Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.159, #2)

2D Polarization Imaging of Turbid Media by Nidhi Mathur; Naresh K. Chaudhary; Purnima Bharti (pp. 295-298).

Cancer normally tends to result in the decrease of tissue elasticity; i.e., the cancerous region is more rigid than the normal surrounding areas. This would appear as differences in the distribution of internal birefringence that could be used to improve image contrast between the cancerous and normal tissue structures. Different filtering techniques are used to enhance the image to help us identify, locate, and diagnose an “object,” such as a tumor inside a biological tissue.

Keywords: Mueller matrix; Imaging; Polarization; Turbid media

Synthesis of Poly(o-anisidine)/H₂SO₄ Film for the Development of Glucose Biosensor by P. A. Savale; M. D. Shirsat (pp. 299-309).

The poly(o-anisidine)–sulfuric acid–glucose oxidase (POA–H₂SO₄–GOx) electrode has been investigated in the present work. Platinum electrode was used for the synthesis of poly (o-anisidine)–sulfuric acid (POA–H₂SO₄) film using galvanostatic method with 0.2 M o-anisidine, 1.0 M H₂SO₄ solution, 1.0 pH and 2 mA/cm² applied current density. The synthesized film was characterized using electrochemical technique, conductivity measurement, UV–visible spectroscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy. GO_X was immobilized on synthesized POA–H₂SO₄ film by cross-linking via glutaraldehyde in phosphate and acetate buffer. The Michaelis–Menten constant ( $$K_{ ext{m}}^prime $$ ) was determined for the immobilized enzyme. The glucose oxidase electrode shows the maximum current response at pH 5.5 and potential 0.6 V. The sensitivity of POA–H₂SO₄–GO_X electrode in phosphate and acetate buffer has been recorded. The results of this study reveal that the phosphate buffer gives fast response as compared to acetate buffer in amperometric measurements.

Keywords: Galvanostatic; Poly(o-anisidine); Glucose oxidase; Cross-linking; Biosensor

Study of Optical Properties of Macrophomina phaseolina Impregnated Sol-gel Derived Silica Matrices by Sunita Sharma; Vandana; S. K. Ghoshal; Pooja Arora; Neeraj Dilbaghi; Ashok Chaudhury (pp. 310-316).

In the present frame of work, Macrophomina phaseolina is encapsulated in silica matrices at various concentrations by low temperature sol-gel technique using tetraethylorthosilicate (TEOS) as precursor. The optical and photophysical properties of these samples have been studied by second harmonics of Nd:YAG laser at 532 nm. UV-visible absorption spectra of samples have been recorded and it is found that the absorption increases with increase in concentration of fungus. Further, a decrease in output transmission intensity of the laser has been observed with increase in fungus concentration. The temporal response of these samples has also been examined. The results show that the fungus concentration can be measured within ∼15–20 min. This method of optical sensing of fungus in test sample is faster than other techniques, such as the conventional colorimetric method which takes about 1 h.

Keywords: Sol-gel; Fungal biosensor; Porous silicon; Immobilization; M. phaseolina

Multiple Responses Optimization and Modeling of Lipase Production by Rhodotorula mucilaginosa MTCC-8737 Using Response Surface Methodology by Subhakar Chennupati; Ravichandra Potumarthi; M. Gopal Rao; P. Lakshmi Manga; M. Sridevi; Annapurna Jetty (pp. 317-329).

Response surface methodology was employed to optimize culture medium for production of lipase with Rhodotorula sp. MTCC 8737. In the first step, a Plackett–Burman design was used to evaluate the effects of different inducers qualitatively. Of all the seven inducers tested, soybean oil showed significant influence on the lipase production. Further, response surface studies were conducted to quantitatively optimize by considering linear, interactive, and quadratic effects of test variables. A novel approach was proposed to optimize the lipase production system by optimizing the responses in terms of yield kinetics rather than optimizing the direct responses like lipase titer and biomass growth. The coefficient of determination (R ²) calculated for Y _P/S (0.769), Y _P/X (0.799), and Y _X/S (0.847) indicated that the statistical model could explain 76.9%, 79.99%, and 84.7% of variability in the response.

Keywords: Lipase; Yield kinetics; Central composite design (CCD); Optimization and modeling; Rhodotorula sp.

Isolation of Phosphate-Solubilizing Fungi from Phosphate Mines and Their Effect on Wheat Seedling Growth by Chunqiao Xiao; Ruan Chi; Huan He; Guanzhou Qiu; Dianzuo Wang; Wenxue Zhang (pp. 330-342).

Three phosphate-solubilizing fungi, identified as Penicillium expansum, Mucor ramosissimus, and Candida krissii, were isolated from phosphate mines (Hubei, People’s Republic of China) and characterized. All the isolates demonstrated diverse levels of phosphate-solubilizing capability in National Botanical Research Institute’s phosphate growth medium containing rock phosphate as sole phosphate source. Acidification of culture medium seemed to be the main mechanism for rock phosphate solubilization. Indeed, citric acid, oxalic acid, and gluconic acid were shown to be present in the culture medium inoculated with these isolates. Moreover, the isolates produced acid and alkaline phosphatases in culture medium, which may also be helpful for RP solubilization. A strong negative correlation between content of soluble phosphorus and pH (r = – 0.89; p < 0.01) in culture medium was observed in this study. All the isolates promoted growth, soil available phosphorus, phosphorus, and nitrogen uptake of wheat seedling in field soil containing rock phosphate under pot culture conditions, thus demonstrating the capability of these isolates to convert insoluble form of phosphorus into plant available form from rock phosphate, and therefore hold great potential for development as biofertilizers to enhance soil fertility and promote plant growth.

Keywords: Isolation; Phosphate-solubilizing fungi; Phosphate mines; Rock phosphate; Soluble phosphorus; Wheat

Exploring the C–H…O Interactions in Glycoproteins by Sudha Anand; Anand Anbarasu; Rao Sethumadhavan (pp. 343-354).

Glycoproteins are an important class of proteins that play a significant role in many cellular events. In the present study, we analyze the influence of C–H…O interactions in relation to other environmental preferences in glycoproteins. CH…O interactions are now accepted as a genuine hydrogen bond. Main chain–main chain interactions are predominant. Proline residues stabilize strands by C–H…O interactions in glycoproteins. Majority of the C–H…O interacting residues were conserved and had one or more stabilization centers. CH…O interactions might be responsible for the global conformational stability, since long-range CH…O contacts were predominant. The results presented in this study might be useful for structural stability studies in glycoproteins.

Keywords: Glycoproteins; C–H…O interactions; Stabilization centers; Conservation score

Cloning and Expression of Pseudomonas fluorescens 26-2 Lipase Gene in Pichia pastoris and Characterizing for Transesterification by Jiangke Yang; Bo Zhang; Yunjun Yan (pp. 355-365).

Pseudomonas lipases are important biocatalysts widely used in a variety of industrial fields. An extracellular lipase gene lipA with 1,854-bp open reading frame was cloned from Pseudomonas fluorescens 26-2. The multialignment assay of the putative amino acid and the secondary structure prediction revealed this enzyme could be classified into the lipolytic subfamily I.3 and secreted via adenosine-triphosphate-binding cassette pathway. The lipA gene was integrated into Pichia pastoris GS115, and the methanol-inducible recombinants with Mut^S and Mut⁺ phenotypes were acquired. The characteristics and the transesterification capacity shown by this enzyme suggested it is a useful biocatalyst for biodiesel preparation.

Keywords: Pseudomonas fluorescens ; Lipase; Pichia pastoris ; Transesterification; Biodiesel

Optimization Strategies Based on Sequential Quadratic Programming Applied for a Fermentation Process for Butanol Production by Adriano Pinto Mariano; Caliane Bastos Borba Costa; Dejanira de Franceschi de Angelis; Francisco Maugeri Filho; Daniel Ibraim Pires Atala; Maria Regina Wolf Maciel; Rubens Maciel Filho (pp. 366-381).

In this work, the mathematical optimization of a continuous flash fermentation process for the production of biobutanol was studied. The process consists of three interconnected units, as follows: fermentor, cell-retention system (tangential microfiltration), and vacuum flash vessel (responsible for the continuous recovery of butanol from the broth). The objective of the optimization was to maximize butanol productivity for a desired substrate conversion. Two strategies were compared for the optimization of the process. In one of them, the process was represented by a deterministic model with kinetic parameters determined experimentally and, in the other, by a statistical model obtained using the factorial design technique combined with simulation. For both strategies, the problem was written as a nonlinear programming problem and was solved with the sequential quadratic programming technique. The results showed that despite the very similar solutions obtained with both strategies, the problems found with the strategy using the deterministic model, such as lack of convergence and high computational time, make the use of the optimization strategy with the statistical model, which showed to be robust and fast, more suitable for the flash fermentation process, being recommended for real-time applications coupling optimization and control.

Keywords: Flash fermentation; Biobutanol; Mathematical modeling; Factorial design; Optimization; SQP

Characterization of a Recombinant Thermostable Dehalogenase Isolated from the Hot Spring Thermophile Sulfolobus tokodaii by Philip G Bachas-Daunert; Stacy A. Law; Yinan Wei (pp. 382-393).

A putative dehalogenase, l-HAD_ST, from the thermophile Sulfolobus tokodaii, was cloned and expressed in Escherichia coli. The recombinant enzyme catalyzes the stereospecific dehalogenation of l-2-haloacids with similar levels of activity as its homolog from mesophiles. l-HAD_ST remains fully active after being incubated for 4 h at 70 °C and tolerates extreme pH conditions ranging from 4 to 10. Furthermore, it can be purified conveniently without the usage of any chromatography method. The high expression yield and easy purification procedure make the recombinant dehalogenase an excellent candidate for biotechnological applications.

Keywords: Thermostable; Extremophile; l-2 Haloacid dehalogenase; Recombinant

Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from Bacillus pumilus by Min-Yuan Wan; Hai-Yan Wang; Yi-Zheng Zhang; Hong Feng (pp. 394-403).

An alkaline protease (DHAP) from Bacillus pumilus has shown great potential in hide dehairing. To get better insights on its catalytic properties for application, the substrate specificity and thermostability were investigated using five natural proteins and nine synthetic peptides. The results showed that DHAP could hydrolyze five proteins tested here in different specificity. Collagen, a component of animal skin, was more resistant to hydrolysis than casein, fibrin, and gelatin. Among the synthetic peptides, the enzyme showed activity mainly with tetrapeptide substrates with the catalytic efficiency in order of Phe>Leu>Ala at P1 site, although k _m value for AAVA-pN is much lower than that for AAPL-pN and AAPF-pN. With tripeptide substrates, smaller side-chain group (Gly) at P1 site was not hydrolyzed by DHAP. The enzyme showed good thermostability below 60 °C, and lost activity so quickly above 70 °C. The thermostability was largely dependent on metal ion, especially Ca²⁺, although other ions, like Mg²⁺, Mn²⁺, and Co²⁺, could sustain stability at certain extent within limited time. Cu²⁺, Fe²⁺, as well as Al³⁺, did not support the enzyme to retain activity at 60 °C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.

Keywords: Bacillus pumilus ; Alkaline protease; Substrate specificity; Thermostability; Metal ion; Dehairing

Use of a Bacterially Expressed Human Factor IX Light Chain to Develop Polyclonal Antibody Anti-hFIX by Sedigheh Safari; Alireza Zomorodipour; Nour Amirmozaffari; Morteza Daliri Choopari (pp. 404-414).

Hemophilia B is an X-linked recessive bleeding disorder caused by deficiency or malfunctioning of human coagulation factor IX (hFIX). Hemophilia B patients are treated at present by infusion of plasma derived hFIX which is not always efficient, because development of anti-hFIX antibodies (alloantibodies) in some cases inhibits the activity of the infused hFIX. The hFIX alloantibodies are directed against γ-carboxyglutamic acid residues (Gla-domain) or protease domain in hFIX light chain. An epitope-containing fragment of hFIX light-chain was expressed in a T7-based Escherichia coli expression system and after purification, it was used for the immunization of rabbit to develop specific antibodies anti-hFIX. The plasma, derived from the immunized rabbit, was shown to be able to detect the normal hFIX, which indicates for the presence of a specific anti-hFIX antibody and supporting that a bacterially expressed hFIX subfragment might be able to neutralize the alloantibodies. Considering the importance of hFIX and its related investigations, both the produced hFIX antigen and its corresponding antibody will play important roles for experiments dealing with the production of hFIX and studies involved in the neutralization of the hFIX inhibitors in hFIX-related disorders and other clinical applications.

Keywords: Human coagulation factor IX (hFIX); hFIX light chain Gla-domain; Escherichia coli ; T7 expression system; Polyclonal antibody anti-hFIX

Rosmarinic Acid and Antioxidant Enzyme Activities in Lavandula vera MM Cell Suspension Culture: A Comparative Study by Milen Georgiev; Radoslav Abrashev; Ekaterina Krumova; Klimentina Demirevska; Mladenka Ilieva; Maria Angelova (pp. 415-425).

The growth and intracellular protein content of lavender (Lavandula vera MM) cell suspension culture was followed along with some antioxidant defense system members—non-enzymatic (rosmarinic acid) and enzymatic [superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6)]. It was found that the media content and the cultivation mode strongly influenced the production of plant defense compounds as well as the ratio between non-enzymatic and enzymatic ones. The bioreactor culture contains about two times more rosmarinic acid, superoxide dismutase, and catalase compared to the shake-flask cultivation. These findings are discussed with respect to the relative stress levels and plant antioxidant orchestra system. It was concluded that investigated defense system components (enzymatic and non-enzymatic) were closely associated in a complex balance. The three isoenzyme forms of SOD (Cu/ZnSOD, FeSOD, and MnSOD) in the cells of Lavandula vera were revealed by polyacrylamide gel electrophoresis analysis, and the FeSOD isoform exhibited highest activity.

Keywords: Bioreactor; Catalase; Cell suspension culture; Lavandula vera ; Rosmarinic acid; Superoxide dismutase

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers by Magdy M. M. Elnashar; Mohamed A. Yassin (pp. 426-437).

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.

Keywords: Biopolymers; Hydrogels; Covalent immobilization; β-Galactosidase; Biotechnology; Lactose intolerance

Production of Oligosaccharide from Alginate Using Pseudoalteromonas agarovorans by DuBok Choi; Yu Lan Piao; Woon-Seob Shin; Hoon Cho (pp. 438-452).

A marine bacterium was isolated from seawater near the Korean south coast for efficient saccharification from alginate. Based on 16S rDNA sequence, the isolated strain was identified as Pseudoalteromonas agarovorans. Various environmental factors affecting saccharification of alginate using P. agarovorans CHO-12 have been investigated in flask cultures. The optimum concentration of sugar was obtained at 30 rpm and 29 °C. Among various NaCl concentrations, when NaCl concentration was increased from 10 to 30 g/l, the cell concentration sharply increased, while there is no increase at above 40 g/l. The maximum sugar concentration was obtained at 13.8 when 30 g/l of NaCl was used. Yeast extract and corn steep liquor were the best nitrogen source for efficient saccharification. Especially, the sugar concentration of 14.9 g/l was obtained after 3 days of culture using a mixture of 1.0 g/l of yeast extract and 1.5 g/l of corn steep liquor. Scale up was carried out at 50 l of reactor for 3 days using P. agarovorans CHO-12 and Stenotrophomonas maltophilia sp. When S. maltophilia was used, cell concentration was about twofold higher than that of P. agarovorans CHO-12. On the other hand, when P. agarovorans CHO-12 was used, the maximum saccharification rate was obtained, 7.5 g/l/day after 2 days of culture, which was about tenfold higher than that of S. maltophilia.

Keywords: Alginate; Oligosaccharide; Saccharification; Pseudoalteromonas agarovorans

Enhanced Production of an Extracellular β-d-Fructofuranosidase Fructohydrolase from a 2-Deoxy-d-glucose Stabilized Mutant of Candida utilis by S. Ali; M. Ashiq (pp. 453-463).

The enzyme β-d-fructofuranosidase fructohydrolase (FFH) cleaves the α-1,4 glycosidic linkage between α-d-glucose and β-d-fructose molecules of sucrose, releasing monosaccharides by hydrolysis. In the present study, FFH production in Candida utilis GC-46, a lipolytic wild yeast strain was improved by exposure to N-methyl N-nitro N-nitroso guanidine (NG) and 2-deoxy-d-glucose (2dg) at various levels. The mutant strain NG-5 was obtained after exposure to 0.06 mg/ml of NG for 20 min. NG-5 offers improved extracellular FFH production (34 ± 2.6 U/ml/min) when compared to the wild strain (1.15 ± 0.01 U/ml/min). A 40-fold increase of FFH (45.65 ± 2.0 U/ml/min) was achieved when the process parameters, including incubation period (48 h), sucrose concentration (5.0 g/l), initial pH (6.0), inoculum size (2.0% v/v, 16 h old), and urea concentration (0.2%, w/v) were identified using Plackett–Burman design. The kinetic parameters viz. Q _p (0.723 U/g/h), Y _p/s (2.036 U/g), and q _p (0.091 U/g yeast cells/h) indicate that NG-5 is a hyperproducer of extracellular FFH with a concomitant increase in growth rate. The volumetric productivity of NG-5 was over sixfold improved over the parental strain. The enzyme production improvement is highly significant (HS, LSD 0.042, p ≤ 0.05), indicating commercial utility.

Keywords: Plackett–Burman design; Candida utilis ; β-d-Fructofuranosidase fructohydrolase; Microbial fermentation; Kinetic study; Two factorial experimental design

Hyperthermostable, Ca²⁺-Independent, and High Maltose-Forming α-Amylase Production by an Extreme Thermophile Geobacillus thermoleovorans: Whole Cell Immobilization by J. L. Uma Maheswar Rao; T. Satyanarayana (pp. 464-477).

The synthesis of extracellular α-amylase in Geobacillus thermoleovorans was constitutive. The enzyme was secreted in metabolizable carbon sources as well as non-metabolizable synthetic analogues of glucose, but the titers were higher in the former than that in the latter. G. thermoleovorans is a fast-growing facultatively anaerobic bacterium that grows under both aerobic and anaerobic conditions and produces an extracellular amylolytic enzyme α-amylase with the by-product of lactic acid. G. thermoleovorans is a rich source of various novel thermostable biocatalysts for different industrial applications. α-Amylase synthesis was subject to catabolite repression in the presence of high concentrations of glucose. The addition of cAMP to the medium containing glucose did not result in the repression of α-amylase synthesis. The addition of maltose (1%) to the starch arginine medium resulted in a twofold enhancement in enzyme titers. Polyurethane foam (PUF)-immobilized cells secreted α-amylase, which was higher than that with the free cells. PUF appeared to be a better matrix for immobilization of the thermophilic bacterium than the other commonly used matrices. The repeated use of PUF-immobilized cells was possible over 15 cycles with a sustained α-amylase secretion. The use of this enzyme in starch saccharification eliminates the addition of Ca²⁺ in starch liquefaction and its subsequent removal by ion exchangers from the product streams.

Keywords: Ca²⁺-independent α-amylase; Whole cell immobilization and production

Raman Spectroscopy for Intracellular Monitoring of Carotenoid in Blakeslea trispora by Emmanouil H. Papaioannou; Maria Liakopoulou-Kyriakides; Dimitrios Christofilos; Ioannis Arvanitidis; Gerasimos Kourouklis (pp. 478-487).

In the present study, we explore the feasibility of Raman spectroscopy for intracellular monitoring of carotenoid in filamentous fungi Blakeslea trispora. Although carotenoid production from this fungus has been extensively studied through various chromatographic methods and ultraviolet-visible spectroscopy, no intracellular monitoring has been demonstrated until now. The intensity of the Raman spectrum, and more conveniently that of the strongest ν ₁ carotenoid band at ∼1,519 cm⁻¹, exhibits a good linear correlation with the carotenoid content of the sample as determined by high-performance liquid chromatography (HPLC) and ultraviolet-visible (UV-Vis) spectroscopy. Our results suggest that Raman spectroscopy can serve as an alternative method for the study and quantification of carotenoid in batch-mated submerged cultivations of B. trispora and similar organisms. Although not as accurate as HPLC, it allows a rapid sampling and analysis, avoiding the prolonged and tedious classical isolation procedures required for carotenoid determination by HPLC and UV-Vis spectroscopy.

Keywords: β-carotene; Lycopene; Carotenoid; Blakeslea trispora ; Raman spectroscopy; RP-HPLC-DAD

Determination of Kinetic Parameters in the Biosorption of Cr (VI) on Immobilized Bacillus cereus M¹ ₁₆ in a Continuous Packed Bed Column Reactor by Soumen K. Maiti; Debabrata Bera; Parimal Chattopadhyay; Lalitagauri Ray (pp. 488-504).

Due to technological advancement, environment suffers from untreated toxic heavy metal bearing effluent coming from different industries. Chromium (VI) is one of those heavy metals having adverse impact on ecological balance, human, and plant health because of its carcinogenic properties. Biosorption is presented as an alternative to traditional technologies which are costly and inefficient for treatment of industrial wastes containing low amount of heavy metals. In this study, bioremediation of Cr (VI) ions by immobilized Bacillus cereus M¹ ₁₆ was investigated in a laboratory scale packed bed up-flow column reactor. The effect of important parameters, such as the inlet flow rate, influent concentration, and effective bed height, has been studied. External mass transfer, surface adsorption, and intrabead mass transfer were also studied to conclude the rate limiting step for removal of Cr (VI) and to determine the process parameters which are important for biosorption optimization. The external mass transfer coefficient was calculated at different flow rates (6.51 × 10⁻² to 7.58 × 10⁻² cm/min). Using the model, the surface adsorption rate constant (k _ad) and the intrabead mass transfer coefficient (k _i) were predicted as 0.0267 × 10⁻³ and 0.7465 × 10⁻³ l/g/min, respectively. Both are much lower than the external mass transfer coefficient (k _e). The surface adsorption phenomenon is acting as the rate-limiting step due to its high resistance for removal of Cr (VI).

Keywords: Biosorption; Immobilized Bacillus cereus M¹ ₁₆ ; Continuous reactor; Packed bed; Kinetic parameters

Compactin Production Studies Using Penicillium brevicompactum Under Solid-State Fermentation Conditions by N. S. Shaligram; S. K. Singh; R. S. Singhal; A. Pandey; G. Szakacs (pp. 505-520).

In the present study, compactin production by Penicillium brevicompactum WA 2315 was optimized using solid-state fermentation. The initial one factor at a time approach resulted in improved compactin production of 905 μg gds⁻¹ compared to initial 450 μg gds⁻¹. Subsequently, nutritional, physiological, and biological parameters were screened using fractional factorial and Box–Behnken design. The fractional factorial design studied inoculum age, inoculum volume, pH, NaCl, NH₄NO₃, MgSO₄, and KH₂PO₄. All parameters were found to be significant except pH and KH₂PO₄. The Box–Behnken design studied inoculum volume, inoculum age, glycerol, and NH₄NO₃ at three different levels. Inoculum volume (p = 0.0013) and glycerol (p = 0.0001) were significant factors with greater effect on response. The interaction effects were not significant. The validation study using model-defined conditions resulted in an improved yield of 1,250 μg gds⁻¹ compactin. Further improvement in yield was obtained using fed batch mode of carbon supplementation. The feeding of glycerol (20% v/v) on day 3 resulted in further improved compactin yield of 1,406 μg gds⁻¹. The present study demonstrates that agro-industrial residues can be successfully used for compactin production, and statistical experiment designs provide an easy tool to improve the process conditions for secondary metabolite production.

Keywords: Compactin; Penicillium brevicompactum WA 2315; Solid-state fermentation; Fractional factorial design; Box–Behnken design

Cloning, Expression, and Characterization of a New Streptomyces sp. S27 Xylanase for Which Xylobiose is the Main Hydrolysis Product by Ning Li; Pengjun Shi; Peilong Yang; Yaru Wang; Huiying Luo; Yingguo Bai; Zhigang Zhou; Bin Yao (pp. 521-531).

A xylanase gene, xynBS27, was cloned from Streptomyces sp. S27 and consisted of 693 bp encoding a 230-residue protein, including a putative 41-residue signal peptide. Belonging to the glycoside hydrolase family 11, XynBS27 exhibits the maximum identity (75.9%) to the xylanase from Streptomyces sp. zxy19. Recombinant XynBS27 was overexpressed in Pichia pastoris, and the xylanase activity was 7624.0 U/ml after high-cell-density fermentation in 3.7-L fermenter. The purified recombinant XynBS27 had a high specific activity of 3272.0 U/mg. The optimum temperature and pH for XynBS27 activity was 65 °C and pH 6.5, respectively. XynBS27 showed good pH stability and retained more than 80% of the maximum activity after incubation in buffers with pH ranging between 4.0 and 12.0 at 37 °C for 1 h. The main hydrolysis product of xylan by XynBS27 was xylobiose (>75%), which was good for human health derived from its ability to modulate the intestinal function. The attractive biochemical characteristics of XynBS27 suggest that it may be a good candidate in a variety of industrial applications.

Keywords: Streptomyces sp. S27; Xylanase; Overexpression; Pichia pastoris ; Xylobiose

Biotreatment on Cellulose Fluff Pulp: Quaternary Ammonium Salts Finish and Grafting with β-cyclodextrin by Djamila Ghemati; Atika Oudia; Djamel Aliouche; Saad Lamouri (pp. 532-544).

For its potential performances to be expanded, cellulose needs to be processed in different ways. Therefore, an object of the present work was to provide a chemical modification of cellulose through: a specific finish with two quaternary ammonium salts (namely Aliquat 336 and Aliquat 1529, respectively). Chemical grafting of β-cyclodextrin derivative (β-CD) onto fibers followed by the inclusion of benzoic acid in the grafted CD cavities as a probe chemical. Physicochemical properties and performances of the untreated and treated fibers have been determined with infrared spectra, microscopy, swelling measurements, antimicrobial finishing tests, and dye adsorption. Our results show that cellulose fibers can be efficiently modified with no significant changes in its structural and surface properties; the treated fibers show an attractive behavior in swelling, dye adsorption and antibacterial activity.

Keywords: Cellulose fluff pulp; Quaternary ammonium salts; Grafting; β-cyclodextrin; Fiber swelling; Antimicrobial activity; Dye adsorption

Accumulation of Intracellular Polyhydroxybutyrate in Alcaligenes sp. d₂ Under Phenol Stress by Indu C. Nair; S. Pradeep; M. S. Ajayan; K. Jayachandran; Shankar Shashidhar (pp. 545-552).

Alcaligenes sp. d₂ isolated from soil was earlier reported as a potent phenol-degrading organism. In the Fourier transform/infrared spectroscopic analysis of the biodegraded sample, the aromatic stretching was missing and the spectrum gave evidence for the presence of polyhydroxybutyric acid along with its depolymerized products. In the gas chromatogram of the biodegraded sample, the peak of phenol at 14.997 min was absent and there were many peaks after 20 min. The organism could carry out 100% degradation of phenol in 32 h and could progressively result in early accumulation of polyhydroxybutyrate (PHB) intracellularly from 8 h onwards. The various conditions optimized for the maximum accumulation of intracellular PHB were pH 7.0, incubation time 24 h, phenol concentration 15 mg/100 ml, and ammonium sulfate concentration 25 mg/100 ml.

Keywords: Alcaligenes sp. d₂ ; Phenol; Biodegradation; Spectroscopic analysis; Polyhydroxybutyrate accumulation

Stagewise Dilute-Acid Pretreatment and Enzyme Hydrolysis of Distillers’ Grains and Corn Fiber by Hossein Noureddini; Jongwon Byun; Ta-Jen Yu (pp. 553-567).

Distillers’ grains and corn fiber are the coproducts of the corn dry grind and wet milling industries, respectively. Availability of distillers’ grains and corn fiber at the ethanol plant and their high levels of lignocellulosic material make these coproducts attractive feedstocks for conversion to ethanol. In this study, dilute sulfuric acid hydrolysis of these coproducts was investigated in a multistage scheme. After the completion of each pretreatment stage, the liquid substrate was separated and reused in the succeeding pretreatment stage with a fresh substrate. The substrate from each stage was also subjected to enzyme hydrolysis in a separate experiment. The sulfuric acid concentration and the substrate loading were maintained at 1.0 vol% and 15.0 wt.%, respectively, and the temperature was maintained at 120 °C in all the experiments. Experiments were also performed to study the effect of removing oil from the samples prior to the pretreatment. The highest concentration of monomeric sugars (MS) was observed when three stages of pretreatment were followed by the enzyme reaction. The enzyme hydrolysis of the three-stage pretreated dried distillers’ grains and corn fiber yielded 122.6 ± 5.8 and 184.5 ± 4.1 mg/mL of MS, respectively. The formation of inhibitory products was also monitored.

Keywords: Distillers’ grains; Whole stillage; DDG; Corn fiber; Dilute sulfuric acid pretreatment; Enzyme hydrolysis; Monomeric sugars; Inhibitors; Ethanol

Oil Production Towards Biofuel from Autotrophic Microalgae Semicontinuous Cultivations Monitorized by Flow Cytometry by Teresa Lopes da Silva; Alberto Reis; Roberto Medeiros; Ana Cristina Oliveira; Luisa Gouveia (pp. 568-578).

Two microalgae species (Scenedesmus obliquus and Neochloris oleoabundans) were cultivated in closed sleeve photobioreactors in order to select the best oil producer for further large-scale open raceway pond cultivations, aiming at biofuel production. Scenedesmus obliquus reached a higher maximum biomass concentration (1.41 g l⁻¹) with a lower lipid content (12.8% w/w), as compared to N. oleoabundans [maximum biomass concentration of 0.92 g l⁻¹ with 16.5% (w/w) lipid content]. Both microalgae showed adequate fatty acid composition and iodine values as substitutes for diesel fuel. Based on these results, N. oleoabundans was selected for further open raceway pond cultivations. Under these conditions, N. oleoabundans reached a maximum biomass concentration of 2.8 g l⁻¹ with 11% (w/w) of lipid content. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional gravimetric lipid analysis was found for both microalgae, making this method a suitable and quick technique for the screening of microalgae strains for lipid production and optimization of biofuel production bioprocesses. Medium growth optimization for enhancement of microalgal oil production is now in progress.

Keywords: Scenedesmus obliquus ; Neochloris oleoabundans ; Lipids; Biofuel; Nile red; Flow cytometry

Continuous Production of Oxytetracycline in Fluidized Bed Bioreactor by Immobilized Cells of Streptomyces varsoviensis MTCC-1537: Effect of Dilution and Glucose Loading Rates by Annapurna Jetty; Ravichandra Potumarthi; A. Gangagni Rao; B. Sarva Rao; S. V. Ramakrishna (pp. 579-588).

Oxytetracycline (OT) production using glutaraldehyde cross-linked calcium alginate immobilized cells of Streptomyces varsoviensis in continuous fluidized bed reactor (FBR) was investigated. Initially, batch experiments were carried in stirred tank reactor (STR) and FBR using calcium alginate immobilized cells. Higher OT production of 0.45 gm/L was achieved by FBR when compared with 0.33 g/L of OT in STR. All subsequent studies were carried out in continuous mode of operation in FBR. During 21 days of operation, effect of glucose concentration and different dilution rates were studied. A maximum of 0.75 g/L OT was achieved in the medium having 10 g/L of glucose concentration. The highest OT concentration of 0.92 g/L and the highest yield of OT with respect to biomass at 0.1713 g/g were obtained at the dilution rate of 0.25 day⁻¹.

Keywords: Yield kinetics; Cross-linking; Antibiotics production; Glucose concentration; Continuous process; Stirred tank reactor

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.159, #2)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.159, #2)