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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.95, #2)
Resin selection and single-step production and recovery of lactic acid from pretreated wood by Ana B. Moldes; José L. Alonso; Juan C. Parajó (pp. 69-81).
Four ion-exchange resins (Amberlite IRA 900, IRA 400, IRA 96, and IRA 67) were employed for lactic acid recovery from simultaneous saccharification and fermentation (SSF) media. The best resins (Amberlite IRA 900 and IRA 400) were assayed for capacity, regenerant consumption, percentage of lactic acid recovery, and product concentration. Almost quantitative lactic acid recoveries at constant capacities were achieved in four sequential loading/regeneration cycles. A strong-base resin (Amberlite IRA 400) was selected for intermittent lactic acid separation in a typical SSF process, in which pretreated wood was saccharified by cellulases in the presence of Lactobacillus delbrueckii. The dynamics of lactic acid generation and lactic acid recovery were established.
Keywords: Ion-exchange resin; lactic acid; lactobacillus delbrueckii ; separation; simultaneous saccharification and fermentation
Simultaneous purification and reversible immobilization of d-amino acid oxidase from Trigonopsis variabilis on a hydrophobic support by Stanislaus F. D’Souza; Ashwini Deshpande (pp. 83-92).
Purification and reversible immobilization of d-amino acid oxidase from Trigonopsis variabilis could be simultaneously accomplished by hydrophobic interaction on Phenyl Sepharose CL-4B in the presence of 50 mM pyrophosphate buffer (pH 8.5). The presence of a high salt concentration of 2M, which is generally required for the hydrophobic interactions, was not essential for the hydrophobic immobilization. The enzyme in free as well as immobilized form was optimally active between pH 7.0 and 9.0. The immobilized preparation could be reused in a batch process for the conversion of d-amino acids to α-keto acids. When the activity of the preparation dropped below practical limits, the gel could be regenerated by water wash and recharged with fresh crude extract from yeast.
Keywords: Immobilization; Trigonopsis variabilis ; hydrophobic support; Phenyl Sepharose CL-4B; reversible immobilization; α-keto acids; d-amino acid oxidase
Cloning and expression of L-asparaginase gene in Escherichia coli by Yingda Wang; Shijun Qian; Guangzhen Meng; Shuzheng Zhang (pp. 93-101).
The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The recombinant plasmid in E. coli AS1.357 remained stable after 72h of cultivation and 5h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data.
Keywords: Escherichia coli ; L-asparaginase; gene expression; plasmid stability; DNA sequence
Multipulse feed strategy for glycerol fed-batch fermentation by Dongming Xie; Dehua Liu; Haoli Zhu; Tianzhong Liu (pp. 103-112).
During glycerol fed-batch fermentation, the process could be divided into multiple equal subintervals, and the feed operation was performed in pulse form at the start of each subinterval. Based on the macrokinetic models, the multipulse feed strategy for both glucose and corn steep slurry was determined by a general nonlinear optimization approach to maximize the final glycerol productivity and still control the residual glucose at a low concentration. The experimental results in a 600-mL Airlift Loop Reactor showed that the tested data with this strategy agreed well with the corresponding model prediction, and that the feed mode with nonlinear optimization could improve the glycerol productivity significantly compared with those determined just by limited experimental optimization in previous studies.
Keywords: Glycerol; multipulse feed; fed-batch fermentation; nonlinear optimization
Optimization of secondary metabolite production using singular approximation and minimum principle by Jung-Heon Lee (pp. 113-124).
Optimal control profiles as calculated with two control algorithms, singular approximation and minimum principle, are compared in this article. Switching points were determined using the singular approximation by mathematical calculation. The optimal growth rate was calculated using minimum principle. With an increased number of switching points, the calculated optimal control profiles approached the theoretical optimal control profile as calculated using the minimum principle. With three switching times, the product concentration approached 96% of the theoretical optimal control profile. From these results, optimal control can be achieved with more than a three-switching-point approximation.
Keywords: Optimization; singular approximation; minimum principle
Dioxygen reactivity of laccase by Feng Xu (pp. 125-133).
A study was carried out on the dioxygen reactivity of the laccases from Trametes villosa, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum, and Coprinus cinereus. At pH 5.5, these laccases showed an apparently constant K m (about 20–50 µM) for O2 with either 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) or methyl syringate as the reducing substrate, in contrast to the k cat , which varied up to 100-fold. O2 reactivity of the Trametes and Myceliophthora laccases was also studied at various pH and NaF concentrations. The apparent K m of Trametes and Myceliophthora laccases varied only slightly when pH changed from 3.0 to 8.0 or when the laccases were inhibited by F− at pH 5.5, although the apparent k cat were more significantly affected by both factors. The dependence of the apparent K m on the source of laccase, pH, and F− inhibition suggested that the fungal laccases might have a conserved O2 pocket and that the F− or OH− inhibition might affect the O2 reduction step (k cat ) more than the O2 binding step (K m ) under steady-state conditions.
Keywords: Laccase; dioxygen reactivity; pH profile; anion inhibition; fungi
Biodegradation of chlorolignin and lignin-like compounds contained in E1-Pulp bleaching effluent by fungal treatment by Carlos H. L. Soares; Nelson Duran (pp. 135-149).
The ligninolytic system from the fungi Trametes villosa and Panus crinitus can efficiently degrade all fractions of different molecular mass contained in E1-bleaching effluent, but with different degradation rates. The lower-molecular-mass (MM) materials were better characterized when the elution in the size-exclusion high-performance liquid chromatography were monitored at 210 than at 280 nm, which indicates that these compounds may be ring cleavage byproducts from depolymerized chlorolignin. The biodegradation of E1 effluent by both fungi was a multistage process, involving an initial chemical modification of the higher-MM compounds and concomitant oxidation of the lower-MM materials. A subsequent depolymerization of chemically modified polymeric lignin-like compounds also took place. Each stage may require one or several different enzymes. The results suggested that laccase was involved in the initial stage.
Keywords: Effluent biodegradation; E1-pulp bleaching effluent; basidiomycete fungi; laccase; ligninolytic enzymes; size-exclusion high-performance liquid chromatography; chlorolignin