Applied Biochemistry and Biotechnology (v.164, #7)

Isolation, Identification and Optimization of a New Extracellular Lipase Producing Strain of Rhizopus sp. by Jayshree B. Kantak; Aditi V. Bagade; Siddharth A. Mahajan; Shrikant P. Pawar; Yogesh S. Shouche; Asmita Ashutosh Prabhune (969-978).
A lipolytic mesophilic fungus which produces lipase extracellularly was isolated from soil. Based on ITS1-5.8S–ITS4 region sequences of ribosomal RNA, it was concluded that the isolate JK-1 belongs to genus Rhizopus and clades with Rhizopus oryzae. The present paper reports the screening, isolation, identification, and optimization of fermentation conditions for the production of lipase (EC 3.1.1.3). Culture conditions were optimized, and the highest lipase production was observed in basal medium with corn steep liquor as nitrogen source and glucose as carbon source. Maximum lipase production was observed at 72 h, which is about 870 U/ml. Optimization of fermentation conditions resulted in 16-fold enhancement in enzyme production.
Keywords: Rhizopus ; ITS region; Lipase; Medium optimization; Fermentation; Corn steep liquor

Fermentation Characteristics of Mortierella alpina in Response to Different Nitrogen Sources by Jinmiao Lu; Chao Peng; Xiao-Jun Ji; Jiangying You; Leilei Cong; Pingkai Ouyang; He Huang (979-990).
The fermentation characteristics of Mortierella alpina were investigated in response to various nitrogen sources. Influences on nitrogen source and glucose uptake rate, mycelial morphology of M. alpina, and pH of medium in relation to different nitrogen sources were discussed. Effects of different nitrogen sources on cell growth, fatty acid composition, arachidonic acid (ARA), and total lipid concentration were also evaluated. It revealed that the maximum nitrogen source uptake ratio was obtained when corn steep liquor was used as nitrogen source. When yeast extract was used as the sole nitrogen source, glucose was completely exhausted at the end of fermentation. The maximum dry cell weight obtained from medium with yeast extract as nitrogen source had the highest total lipid concentration. Sodium nitrate was the favorable nitrogen source for ARA accumulation, and the highest ARA percentage in total fatty acids was obtained, 35.9%. Urea was identified as the favorable nitrogen source for ARA production, the highest ARA concentration obtained from urea was 5.8 g/l. Compared with inorganic nitrogen sources, organic nitrogen compounds are favorable for both cell growth and total lipids accumulation.
Keywords: Arachidonic acid; Mortierella alpina ; Nitrogen source; Fermentation characteristics

Significant studies on phospholipases optimization, characterization, physiological role and industrial potential have been conducted worldwide. Some of them have been directed for biotechnological advances such as gene discovery and functional enhancement by protein engineering. Others reported phospholipases as virulence factor and major cause of pathophysiological effects. A general overview on phospholipase is needed for the identification of new reliable and efficient phospholipase, which would be potentially used in number of industrial and medical applications. Phospholipases catalyse the hydrolysis of one or more ester and phosphodiester bonds of glycerophospholipids. They vary in site of action on phospholipid which can be used industrially for modification/production of new phospholipids. Catalytically active phospholipase mainly use phosphatidylcholine as major substrate, but they can also show specificity with other phospholipids. Several accurate phospholipase assay methods are known, but a rapid and reliable method for high-throughput screening is still a challenge for efficient supply of superior phospholipases and their practical applications. Major application of phospholipase is in industries like oil refinery, health food manufacturing, dairy, cosmetics etc. All types of phospholipases can be involved as virulence factor. They can also be used as diagnostic markers for microbial infection. The importance of phospholipase in virulence is proven and inhibitors of the enzyme can be used as candidate for preventing the associated disease.
Keywords: Phospholipase types; Assay methods; Industrial uses; Bacterial phospholipase; Fungal phospholipase; Pathogenicity; Virulence factor

Heterologous Expression of Transaldolase Gene Tal from Saccharomyces cerevisiae in Fusarium oxysporum for Enhanced Bioethanol Production by Jin-Xia Fan; Xiao-Xue Yang; Jin-Zhu Song; Xiao-Mei Huang; Zhong-Xiang Cheng; Lin Yao; Olivia S. Juba; Qing Liang; Qian Yang; Margaret Odeph; Yan Sun; Yun Wang (1023-1036).
The filamentous fungus Fusarium oxysporum is known for its ability to ferment xylose-producing ethanol. However, efficiency of xylose utilization and ethanol yield was low. In this study, the transaldolase gene from Saccharomyces cerevisiae has been successfully expressed in F. oxysporum by an Agrobacterium tumefaciens-mediated transformation method. The enzymatic activity of the recombinant fungus (cs28pCAM-Sctal4) was 0.195 times higher than that of the wild-type strain (cs28). The recombinant strain also exhibited a 28.83% increase in ethanol yield on xylose media compared to the parental strain. Enhanced ethanol production and a reduction in the biomass were observed during xylose fermentation. Ethanol yield from rice straw by simultaneous saccharification and fermentation with cs28pCAM-Sctal4 was 0.25 g g−1 of rice straw. The transgenic strain of F. oxysporum cs28pCAM-Sctal4 might therefore have potential applications in industrial bioenergy production.
Keywords: Transaldolase; Fusarium oxysporum ; Xylose fermentation; Bioethanol

Co-overexpression of PpPDI Enhances Secretion of Ancrod in Pichia pastoris by Shou-Tao Zhang; Hui-Min Fang; Li Zhao; Qing-Nan Tian; Yun-Fei Qin; Ping Lu; San-Jun Chen; Zhen-Xia Bao; Feng Liang (1037-1047).
Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, is highly specific for fibrinogen. It causes anticoagulation by defibrinogenation and has been used as a therapeutic anticoagulant for the treatment of moderate to severe forms of peripheral arterial circulatory disorders in a variety of countries. The DNA of ancrod was amplified by recursive PCR with a yeast bias codon and cloned into the pGEM-T Easy vector. In order to achieve a high level secretion and a full activity expression of ancrod in Pichia pastoris (P. pastoris), the P. pastoris protein disulfide bond isomerase (PpPDI) was co-overexpressed in the strain. The secretion characteristics of ancrod with and without PpPDI were examined. With co-overexpression of PpPDI, the production of recombinant ancrod (rAncrod) was increased to 315 mg/L in the culture medium, which is twofold higher than the control strain carrying only the ancrod gene. Through purified by Ni2+ affinity chromatography and phenyl Sepharose column, the purity of rAncrod was found to be as high as 95.2%. The fibrinogenolytic and zymographic activities of the rAncrod were determined and found to be similar to that of the native protein. This improved expression system can facilitate further studies and the industrial production of ancrod.
Keywords: Co-overexpression; Pichia pastoris ; PpPDI; Purification; rAncrod

The display of proteins to cyanobacterial cell surface is made complex by combination of Gram-positive and Gram-negative features of cyanobacterial cell wall. Here, we showed that Synechococcus outer membrane protein A (SomA) can be used as an anchoring motif for the display of organophosphorus hydrolase (OPH) on cyanobacterial cell surface. The OPH, capable of degrading a wide range of organophosphate pesticides, was fused in frame to the carboxyl-terminus of different cell-surface exposed loops of SomA. Proteinase K accessibility assay and immunostaining visualized under confocal laser scanning microscopy demonstrated that a minor fraction of OPH with 12 histidines fused in frame with the third cell-surface exposed loop of SomA (SomAL3-OPH12H) was displayed onto the outermost cell surface with a substantial fraction buried in the cell wall, whereas OPH fused in frame with the fifth cell-surface exposed loop of SomA (SomAL5-OPH) was successfully translocated across the membrane and completely displayed onto the outermost surface of Synechococcus. The successful display of the functional heterologous protein on cell surface provides a useful model for variety of applications in cyanobacteria including screening of polypeptide libraries and whole-cell biocatalysts by immobilizing enzymes.
Keywords: Cyanobacteria; Synechococcus PCC 7942; Organophosphorus hydrolase; SomA; Porin; Surface display

Onsite Enzyme Production During Bioethanol Production from Biomass: Screening for Suitable Fungal Strains by Annette Sørensen; Philip J. Teller; Peter S. Lübeck; Birgitte K. Ahring (1058-1070).
Cellulosic ethanol production from biomass raw materials involves process steps such as pre-treatment, enzymatic hydrolysis, fermentation, and distillation. Use of streams within cellulosic ethanol production was explored for onsite enzyme production as part of a biorefinery concept. Sixty-four fungal isolates were in plate assays screened for lignocellulolytic activities to discover the most suitable fungal strain with efficient hydrolytic enzymes for lignocellulose conversion. Twenty-five were selected for further enzyme activity studies using a stream derived from the bioethanol process. The filter cake left after hydrolysis and fermentation was chosen as substrate for enzyme production. Five of the 25 isolates were further selected for synergy studies with commercial enzymes, Celluclast 1.5L and Novozym 188. Finally, IBT25747 (Aspergillus niger) and strain AP (CBS 127449, Aspergillus saccharolyticus) were found as promising candidates for onsite enzyme production where the filter cake was inoculated with the respective fungus and in combination with Celluclast 1.5L used for hydrolysis of pre-treated biomass.
Keywords: Onsite enzyme production; Fungal screening; Beta-glucosidase; Cellulase; Bioethanol

Enriched Microbial Community in Bioaugmentation of Petroleum-Contaminated Soil in the Presence of Wheat Straw by Zuojun Wu; Hongjun Dong; Liangdong Zou; Diannan Lu; Zheng Liu (1071-1082).
The bioaugmentation of petroleum-contaminated soil using Enterobacter cloacae was profiled from the evolution of microbial community, soil dehydrogenase activity, to the degradation of petroleum contaminants. The seeding and proliferation of inoculant and the consequential microbial community were monitored by denaturing gradient gel electrophoresis analysis of the amplification of V3 zone of 16S rDNA. Degradation process kinetics was characterized by the degradation ratio of nC17 to nC18. The dehydrogenase activity was also determined during the degradation process. An abrupt change in the microbial community after inoculation was illustrated as well as successive changes in response to degradation of the petroleum contaminants. Seeding with E. cloacae stimulated the growth of other degrading stains such as Pseudomonas sp. and Rhodothermus sp. The application of wheat straw as a representative lignin waste, at 5% (w/w), induced an increase in the total dehydrogenase activity from 0.50 to 0.79, an increase in the microbial content of 130% for bacteria and 84% for fungi, and an increase of the overall degradation ratio from 44% to 56% after 56 days of treatment. The above mentioned results have provided a microbial ecological insight being essential for the design and implementation of bioaugmentation processes.
Keywords: Bioremediation; Bioaugmentation; Microbial ecology; Petroleum-contaminated soil; Enterobacter cloacae ; Wheat straw

The elimination of spores from low-acid foods presents food-processing and food-safety challenges to high-pressure processing (HPP) developers as bacterial spores are extremely resistant to pressure. Therefore, the effects of pressure (400–800 MPa), temperature (35–95°C), and nisin (0–496 IU/mL) on the inactivation of Clostridium perfringens AS 64701 spores at various pressure-holding times (7.5–17.5 min) were explored. A second-order polynomal equation for HPP- and nisin-induced inactivation of C. perfringens spores was constructed with response surface methodology. Experiment results showed that the experimental values were shown to be significantly in agreement with the predicted values because the adjusted determination coefficient (R Adj 2 ) was 0.9708 and the level of significance was P < 0.0001. The optimum process parameters (obtained by solving the quadratic polynomal equation) for a six-log cycle reduction of C. perfringens AS 64701 spores were pressure of 654 Mpa, temperature of 74°C, pressure-holding time of 13.6 min, and nisin concentration of 328 IU/mL. The validation of the model equation for predicting the optimum response values was verified effectively by ten test points that were not used in the establishment of the model. Compared with conventional HPP techniques, the main process advantages of HPP–nisin combination sterilization in the UHT milk are, lower pressure, temperature, natural preservative (nisin), and in a shorter treatment time. The synergistic inactivation of bacteria by HPP–nisin combination is a promising and natural method to increase the efficiency and safety of high-pressure pasteurization.
Keywords: High pressure; Nisin; Clostridium perfringens spore; Central composite rotatable design; Response surface model

Alkaline proteases from the viscera of the striped seabream (Lithognathus mormyrus) were extracted and characterized. Interestingly, the crude enzyme was active over a wide range of pH from 6.0 to 11.0, with an optimum pH at the range of 8.0–10.0. In addition, the crude protease was stable over a broad pH range (5.0–12.0). The optimum temperature for enzyme activity was 50 °C. The crude alkaline proteases showed stability towards various surfactants and bleach agents and compatibility with some commercial detergents. It was stable towards several organic solvents and retained more than 50% of its original activity after 30 days of incubation at 30 °C in the presence of 25% (v/v) dimethyl sulfoxide, N,N-dimethylformamide, diethyl ether, and hexane. The crude enzyme extract was also tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio enzyme/substrate of 10 was about 79%.
Keywords: Lithognathus mormyrus ; Alkaline proteases; Biochemical characterization; Detergent; Organic solvent-stable proteases; Enzymatic deproteinization

Purification and Characterization of Aspergillus terreus α-Galactosidases and Their Use for Hydrolysis of Soymilk Oligosaccharides by Joana Gasperazzo Ferreira; Angélica Pataro Reis; Valéria Monteze Guimarães; Daniel Luciano Falkoski; Lílian da Silva Fialho; Sebastião Tavares de Rezende (1111-1125).
α-Galactosidases has the potential to hydrolyze α-1-6 linkages in raffinose family oligosaccharides (RFO). Aspergillus terreus cells cultivated on wheat bran produced three extracellular forms of α-galactosidases (E1, E2, and E3). E1 and E2 α-galactosidases presented maximal activities at pH 5, while E3 α-galactosidase was more active at pH 5.5. The E1 and E2 enzymes showed stability for 6 h at pH 4–7. Maximal activities were determined at 60, 55, and 50°C, for E1, E2, and E3 α-galactosidase, respectively. E2 α-galactosidase retained 90% of its initial activity after 70 h at 50°C. The enzymes hydrolyzed ρNPGal, melibiose, raffinose and stachyose, and E1 and E2 enzymes were able to hydrolyze guar gum and locust bean gum substrates. E1 and E3 α-galactosidases were completely inhibited by Hg2+, Ag+, and Cu2+. The treatment of RFO present in soy milk with the enzymes showed that E1 α-galactosidase reduced the stachyose content to zero after 12 h of reaction, while E2 promoted total hydrolysis of raffinose. The complete removal of the oligosaccharides in soy milk could be reached by synergistic action of both enzymes
Keywords: α-Galactosidase; Aspergillus terreus ; Raffinose; Stachyose; Soybean; Anti-nutritional factors

South Africa has a rich microalgal biodiversity which has the potential to be used for renewable bio-fuel production in the region. Bioprospecting for oleaginous microalgae in KwaZulu Natal Province, South Africa, resulted in the establishment of a microalgal culture collection system for alternative energy research in the country. A potential hyper-lipid-producing Chlorella spp. strain was isolated, purified, and cultured in supplemented post-chlorinated wastewater for biomass and lipid production at the laboratory scale under batch mode. The microalgal strain was cultivated in different strengths of BG-11 media supplemented with wastewater from a local municipal domestic wastewater treatment plant. The Chlorella spp. was grown using ambient dissolved carbon dioxide in shake flasks under photosynthetically active radiation (±120 μmolm−2s−1). Microalgal biomass and lipid productivity were monitored at 24-h intervals in the batch mode. The microalgal biomass was analyzed by direct light microscopy and indirectly by spectrophotometry at 600 nm, and the lipids were extracted and quantified. The growth rate of the Chlorella spp. was enhanced in post-chlorinated wastewater supplemented with 5 mM NaNO3 with maximal biomass productivity. A dramatic increase in lipid yield was achieved with the post-chlorinated wastewater supplemented with 25 mM NaNO3. Low dosages of free chlorine were found to enhance microalgal growth. These findings serve as a basis for further scale-up trials using municipal wastewater as a medium for microalgal biomass and lipid production.
Keywords: Microalgae; Wastewater; Biomass; Raceway; Biodiesel; Chlorine

Study on the Decreased Sugar Yield in Enzymatic Hydrolysis of Cellulosic Substrate at High Solid Loading by Wei Wang; Li Kang; Hui Wei; Rajeev Arora; Y. Y. Lee (1139-1149).
Current technology for conversion of biomass to ethanol is an enzyme-based biochemical process. In bioethanol production, achieving high sugar yield at high solid loading in enzymatic hydrolysis step is important from both technical and economic viewpoints. Enzymatic hydrolysis of cellulosic substrates is affected by many parameters, including an unexplained behavior that the glucan digestibility of substrates by cellulase decreased under high solid loadings. A comprehensive study was conducted to investigate this phenomenon by using Spezyme CP and Avicel as model cellulase and cellulose substrate, respectively. The hydrolytic properties of the cellulase under different substrate concentrations at a fixed enzyme-to-substrate ratio were characterized. The results indicate that decreased sugar yield is neither due to the loss of enzyme activity at a high substrate concentration nor due to the higher end-product inhibition. The cellulase adsorption kinetics and isotherm studies indicated that a decline in the binding capacity of cellulase may explain the long-observed but little-understood phenomenon of a lower substrate digestibility with increased substrate concentration. The mechanism how the enzyme adsorption properties changed at high substrate concentration was also discussed in the context of exploring the improvement of the cellulase-binding capacity at high substrate loading.
Keywords: Biomass; Enzymatic hydrolysis; High substrate concentration; Cellulase adsorption; Binding capacity

gTME for Improved Adaptation of Saccharomyces cerevisiae to Corn Cob Acid Hydrolysate by Hongmei Liu; Kai Liu; Ming Yan; Lin Xu; Pingkai Ouyang (1150-1159).
Global transcription machinery engineering was employed to engineer xylose metabolism, tolerance and adaptation to lignocellulosic hydrolysates. Mutation of the transcription factor spt15 was introduced by error-prone PCR, then transformed the recombinant plasmid pYX212-spt15 into Saccharomyces cerevisiae YPH499 which was not able to grow on xylose, and screened on media using lignocellulosic hydrolysates as the sole carbon source. The maximum sugar yield was obtained by the hydrolysis form with 3% HCl (m/v) using autoclaving at 120°C, for 2 h with a solid to liquid ratio of 1:10. The corn cob hydrolysate contained 68.41 g/L xylose and 7.67 g/L glucose. The recombinant strain showed modest growth rate when cultured in the cellulosic hyrolysates with different pretreatment and pH conditions. The results showed that spt15-29 reached the maximum xylose and glucose utilization of 65.7% and 87.0%, respectively, the maximum ethanol concentration was 11.9 g/L, after 71 h, using the acid hydrolysate with the initial pH of 5.
Keywords: gTME; Corn cob acid hydrolysate; Saccharomyces cerevisiae ; Cofermenting xylose and glucose

d(−)-Lactic Acid Production by Leuconostoc mesenteroides B512 Using Different Carbon and Nitrogen Sources by Luciana Fontes Coelho; Cristian J. Bolner de Lima; Marcela Piassi Bernardo; Jonas Contiero (1160-1171).
Sugar concentration from sugarcane juice and yeast autolysate increased lactic acid production more than the other agro-industrial substrates tested. The concentrations of these two components were further optimized using the Plackett–Burman design and response surface method. A second-order polynomial regression model estimated that a maximal lactic acid production of 66.11 g/L would be obtained when the optimal values of sugar and yeast autolysate were 116.9 and 44.25 g/L, respectively. To validate the optimization of the medium composition, studies were carried out using the optimized conditions to confirm the result of the response surface analysis. After 48 h, lactic acid production using the shake-flask method was at 60.2 g/L.
Keywords: Response surface method; Medium optimization; d(−)-Lactic acid; Residues

Efficiency of DNA Transfection of Rat Heart Myoblast Cells H9c2(2-1) by Either Polyethyleneimine or Electroporation by Yu Chia Liu; Win Yu Lin; Ya Ru Jhang; Sing Hui Huang; Chean Ping Wu; Hsi Tien Wu (1172-1182).
Expression of exogenous DNA in vitro is significantly affected by the particular transfection method utilized. In this study, we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine (PEI) treatment and physically mediated electroporation, on a rat heart myoblast cell line, H9c2(2-1). After PEI transfection of pPgk-1/EGFP into H9c2(2-1) cells, EGFP expression could be easily detected by fluorospectrometer after 48 h (210 ± 12 RFU) and continued to increase after 72 h (243 ± 14 RFU). However, when H9c2(2-1) cells were transfected by electroporation (200 V, 500 μF, and one pulse), low level EGFP expression was observed after 48 h (49 ± 4 RFU) or 72 h (21 ± 14 RFU). In contrast, the easily transfectable control CHO-K1 cell line displayed a stronger EGFP expression than the H9c2(2-1) cells either by PEI or electroporation transfection. When transfection efficiencies were assayed by flow cytometry after 72 h, 13.6 ± 2.2% of PEI and 10.1 ± 1.5% of electroporation (250 V, 500 μF, and two pulses) transfected cells of H9c2(2-1) expressed EGFP, and PEI-transfected cells appeared to be less damaged (viability 93.6%) as compared to electroporation-transfected cells (39.5%). However, both PEI and electroporation (580 V, 50 Ω, and 50 μF) were effective for transfection of CHO-K1 with a higher efficiency, cell viability, and EGFP expression than H9c2(2-1). Our results indicate that the transfection efficiency of different methods varies among cell lines and that PEI is more efficient than electropolation for transfection of H9c2(2-1) whereas both PEI and electroporation are suitable for CHO-K1 transfection.
Keywords: DNA transfection; Polyethyleneimine; Electroporation; H9c2(2-1)

Pretreatment of Rice Straw by Proton Beam Irradiation for Efficient Enzyme Digestibility by Sung Bong Kim; Jun Seok Kim; Jong Ho Lee; Seong Woo Kang; Chulhwan Park; Seung Wook Kim (1183-1191).
Biomass was pretreated with proton beam irradiation (PBI) in order to enhance enzyme digestibility. Rice straw and soaking in aqueous ammonia (SAA)-treated rice straw were treated with 1–25 kGy doses of PBI at a beam energy of 45 MeV. The optimal doses of PBI for efficient sugar recovery were 15 and 3 kGy for rice straw and SAA-treated rice straw, respectively. When PBI was applied to rice straw at 15 kGy, the glucose conversion reached 68% of the theoretical maximum at 72 h. When 3 kGy of PBI was applied to SAA-treated rice straw, approximately 90% of the theoretical glucose conversion was obtained at 12 h compared to a 89% conversion at 48 h. After 2 h of enzymatic saccharification, the initial reaction rates of raw rice straw pretreated with 15 kGy of PBI and SAA-treated rice straw pretreated with 3 kGy of PBI were 1.4 × 10−4 and 9.7 × 10−4 g L−1 s−1, respectively. Further, the results of X-ray diffractometry support the effect of PBI on sugar recovery, whereas scanning electron microscopy images revealed a more rugged rice straw surface.
Keywords: Proton beam irradiation; Pretreatment; Rice straw; Saccharification; Biomass

Phytochemicals such as polyphenols and carotenoids are gaining importance because of their contribution to human health and their multiple biological effects such as antioxidant, antimutagenic, anticarcinogenic, and cytoprotective activities and their therapeutic properties. Banana peel is a major by-product in pulp industry and it contains various bioactive compounds like polyphenols, carotenoids, and others. In the present study, effect of ripening, solvent polarity on the content of bioactive compounds of crude banana peel and the protective effect of peel extracts of unripe, ripe, and leaky ripe banana fruit on hydrogen peroxide-induced hemolysis and their antioxidant capacity were investigated. Banana (Musa paradisica) peel at different stages of ripening (unripe, ripe, leaky ripe) were treated with 70% acetone, which were partitioned in order of polarity with water, ethyl acetate, chloroform (CHCl3), and hexane sequentially. The antioxidant activity of the samples was evaluated by the red cell hemolysis assay, free radical scavenging (1,1-diphenyl-2-picrylhydrazyl free radical elimination) and superoxide dismutase activities. The Folin–Ciocalteu's reagent assay was used to estimate the phenolic content of extracts. The findings of this investigation suggest that the unripe banana peel sample had higher antioxidant potency than ripe and leaky ripe. Further on fractionation, ethyl acetate and water soluble fractions of unripe peel displayed high antioxidant activity than CHCl3 and hexane fraction, respectively. A positive correlation between free radical scavenging capacity and the content of phenolic compound were found in unripe, ripe, and leaky ripe stages of banana peel.
Keywords: Musa paradisica ; Erythrocyte lysis; Superoxide dismutase; Radical scavenging activity; Phenolic content; Ripening

In Vitro Refolding of Triosephosphate Isomerase from L. donovani by Kishore Kumar; Prachi Bhargava; Uma Roy (1207-1214).
The triosephosphate isomerase of Leishmania donovani (LdTIM) was expressed at high level in Escherichia coli. The TIM gene was cloned in expression vector pET-23(a) with C-terminal 6× His tag fused in frame, and expressed as a 27.6-kDa protein in E. coli as inclusion bodies. The recombinant LdTIM from E. coli lysate was solubilized in 6 M guanidine hydrochloride and purified by Ni-NTA chromatography. In the present study, the effect of bovine serum albumin on the reactivation of TIM was investigated. Furthermore, 8-anilino-1-naphthalene sulfonic acid was used to detect the structural changes induced by bovine serum albumin (BSA). Here, we conclude that BSA assists in the refolding and regain of LdTIM enzyme activity by providing framework for structure formation. This study indicates that numerous protein–protein contacts are constantly occurring inside the cell that leads to the formation of native protein.
Keywords: Leishmania donovani ; Triosephosphate isomerase; Guanidine hydrochloride; Isopropyl β-d-thiogalactopyranoside; 8-Anilino-1-naphthalene sulfonic acid

A comparative evaluation of different cell disruption methods for the release of lipids from marine Chlorella vulgaris cells was investigated. The cell growth of C. vulgaris was observed. Lipid concentrations from different disruption methods were determined, and the fatty acid composition of the extracted lipids was analyzed. The results showed that average productivity of C. vulgaris biomass was 208 mg L−1 day−1. The lipid concentrations of C. vulgaris were 5%, 6%, 29%, 15%, 10%, 7%, 22%, 24%, and 18% when using grinding with quartz sand under wet condition, grinding with quartz sand under dehydrated condition, grinding in liquid nitrogen, ultrasonication, bead milling, enzymatic lysis by snailase, enzymatic lysis by lysozyme, enzymatic lysis by cellulose, and microwaves, respectively. The shortest disruption time was 2 min by grinding in liquid nitrogen. The unsaturated and saturated fatty acid contents of C. vulgaris were 71.76% and 28.24%, respectively. The extracted lipids displayed a suitable fatty acid profile for biodiesel [C16:0 (~23%), C16:1 (~23%), and C18:1 (~45%)]. Overall, grinding in liquid nitrogen was identified as the most effective method in terms of disruption efficiency and time.
Keywords: Microalga; Lipid concentration; Cell disruption