Leuconostoc mesenteroides;Cashew apple juice; Ammonium sulfate

Enhanced bioaccumulation of astaxanthin in Phaffia rhodozymaby utilising low-cost agro products as fermentation substrate

Prakash Chandra Bhatt,Makhmur Ahmad,Bibhu Prasad Panda^,

 

                                                                                                                 

Abstract

Low-cost medium component for fermentation and the down-streaming process for intracellular astaxanthin production by Phaffia rhodozyma MTCC 7536 were optimized using statistical methods. From a total of fifteen, four important natural media components were screened based on biomass, total carotenoids and astaxanthin production. The statistical model was constructed via Box–Behnken design using selected medium parameters (Calendula officinalis flower, Zea mays seed flour, Triticum aestivum seed flour,Pennisetum glaucum seed flour). Under optimized condition, C. officinalis (dried and crushed marigold flower) 2.5 g l⁻¹, Z. mays (corn flour) 2.38 g l⁻¹, T. aestivum (wheat flour) 1.89 g l⁻¹ and P. glaucum (pearl millet flour) 2.47 g l⁻¹, astaxanthin production was 1448.38 μg g⁻¹ (dry cell weight) with volumetric yield of 1333.96 μg ml⁻¹ (R²=0.908, p-value<0.0001 and validly=94.9%). A successful and significant improvement (12-fold) in the production of intracellular free astaxanthin by the P. rhodozyma was achieved by adding low-value agro products in fermentation medium, which may result in a significant reduction in the cost of fermentation process.

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Keywords

• Low-cost fermentation medium;Astaxanthin;Phaffia rhodozyma;Carotenoids

Exploitation of chicken feather waste as a plant growth promoting agent using keratinase producing novel isolate Paenibacillus woosongensis TKB2

Tanmay Paul^a,Suman K. Halder^a,Arpan Das^a,Surojit Bera^a,Chiranjit Maity^a,Arpita Mandal^b,Partha S. Das^a,Pradeep K. Das Mohapatra^a,Bikas R. Pati^a,Keshab C. Mondal^a

 

                                                                                                              

Abstract

A potent feather degrading bacterium was isolated from soil and it was identified as Paenibacillus woosongensis TKB2 on the basis of morphological, biochemical and as well as 16S rDNA sequence characteristics. The physico-chemical cultural condition for keratinase (principal feather degrading enzyme) production by the isolate was optimized following OVAT (one variable at a time) protocol. It was found that the bacterium produced highest amount of keratinase at 0.75% (w/v) raw feather as sole substrate, 2% (v/v) inoculums, pH 8.5, incubation temperature 30 °C, 5% (w/v) sodium chloride and other mineral components like 0.05% K₂HPO₄, 0.025% MgSO₄ and 0.02% CaCO₃ (w/v). The filter sterilized fermented hydrolysate has the ability to promote significantly the germination of seeds (germination rate 87.5%) and growth of Bengal gram (Cicer arietinum) seedlings. It induces the nodule formation (3 fold) and increased soil fertility by altering N, P, K and the C/N ratio by 1.2 fold. This also enhanced the quantity of free living nitrogen fixers (2 fold) and phosphate solubilizers (5.8 fold) in comparison to the control soil. Thus the enzymatic feather hydrolysate can be exploited as a useful biological fertilizer in future.

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Keywords:Chicken feather;Paenibacillus;Keratinase;Filter sterilized;Fertilizer

Production and partial characterization of extracellular glucose isomerase using thermophilic Bacillus sp. isolated from agricultural land

MohanaSundaram Sukumar^, ,Arun Jeyaseelan,Thirumalai Sivasankaran,Pennarasi Mohanraj, Prasanna Mani,Gowthamraj Sudhakar,Vimalraj Arumugam,Sasirekha Bakthavachalu,Alagulakshmi Ganeshan,Monika Susee

 

                                                                                                                   

Abstract

In this investigation, glucose isomerase was extracted from Bacillus sp., isolated from agricultural soil sample and purified further by ammonium sulfate fractionation followed by gel filtration chromatography using Sephadex G-25. The purified enzyme over crude was further subjected to partial characterization which includes the assessment of influence of pH, temperature, incubation time and metal ions on the stability and activity of glucose isomerase was observed. As a result, it was found that, glucose isomerase isolated from the thermophilic Bacillus sp. enumerated from the agricultural soil showed maximal stability at pH 8.0 and at temperature 70 °C within 35 min. It was also found that, at the minimum concentration of 5 mM Mg²⁺, Co²⁺and Mn²⁺ showed 41%–59% of enzyme activity.

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Keywords

• Extracellular glucose isomerase;Thermostable;Bacillus sp.;Fermentation;Stability