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

Thermostable alpha-amylase enzyme production from Bacillus laterosporus: Statistical optimization, purification and characterization

N. Manoj Kumar^a, ,S. Karthikeyan^a,G. Jayaraman^b

 

                                                                                                                            

 

Abstract

Production and characterization of a thermostable alpha-amylase enzyme from Bacillus laterosporus isolate was investigated in this study. Box-Behnken design (BBD) of response surface methodology (RSM) was applied to evaluate the optimal level of four medium components (yeast extract, starch, peptone and NaCl).R² value of 0.867 showed that model was well fitted with the experimental data and linear effect of starch, yeast extract and peptone were found significant on alpha-amylase production. Optimum values of yeast extract, starch, peptone and NaCl were predicted as 0.58%, 2.44%, 2.34% and 0.11%, respectively, with maximum enzyme activity of 4.838 U/ml. Enzyme purification was carried out by ammonium sulphate precipitation and size exclusion chromatography (SEC). Maximum purification was obtained by SEC with 4.71 fold. Effect of temperature and pH were studied on purified enzyme and the maximum activity was observed at optimal conditions of temperature (60 °C) and pH 7. Presence of Ca²⁺ ions and EDTA does not affect the enzyme activity, however reduced activity of the enzyme was observed in presence of Mg²⁺ ions, SDS and β-mercaptoethanol.

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Keywords

• Thermostable;Bacillus laterosporus;Box-Behnken design (BBD);Response surface methodology (RSM); Size exclusion chromatography (SEC)

Purification and characterization of Pseudomonas aeruginosalipase produced by SSF of deoiled Jatropha seed cake

Chetna Joshi,S.K. Khare^,

                                                                                                   

Abstract

A solvent stable lipase from Pseudomonas aeruginosa PseA is described. The lipase was produced by solid-state fermentation of deoiled Jatropha seed cake and purified by ultrafiltration and gel filtration chromatography using Sephadex G-100. The purified lipase was characterized for its enzymatic properties and stability. It exhibited remarkable stability in a wide range of organic solvents up to 75% (v/v) concentrations. Circular dichroism and intrinsic fluorescence spectra were recorded to investigate the effect of organic solvents on the secondary and tertiary structures. The secondary structure of the lipase was predominantly α-helical which remained unperturbed even at high concentration of organic solvents after 24 h of exposure. Highly hydrophobic tetradecane caused loss of activity and structure of lipase. The solvent stable lipase was exploited for an efficient synthesis of ethyl butyrate in n-hexane medium.

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Keywords

• Lipase;Organic solvent tolerant enzyme;Pseudomonas aeruginosa;Ethyl butyrate synthesis

Pleurotus ostreatus as a source of enoate reductase

Andrzej Skrobiszewski^a, ,Rafał Ogórek^b,Elżbieta Pląskowska^b, ,Witold Gładkowski^a,

                                                                                                            

 

 

Abstract

(E)-4-Phenylbut-3-ene-2-one (2a) and its derivatives with different substituents at phenyl ring (2b–e) have been subjected to biotransformation mediated by whole cells of Pleurotus ostreatus. The strain showed enoate reductase activity towards all the substrates tested. Saturated ketones with p-isopropyl-, p-methoxy- and 2,4-dimethoxyphenyl ring (3c–e) were obtained with 100% chemoselectivity. In case of (E)-4-phenylbut-3-ene-2-one (2a) and (E)-4-(benzo[1,3]dioxol-5-yl)but-3-en-2-one (2b) CC bond reduction was slowly followed by CO bond reduction, which afforded corresponding saturated alcohols 4a,b, respectively with low or moderate predominance of (S)-isomers. The presence of enoate reductase in P. ostreatus as well as enantiomerically enriched alcohol 4b has not been reported so far.

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Keywords

• Pleurotus ostreatus;Bioreduction;α,β-unsaturated aryl ketone;Enoate reductase;Carbonyl reductase

Effect of ultrasound-assisted irradiation on the activities of α-amylase and amyloglucosidase

Eloisa X. Leaes,Dirléia Lima,Luiza Miklasevicius,Adriana P. Ramon,Valéria Dal Prá,Mariana M. Bassaco,Lisiane M. Terra,Marcio A. Mazutti^,

                                                                                                                            

Abstract

This work is focused on the evaluation of the effects of ultrasound irradiation on the activity of α-amylase and amyloglucosidase. A central composite rotational design was carried out for each of the enzymes to evaluate the effects of temperature and pH on the activities in the presence and absence of ultrasound irradiation. The activation energy was also determined for both enzymes in the presence and absence of ultrasound irradiation. The results obtained for α-amylase and amyloglucosidase indicated that the ultrasound alters the behavior of enzymes, since they responded differently to alterations of pH and temperature in the presence of ultrasound comparing with effects in the absence. For temperatures up to 50 °C the activities of enzymes were always higher in the presence of ultrasound irradiation than in the absence. The activation energy of both enzymes in the presence of ultrasound was considerably reduced. The ultrasound showed to be a promising alternative to improve the enzyme activity, presenting potential for industrial application in enzyme reactions.

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Keywords

• α-Amylase;Amyloglucosidase;Ultrasound irradiation;Activation energy

Enhancement of specific activity of Xylanase produced by Ca-alginate entrapped cells of Aspergillus candidus: Optimization by Box–Behnken design

Debabrata Garai,Vineet Kumar^,

 

                                                                                                                     

Abstract

Calcium -alginate entrapment method for the production of xylanase by Aspergillus candidus was optimized by statistical design. Influential parameters like xylan concentration, bead loading and alginate concentration were optimized by Box–Behnken design and subsequent analysis and model validation by second order regression equation. The level of three variables such as xylan concentration, 19.0 g/l; number of bead loading, 112 and alginate concentration, 3.09% were found to be the optimum for maximum xylanase production. Under optimized condition maximum xylanase activity observed 100.23 IU/ml, which was 9% higher than predicted by the model. Organic nitrogen sources were found suitable for xylanase biosynthesis and mixture of yeast extract and peptone was emerged as best among all nitrogen sources tested. Specific activity of xylanase produced under immobilized condition was found 2.8 times higher than that produced under free cells. In repeated batch process maximum xylanase produced at second cycle and it retained more than 50% operational efficiency at the end of 4th cycle. Operational efficiency was found to increase when reused bead loading was reduced up to 50% after the end of 4th cycle.

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Keywords

• Aspergillus candidus;Immobilization;Box–Behnken design;Xylanase;Repeated batch fermentation; Calcium alginate

Thermal stability of Candida antarctica lipase B immobilized on macroporous acrylic resin particles in organic media

Yadagiri Poojari^, ,Stephen J. Clarson

 

                                                                                                                   

Abstract

Lipase B from Candida antarctica (CALB) has been exploited by many researchers for synthesis of a variety of organic compounds through esterification or transesterification reactions. Among the various immobilization media reported in the literature, the porous acrylic resin utilized in Novozym-435 has been widely studied. However, the thermal stability of free CALB and immobilized CALB (Novozym-435) in organic media at elevated temperatures for prolonged periods of time is largely unexplored. Here, we present the thermal stability and swelling of the Novozyme-435 in organic solvents (toluene and diphenyl ether) where the enzyme activity was quantified using an octyl laurate assay.

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Keywords

• Candida antarctica Lipase B;Thermal stability;Swelling;Organic media;Biocatalysis;Esterification; Transesterification

Methods for microbial screening and production of polyol oils from soybean oil through bioprocess

Ching T. Hou^a, ,Jiann-Tsyh Lin^b

 

                                                                                                                     

Abstract

Soypolyol oils (oxygenated acylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. Currently, they are produced by a two-step chemical process involving epoxidation and the subsequent opening of the oxirane ring. The objective of this study is to develop a bioprocess to produce polyol oils directly from soybean oil. For product separation, we found that TLC with a two step development solvent systems could separate the polyol products from substrate soybean oil. The products and substrates were separated in the following order: substrate triacylglycerols (R_f 0.8), free fatty acids (FA, R_f 0.7), product dihydroxy TAG (R_f 0.4), trihydroxy TAG (R_f 0.3), monohydroxy FA (R_f 0.1) and dihydroxy FA (R_f 0.05). We also found that HPLC with a C18 reverse phase column and a linear gradient of 100% methanol to 100% 2-propanol over 60 min at 1 mL/min flow rate was able to separate product polyol oils and substrate soybean oil. Free FA and polyol oils (diacylglycerols (DAG) containing hydroxyl FA) were eluted between 5 min and 15 min. DAG containing two normal FA was eluted between 15 min and 28 min and the substrate soybean oil was eluted between 36 min and 45 min. A total of 400 microbial cultures were screened and we identified 25 hits. Polyol oils' products were purified through a silica gel column chromatography, fractionated by HPLC and then analyzed by MS. A total of 57 molecular species of DAG containing tri-, di-, monohydroxy FA and normal FA were identified by MS. HPLC chromatogram of evaporative light scattering detector was used for semi-quantification of these DAG in the purified polyol oils. The total content of the DAG containing two normal FA was about 25% and the total content of DAG containing hydroxy FA might be about 75%. The yield of bioconversion by culture A01-35 from soybean oil to polyol oil products (DAG containing hydroxyl FA) plus DAG containing normal FA was 31% by weight. Bioconversion of soybean oil to polyol oils is a new research area without available methodology to follow. Here we report a new bioprocess for the production of soypolyol oils directly from soybean oil.

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Keywords

• Soypolyol oils,Soybean oil;Bioconversion;Hydroxy diacylglycerols;Hydroxy fatty acids;Oils separation

Substrate preference and oxygen requirement for cyanophycin synthesis by recombinant Escherichia coli

Daniel K.Y. Solaiman^, ,Richard D. Ashby,Jonathan A. Zerkowski

 

  

                                                                                                          

Abstract

Cyanophycin (CGP) is a bacterial bioproduct having a straight-chain poly(aspartic acid) as a backbone with arginine pendant groups attached to it. It has many potential industrial applications in the areas of water softening, hydrogel, metal–ion chelation, and nutriceuticals. Biotechnological production of CGP employs as producing strains the recombinant organisms that express heterologous cyanophycin synthase (cph) gene. A systematic study of fermentation parameters influencing CGP synthesis by a recombinant Escherichia coliexpressing a cphA of Synechocystis sp. showed that high aeration conditions as provided by 400 rpm stirrer speed and 1.0 L/min air flow in a Sixfors vessel (450 mL culture working-volume) resulted in high yields of cell biomass and crude CGP product. Glycerol substrate was found to yield 1.8-times higher crude CGP than glucose did under similar conditions. With glycerol as substrate, we found that a simplified fermentation scheme consisting of a straight 48-h fermentation at 37 °C (without a 30–37 °C temperature-shifting induction step) yielded compatible or higher amounts of crude CGP as those obtained under various temperature-shifting conditions. By studying the effects of glycerol concentration on CGP yields and analyzing glycerol consumption patterns, we demonstrated that substrate-to-product conversion could be increased by at least 15% and that costly leftover of unused substrate could be alleviated. The results yielded valuable information for optimization of fermentative production of CGP using glycerol that could be obtained as surplus coproduct from biodiesel production.

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Keywords

• Bioglycerol;Biopolymer;Poly(amino acids)

Functional diversification of the Tubby-like protein gene families (TULPs) during eukaryotic evolution

Chia-Ping Lai^a, Po-Hsuan Chen^b, Jen-Pan Huang^{c, d}, Yun-Huei Tzeng^{c, e}, Shu-Miaw Chaw^{c, ,} ,Jei-Fu Shaw^{f, g}

 

                                                                                                                      

 

Abstract

The Tubby-like protein (TULP) is ubiquitous among multicellular organisms but has extremely diversified functions. Although functional analyses of certain mammalian and model plant TULPs are available and phylogenies separately based on animal or plant TULPs have been reconstructed, the origin, evolutionary history, and functional divergence of TULPs remain obscure. The existence of TULP in unicellular organisms is also not found yet. In this study, 145 TULP homologs were identified from four eukaryotic supergroups—Excavata, Chromalveolata, Archaeplastida (plants), and Opistokonta (fungi and animals). In addition to the tubby domain, many TULPs also contain different motifs (F-box, WD40 repeat, or SOCS box) in their N-terminus regions, leading to their groupings into three classes, in which TULPs with no N-terminal modifications (class II) locate at the basal positions on the phylogeny. Our comparison data reveals that phosphatidylinositol 4,5-bisphosphate (PIP₂) binding sites are conserved in class II TULPs, especially those that are animal derived, but not in TULPs from classes I and III. Differentiation at the three PIP2 binding sites implies that the function of class II TULPs differs from those of other two classes. We further demonstrate that only class II TULPs can be targeted to the plasma membrane, suggesting functional divergence and differentiation of TULPs. Functional divergence and differentiation of TULPs should have occurred after duplications and structural modifications around 1100 MYA as eukaryotic organisms started diversifying.

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Keywords

• Tubby;TULP gene family;Arabidopsis;F-box domain;Evolution

Effect of corn quality on bioethanol production

Vijay Singh

 

                                                                                                                  

 

Abstract

Corn quality affects dry grind ethanol yields. Average loss in ethanol yield due to grain quality can range from 3 to 23%. Corn quality factors (kernel composition, test weight, endosperm hardness, planting location, crop year and mycotoxins) affected or controlled by genetics, environment and postharvest practices were evaluated for dry grind ethanol yield. Significant effects of kernel composition, endosperm hardness, crop year, planting location, harvest moisture content and drying air temperature were observed on ethanol yields. However, effect of alfatoxin B1 (mycotoxins) or Stenocarpella maydis (fungus) on ethanol yield was not significant.

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Keywords

• Corn quality;Bioethanol;Genetics;Environment;Post-harvest practices

Effect of corn bran as dietary fiber addition on baking and sensory quality

Mukti Singh,Sean X. Liu,Steven F. Vaughn

 

                                                                                                                               

 

Abstract

Development of wholesome and nutritious fiber rich food products with acceptable functional and sensory quality is a major industrial concern, seeking to capture consumer's interest in healthy and functional foods. Dietary fiber in corn bran is known for its beneficial effects on human health and nutrition. The main objective was to develop and characterize cakes with added corn bran to increase the dietary fiber intake in the form of purified fine food-grade corn bran (free of germ and endosperm), a byproduct from the grain milling industry that is a good source of dietary fiber replaced flour in cakes at 0%, 5%, 15%, 20%, 25%, and 30% level. The effects of flour replaced with corn bran on batter viscosity, cake volume, crumbgrain, cake color, cake texture cake were examined. Hardness and springiness of cakes were not affected by the increasing levels of corn bran replacement in cake batter. Flour replaced (20%) by corn bran resulted in cakes with acceptable sensory scores based on texture, taste and overall acceptability of the cakes. This study will provide important information to the food industry developing functional ingredients in baked foods and benefit the baking industry by generating potentially new food products with healthful attributes.

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Keywords

• Corn bran; Particle size; Sensory evaluation; Rheology; Physical characteristics; Baking quality

Biochemical, structural and functional characterization of two novel antifungal endoglucanases from Anabaena laxa

Vishal Gupta¹, Radha Prasanna Vidhi Chaudhary, Lata Nain

 

                                                                                                                         

 

Abstract

An investigation was undertaken to analyse the various biochemical and structural facets of two purified endoglucanases (End1 and End2) exhibiting antifungal activity from Anabaena laxa. SDS-PAGE analyses indicated that End1 and End2 proteins were 38 and 74 kDa, respectively. Both the purified End proteins showed activities (high β-1,4 in both End1 and End2 and low β-1,3-endoglucanase activity only in End2) in the zymograms. Thin layer chromatography (TLC) analyses of the hydrolyzed products using different substrates confirmed endo and exo/endo-type nature of End1 and End2, respectively. The highest endoglucanase activity of both the purified End proteins was recorded at pH 6.0 and temperature 30 °C, however, stabilities of End1 and End2 were recorded over a wide range of pH (5.0–7.0 and 5.0–9.0, respectively) up to 12 h incubation; and temperature of 40 and 50 °C, up to 3 h incubation. The predicted three dimensional structures of End1 and End2 were found to be distorted beta-barrel and beta-barrel, respectively. The binding sites of both End proteins were identified and cellotetraose substrate was found to completely span over the active site in the three dimensional cartoon and surface models for End2, confirming the biochemical and kinetic analyses. Site-directed mutagenesis of the end1 and end2 led to the identification of Glu-23 and Cys-11 (present at signal peptide regions) as catalytic residues critical to antifungal activity, respectively. To our knowledge, this study represents a first time in-depth investigation into the structural/functional aspects of purified endoglucanases from cyanobacteria.

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Keywords

• Antifungal activity; Cyanobacteria; Endoglucanase; Site-directed mutagenesis;TLC

Production and characterization of xylanase from Bacillus licheniformis P11(C) with potential for fruit juice and bakery industry

Bijender Kumar Bajaj^, ,Kalpana Manhas

                                                                                             

Abstract

Bacterial isolate Bacillus licheniformis P11(C) efficiently utilized agricultural residues as carbon and nitrogen sources to generate substantial amount of xylanase which exhibited activity and stability over broad pH range (5–11) and over elevated temperatures (40–100 °C), and even in presence of potential inhibitors (triton, SDS, EDTA). Purification of xylanase (4.24-fold) by ammonium sulphate precipitation and DEAE-sepharose chromatography, and analysis by SDS-PAGE and zymography showed that B. licheniformis P11(C) produced two xylanases (17.5 and 23 kDa). Furthermore, xylanase displayed exciting potential for application in fruit juice processing and bakery processes; enzyme was found to be effective in getting enhanced sugar extraction from fruit juices, clarification of fruit juices, and substantial dough-raising in bakery.

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Keywords

• Xylanase; Production; Purification; Characterization; Applications;Bacillus licheniformis

Medium optimization for a novel crude-oil degrading lipase fromPseudomonas aeruginosa SL-72 using statistical approaches for bioremediation of crude-oil

 

Shikha Verma^a,Jyoti Saxena^b,Radha Prasanna^c,Vinay Sharma^a,Lata Nain^c,

                                                                                                                     

Abstract

A meso-alkaliphilic strain of Pseudomonas aeruginosa SL-72 was isolated from the soil contaminated with the effluents from mustard-oil plant. Statistical tools were employed to optimize the medium constituents to enhance lipase-yield. Plackett–Burman design evaluated the effect of six medium constituents which significantly influence lipase-production, cell growth and microbial activity. Variance of analysis (ANOVA) showed that Tween-80, (NH₄)₂HPO₄ and MgSO₄·7H₂O were the most significant variables (p<0.05). Further, Central Composite Design (CCD) was used to optimize the most significant medium constituents and the optimum values estimated by multiple response optimization were 0.5% Tween-80, 1.0% (NH₄)₂HPO₄, and 0.1% MgSO₄·7H₂O, with a predicted value of 5038.86 IU ml⁻¹ lipase-production. Evaluation in a fermenter using the optimized medium constituents resulted in an approximately 1.59-fold increase over the central-point (3155.24 IU ml⁻¹) and 3.68-fold increase under unoptimized culture conditions (1376.60 IU ml⁻¹). This strain had also effectively degraded 82.83% crude-oil within a week, for safe disposal of wastewater.

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Keywords

• Crude oil;Lipase; Pseudomonas aeruginosa; Medium optimization; Response surface methodology

Agricultural residues as potential substrates for production of xylanase from alkali-thermotolerant bacterial isolate

Bijender Kumar Bajaj^, ,Yash Paul Khajuria,Vijay P. Singh

 

                                                                                                                       

Abstract

Xylanolytic Bacillus pumilus SS1 grew well at highly alkaline pH (9–11) and at moderately high temperature (25–55 °C) and successfully utilized wheat bran as the sole carbon source and produced considerable titer of xylanase. Maximum enzyme production occurred at medium pH 8 and at 45 °C. The enzyme purified (2.97-fold) with ammonium sulfate fractionation and carboxymethyl sephadex chromatography, showed molecular weight of 25 kDa, low K_m (2.7 mg/ml) and reasonably good V_max (36 μmol/mg/min). Enzyme showed maximum activity at pH 6–8, and at 40–50 °C. Enzyme was inhibited by HgCl₂, CoCl₂, MnSO₄, MgCl₂, phenylmethylsulphonylfluoride and CaCl₂, while FeCl₂ caused slight stimulation.

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Keywords

• Bacillus pumilus SS1; Fermentation; Purification; Wheat bran; Xylanase; Zymogram

Purification and characterization of solvent tolerant lipase from Bacillus sphaericus MTCC 7542

K. Tamilarasan^{a, ,}M. Dharmendira Kumar^b

 

                                                                                                   

 Abstract

Solvent tolerant extracellular lipase from Bacillus sphaericus MTCC 7542 was purified by DEAE–Sepharose anion exchange chromatography. This purification resulted in 377 U/mg specific activity and 17.33-fold with 5.7% recovery obtained. The molecular weight of the purified lipase was determined to be 69 kDa using SDS polyacrylamide gel electrophoresis and conformed by zymogram. The purified lipase showed optimal activity and stability at pH 8.0 and 40 °C. The purified lipase was stable in the presence of hydrophobic solvents liken-butanol, benzene, hexane and toluene. Lipase activity was promoted in the presence of Mg²⁺, Ca²⁺, Cu²⁺, K⁺ and Tween 80.

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Keywords

• Molecular weight;,Zymogram;Electrophoresis;Stability;Lipase activity

Simulation of the xanthan gum production in continuous fermentation systems

Giovani L. Zabot^a, Marceli F. Silva^a, Lisiane de Marsillac Terra^b, Edson L. Foletto^b, Sérgio L. Jahn^b,  Valéria Dal Prá^a, J. Vladimir Oliveira^c, Helen Treichel^d, Marcio A. Mazutti^b,

                                                                                                            

Abstract

This work is focused on the simulation of the bioproduction of xanthan gum in continuous fermentation systems. We evaluated the cell and gum productivities in a continuous system without cell recycling and in continuous systems with internal and external cell recycling. Results demonstrated that the continuous system can improve the production of gum, since the gum productivity obtained in the systems with external and internal cell recycling were 2.57 and 3.62 g L⁻¹ h⁻¹, respectively, which was about 12–17 times higher than obtained in the reactor without cell recycling.

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Keywords Bioreactor; Recycling; Xanthan gum; Scale up; Xanthomonas campestris

Production of methyl oleate with a lipase from an endophytic yeast isolated from castor leaves

Anne Caroline Defranceschi Oliveira^a, Felipe Miguel Farion Watanabe^a, José Viriato Coelho Vargas^a,  Maria Luiza Fernandes Rodrigues^b, André Bellin Mariano^a,

                                                                                             

 Abstract

This work aims to optimize lipase production through experimental design and to immobilize these biocatalysts for methyl oleate synthesis. For that, it was applied a 2⁶⁻³ experimental design to optimize the lipase production, reaching the production of 25 U mL⁻¹. The enzymes produced by submerged fermentation were obtained from an endophytic yeast Candida guillermondi isolated from castor leaves (Ricinus communisL.). The obtained enzyme was partially purified and freeze-dried before the immobilization process using agarose and silica gel supports. The distribution of the enzyme on the silica gel was homogeneous and verified by atomic force microscopy. The free and the immobilized enzymes were evaluated by the methyl oleate synthesis in order to compare the efficiencies of the reaction processes. The immobilization process increased the conversion rates for the synthesis of methyl oleate in all immobilized enzymes experiments. In addition to the immobilization process ensuring greater efficiency, the immobilized enzyme could also be reused. Therefore, the enzyme used in this work is expected to be a good biocatalyst to be applied in ester synthesis in the food and biofuel industry or laboratorial applications.

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Keywords :Lipase; Esterification; Immobilization; Endophytic yeast; Submerged fermentation; Candida guillermondi

Influence of process parameters on the immobilization of commercial porcine pancreatic lipase using three low-cost supports

• Robison P. Scherer^a, Rogério L. Dallago^b, Fábio G. Penna^b, Francile Bertella^b, Débora de Oliveira^a, , J. Vladimir de Oliveira^a, Sibele B.C. Pergher^c

                                                                                                                        

 

Abstract

The use of lipases as biocatalysts has attracted much attention recently, but industrial application has been hindered mainly due to the high production cost, determined by the production yield and enzyme stability. Immobilization of biocatalysts in inert supports can ensure their use for several batches, making possible to build cost-effective processes. The objective of this work was to investigate the influence of immobilization time and enzyme to support mass ratio on the yield of immobilization and esterification activity of porcine pancreatic lipase. Higher immobilization yields (38.2%) were obtained for pillared montmorillonite, 120 min of immobilization and enzyme to support mass ratio of 2:0.5. The highest esterification activity (1403 U/g) was achieved using the montmorillonite, after 180 min of immobilization and enzyme to support ratio of 2:1. The characterization of the supports, free and immobilized enzyme on different supports made possible to elucidate the immobilization results through the knowledge of support features.

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Keywords

• Porcine pancreatic lipase; Immobilization; KSF; Natural montmorillonite; Pillared montmorillonite

Natural montmorillonite as support for the immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571

 Chaline C. Coghetto^a, Robison P. Scherer^a, Marceli F. Silva^a, Simone Golunski^a, Sibele B.C. Pergher^b,  Débora de Oliveira^{c, 1}, J. Vladimir Oliveira^{c, 1},Helen Treichel^{d, e}

                                                                                                                         

Abstract

The objective of this study was to investigate the process of immobilization of inulinases using natural montmorillonite as inorganic support. The enzyme to buffer ratio of 3:10 and 10 min of immobilization led to the highest specific activity, 375.07 U/mg protein. The immobilized inulinase kept its activity after 1968 h under storage at low temperatures and after 456–1826 h at high temperatures. The pH value of 3.5 led to the highest specific activity. K_m values of 1.46 and 0.38 mM, and v_max of 0.2487 and 0.2396 mol/L min, were obtained, respectively, for sucrose and inulin.

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Keywords

• Kluyveromyces marxianus NRRL Y-7571; Inulinase; Inorganic support; Immobilization

Lipase-catalyzed synthesis of poly(e-caprolactone) in supercritical carbon dioxide

Roseni D. Santos^a, Sibele R. Rosso Comim^a, Débora de Oliveira^a,  Helen Treichel^b, Marco Di Luccio^a, Sandra R.S. Ferreira^a, J. Vladimir Oliveira^a

 

                                                                                                                  

Abstract

In this study, the synthesis of poly(ε-caprolactone) by lipase-catalyzed reactions in supercritical carbon dioxide was performed in a batch reactor in order to evaluate the influence of the operating conditions on polymer chain size and on polydispersity index. Firstly, a fractional 2⁴⁻¹ factorial design was performed to evaluate the effect of temperature, concentration of enzyme, water content and agitation. Then, a full 2³experimental design was carried out to evaluate the effects of pressure, enzyme concentration and water content. The best result was a polymer chain size of 7419.6 g/gmol, obtained in a reaction conducted at 145 bar, 65 °C, 200 rpm, 10 wt% enzyme concentration and at the lowest water content.

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Keywords

• Polycaprolactone; Lipase; Carbon dioxide; Biopolymer

Optimization of reaction parameters for enzymatic glyceride synthesis from fish oil: Ethyl esters versus free fatty acids

Helle C. Ravn^b, Marianne L. Damstrup^a, Anne S. Meyer^b,

                                                                                               

Abstract

Enzymatic conversion of fish oil free fatty acids (FFA) or fatty acid ethyl esters (FAE) into glycerides via esterification or transesterification was examined. The reactions catalyzed by Lipozyme™ 435, a Candida antarctica lipase, were optimized. Influence on conversion yields of fatty acid chain length, saturation degree, temperature, enzyme dosage, molar ratio glycerol:fatty acids, acyl source composition (w/w ratio FFA:FAE), and reaction time was evaluated collectively by multiple linear regression. All reaction variables influenced the conversion into glycerides. Transesterification of FAE produced the highest yields of 94–95% (w/w) conversion yield at 1:3 glycerol:FAE, 25 h, 66 °C, enzyme dosage 3.1%w/w.

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Keywords

• Immobilized Candida antarctica lipase B; Fish oil concentrates; Omega-3 fatty acids; Central composite design; Esterification; Transesterification