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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.135, #1)
Enzyme catalyzed production of biodiesel from olive oil by Fernando Sanchez; Palligarnai T. Vasudevan (pp. 1-14).
Biodiesel (fatty acid methyl esters) was produced by transesterification of triglycerides (triolein) present in olive oil with methanol and Novozym®435.The effect of the molar ratio of methanol to triolein, semibatch (stepwise addition of methanol) vs batch operation, enzyme activity, and reaction temperature on overall conversion was determined. Stepwise methanolysis with a 3∶1 methanol to triolein molar ratio and an overall ration of 8∶1 gave the best results. The final conversion and yield of biodiesel were unaffected by initial enzyme concentrations greater than 500 U/mL olive oil. The optimum reaction temperature was 60°C.Comparison of conversion data between a test-tube scale reactor and a 2-L batch reactor revealed that the difference in conversion was within 10%. Experiments were also carried out with used cooking oil; the conversion with used cooking oil was slightly lower but no major differences were observed.The efficacy of Novozym435 was determined by reusing the enzyme; although the enzyme's relative activity decreased with reuse, it still retained 95% of its activity after five batches and more than 70% after as many as eight batches.
Keywords: Biodiesel; Novozym435; lipase
Chemo-enzymatic production of fuel ethanol from cellulosic materials utilizing yeast expressing β-glucosidases by Toshiyuki Uryu; Masashi Sugie; Satoshi Ishida; Shinya Konoma; Hironori Kato; Kaname Katsuraya; Kohsaku Okuyama; Gereltu Borjihan; Kazuhiro Iwashita; Haruyuki Iefuji (pp. 15-31).
Ethanol was produced in a considerably high yield by fermenting hydrolyzates from cellulosic materials by means of a recombinant laboratory yeast expressing β-glucosidases. Tissue paper, cotton, and sawdust were hydrolyzed by two-step sulfuric acid hydrolysis to give mixtures containing glucose, cellobiose, and higher cello-oligosacc arides. After the cellulosic material was partially hydrolyzed with 80% sulfuric acid, the hydrolysis was continued with 5% sulfuric acid. Except for non-carbohydrate components, all constitutents in the hydrolyzates were fermented by the yeast that was preincubated in the medium that the plasmid encoded by the β-glucosidases gene was kept in the muliplicated yeast. A solution containing 4% hydrolyzates from paper was fermented to give as high as 1.9% maximum ethanol concentration and 70% ethanol conversion. Cotton also gave a similar result. Sawdust was converted into ethanol in approx 22% conversion. Accordingly, it was revealed that the β-glucosidases-expressing yeast can ferment the cello-oligosaccharides obtained by hydrolysis of cellulosic materials into ethanol. In addition, a hydrolyzate containing a high glucose proportion gave a high ethanol concentration in a short time.
Keywords: Recombinant yeast; fuel ethanol fermentation; cellulose; two-step hydrolysis; paper; wood
Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation by Abha Mishra (pp. 33-42).
This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.
Keywords: l-Asparaginase; SSF; Aspergillus niger ; agro-waste; bran
Mutagenesis and analysis of mold Aspergillus niger for extracellular glucose oxidase production using sugarcane molasses by O. V. Singh (pp. 43-57).
Aspergillus niger ORS-4.410, a mutant of A. niger ORS-4, was generated by repeated ultraviolet (UV) irradiation. Analysis of the UV treatment dose on wild-type (WT) A. niger ORS-4, conidial survival, and frequency of mutation showed that the maximum frequency of positive mutants (25.5%) was obtained with a 57% conidial survival rate after the second stage of UV irradiation. The level of glucose oxidase (GOX) production from mutant A. niger ORS-4.410 thus obtained was 149% higher than that for WT strain A. niger ORS-4 under liquid culture conditions using hexacyanoferrate (HCF)-treated sugarcane molasses (TM) as a cheaper carbohydrate source. When subcultured monthly for 24 mo, the mutant strain had consistent levels of GOX production (2.62±0.51 U/mL). Mutant A. niger ORS-4.410 was markedly different from the parent strain morphologically and was found to grow abundantly on sugarcane molasses. The mutant strain showed 3.43-fold increases in GOX levels (2.62±0.51 U/mL) using HCF-TM compared with the crude form of cane molasses (0.762±0.158 U/mL).
Keywords: Glucose oxidase; sugarcane molasses; submerged fermentation; Aspergillus niger ; mutation
Sequential and simultaneous statistical optimization by dynamic design of experiment for peptide overexpression in recombinant Escherichia coli by Kwang-Min Lee; Chang-Hoon Rhee; Choong-Kyung Kang; Jung-Hoe Kim (pp. 59-80).
The production of recombinant anti-HIV peptide, T-20, in Escherichia coli was optimized by statistical experimental designs (successive designs with multifators) such as 24–1 fractional factorial, 23 full factorial, and 22 rotational central composite design in order. The effects of media compositions (glucose, NPK sources, MgSO4, and trace elements), induction level, induction timing (optical density at induction process), and induction duration (culture time after induction) on T-20 production were studied by using a statistical response surface method. A series of iterative experimental designs was employed to determine optimal fermentation conditions (media and process factors). Optimal ranges characterized by %T-20 (proportion of pepttide to the total cell protein) were observed, narrowed down, and further investigated to determine the optimal combination of culture conditions, which was as follows: 9, 6, 10, and 1 mL of glucose, NPK sources, MgSO4, and trace elements, respectively, in a total of 100 mL of medium inducted at an OD of 0.55–0.75 with 0.7 mM isopropyl-β-d-thiogalactopyranoside in an induction duration of 4 h. Under these conditions, up to 14% of T-20 was obtained. This statistical optimization allowed, the production of T-20 to be increased more than twofold (from 6 to 14%) within, a shorter induction duration (from 6 to 4 h) at the shake-flask scale.
Keywords: Anti-HIV peptide; statistical optimization; dynamic experimental design
Statistical medium formulation and process modeling by mixture design of experiment for peptide overexpression in recombinant Escherichia coli by Kwang-Min Lee; Chang-Hoon Rhee; Choong-Kyung Kang; Jung-Hoe Kim (pp. 81-100).
The medium formulation and robust process modeling for anti-HIV peptide (T-20) production by recombinant Escherichia coli overexpression were studied by employing a crossed experimental design. The crossed design, a mixture design combined with process factor (induction duration), was used to find the optimal medium formulation and process time. The optimal settings for three major components, (7.75 mL of NPK sources, 5.5 mL of glucose, and 11.75 mL of MgSO4) characterized by %T-20 (14.45%), the proportion of peptide to the total protein, were observed in a total of 100 mL of medium inducted at an optical density of 0.67 with 0.7 mM isopropyl-β-d-thiogalactopyranoside) for a 3-h induction duration at shake-flask scale. These conditions were further investigated to find robust, process conditions (8.2 mL of NPK sources, 5.6 mL of glucose, and 11.3 mL of MgSO4, and a 3.5-h induction duration time) for T-20 production (13.9%) by applying propagation of error.
Keywords: Anti-HIV peptide; statistical formulation; crossed design; robust process; propagation of error