• Journal of Microbiology and Biotechnology
• /
• v.19 no.4
• /
• pp.378-386
• /
• 2009

A sequential optimization strategy, based on statistical experimental designs, is employed to enhance the production of alkaline protease by a Bacillus pseudofirmus local isolate. To screen the bioprocess parameters significantly influencing the alkaline protease activity, a 2-level Plackett-Burman design was applied. Among 15 variables tested, the pH, peptone, and incubation time were selected based on their high positive significant effect on the protease activity. A near-optimum medium formulation was then obtained that increased the protease yield by more than 5-fold. Thereafter, the response surface methodology(RSM) was adopted to acquire the best process conditions among the selected variables, where a 3-level Box-Behnken design was utilized to create a polynomial quadratic model correlating the relationship between the three variables and the protease activity. The optimal combination of the major medium constituents for alkaline protease production, evaluated using the nonlinear optimization algorithm of EXCEL-Solver, was as follows: pH of 9.5, 2% peptone, and incubation time of 60 h. The predicted optimum alkaline protease activity was 3,213 U/ml/min, which was 6.4 times the activity with the basal medium.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.10 no.5
• /
• pp.408-417
• /
• 2005

Increasing numbers of value added chemicals are being produced using microbial fermentation strategies. Computational modeling and simulation of microbial metabolism is rapidly becoming an enabling technology that is driving a new paradigm to accelerate the bioprocess development cycle. In particular, constraint-based modeling and the development of genome-scale models of industrial microbes are finding increasing utility across many phases of the bioprocess development workflow. Herein, we review and discuss the requirements and trends in the industrial application of this technology as we build toward integrated computational/experimental platforms for bioprocess engineering. Specifically we cover the following topics: (1) genome-scale models as genetically and biochemically consistent representations of metabolic networks; (2) the ability of these models to predict, assess, and interpret metabolic physiology and flux states of metabolism; (3) the model-guided integrative analysis of high throughput 'omics' data; (4) the reconciliation and analysis of on- and off-line fermentation data as well as flux tracing data; (5) model-aided strain design strategies and the integration of calculated biotransformation routes; and (6) control and optimization of the fermentation processes. Collectively, constraint-based modeling strategies are impacting the iterative characterization of metabolic flux states throughout the bioprocess development cycle, while also driving metabolic engineering strategies and fermentation optimization.

• KSBB Journal
• /
• v.11 no.6
• /
• pp.704-711
• /
• 1996

An optimal process design of a foreign protein production system was carried out using a bioprocess flowsheeting software, BioPro Designer, with a capability of economic analysis. The flowsheeting program was applied to a production system of the tailspike protein of Salmonella phage P22, and helped save time and efforts in selecting an optimal process. A wild type tailspike and two types of mutant tailspikes, tsf G244\longrightarrow,R and Su A334\longrightarrowV, were considered in this study to show that the folding characteristics of foreign protein produced inside host influenced the selection of the best production system. An optimal production system for mature tailspike was chosen under the criterion of capital investment per unit mass of mature protein recovered.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.12
• /
• pp.1930-1936
• /
• 2007

A sequential optimization strategy based on statistical experimental designs was employed to enhance the production of cyclosporin A (CyA) by Tolypocladium inflatum DSMZ 915 in a submerged culture. A 2-level Plackett-Burman design was used to screen the bioprocess parameters significantly influencing CyA production. Among the 11 variables tested, sucrose, ammonium sulfate, and soluble starch were selected, owing to their significant positive effect on CyA production. A response surface methodology (RSM) involving a 3-level Box-Behnken design was adopted to acquire the best process conditions. Thus, a polynomial model was created to correlate the relationship between the three variables and the CyA yield, and the optimal combination of the major media constituents for cyclosporin A production, evaluated using the nonlinear optimization algorithm of EXCEL-Solver, was as follows (g/l): sucrose, 20; starch, 20; and ammonium sulfate, 10. The predicted optimum CyA yield was 113 mg/l, which was 2-fold the amount obtained with the basal medium. Experimental verification of the predicted model resulted in a CyA yield of 110 mg/l, representing 97% of the theoretically calculated yield.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.8 no.6
• /
• pp.313-321
• /
• 2003

This review summarizes the recent advances in high-density algal cultures in the field of algal biotechnology. Photobioreactor engineering for economical and effective utilization of algae and its products has made impressive and promising progress. Bioprocess engineers have expedited the design and the operation of algal cultivation systems. Many of them in use today are open systems due to cost considerations, and closed photobioreactors have recently attracted a considerable attention for the production of valuable biochemicals or for special applications. For high-density cultures, the optimization of environmental factors in the photobioreactors have been explored, including light delivery, CO$_2$and O$_2$gas transfer, medium supply, mixing and temperature. It is expected that further advanced photobioreactor engineering will enable the commercialization of noble algal products within the next decade.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.10 no.3
• /
• pp.192-197
• /
• 2005

The effects of macronutrients $(NO_3^-,\; NH_4^+\;and\;PO_4^{3-})$ on cell growth and triterpenoids production in Centella asiatica cell suspension cultures were analyzed using the Box­Behnken response surface model experimental design. In screening and optimization experiments, $PO_4^{3-}$ as a single factor significantly influenced cell growth where increasing the phosphate level from 0.1 to 2.4 or 2.6 mM, elevated cell growth from 3.9 to $14\~16g/L$. The optimum values predicted from the response surface model are 5.05mM $NH_4^+$, 15.0mM $NO_3^-$ and 2.6mM $PO_4^{3-}$, yielding 16.0g/L cell dry weight with $99\%$ fitness to the experimental data. While the $NH_4^+-NO_3^-$ interaction influenced cell growth positively in the optimization experiment, $NH_4^+$ and $NO_3^-$ as single factors; and interactions of $NO_3^--PO_4^{3-},\;NH_4^+-PO_4^{3-}$ and $NH_4^+-NO_3^-$ were all negative in the screening experiment. Cell growth and the final pH level were positively affected by $PO_4^{3-}$, but negatively affected by $NH_4^+\;and\;NH_4^+-PO_4^{3-}$ interactions. The different effects of factors and their interactions on cell growth and final pH are influenced by a broad or narrow range of macronutrient concentrations. The productions of triterpenoids however were lower than 4mg/g cell dry weight.

• Microbiology and Biotechnology Letters
• /
• v.49 no.2
• /
• pp.181-191
• /
• 2021

The present study addresses isolation, optimization, partial purification, and characterization of a haloalkaline serine protease from a newly isolated haloarchaeal strain isolated from Wadi El Natrun in Egypt. We expected that a two-step sequential statistical approach (one variable at a time, followed by response surface methodology) might maximize the production of the haloalkaline serine protease. The enzyme was partially purified using Hiprep 16/60 sephacryl S-100 HR gel filtration column. Molecular identification revealed the newly isolated haloarchaeon to be Natrialba hulunbeirensis strain WNHS14. Among several tested physicochemical determinants, casamino acids, KCl, and NaCl showed the most significant effects on enzyme production as determined from results of the One-Variable-At-A-time (OVAT) study. The BoxBehnken design localized the optimal levels of the three key determinants; casamino acids, KCl, and NaCl to be 0.5% (w/v), 0.02% (w/v), and 15% (w/v), respectively, obtaining 62.9 U/ml as the maximal amount of protease produced after treatment at 40℃, and pH 9 for 9 days with 6-fold enhancement in yield. The enzyme was partially purified after size exclusion chromatography with specific activity, purification fold, and yield of 1282.63 U/mg, 8.9, and 23%, respectively. The enzyme showed its maximal activity at pH, temperature, and NaCl concentration optima of 10, 75℃, and 2 M, respectively. Phenylmethylsulfonyl fluoride (PMSF, 5 mM) completely inhibited enzyme activity.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.10 no.5
• /
• pp.400-407
• /
• 2005

Reverse engineering is defined as the process where the internal structures and dynamics of a given system are inferred and analyzed from external observations and relevant knowledge. The first part of this paper surveys existing techniques for biosystem reverse engineering. Network structure inference techniques such as Correlation Matrix Construction (CMC), Boolean network and Bayesian network-based methods are explained. After the numeric and logical simulation techniques are briefly described, several representative working software tools were introduced. The second part presents our component-based software architecture for biosystem reverse engineering. After three design principles are established, a loosely coupled federation architecture consisting of 11 autonomous components is proposed along with their respective functions.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.5 no.4
• /
• pp.275-287
• /
• 2000

Fermentation strategies for recombinant protein production in Pichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, app58lication of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.5
• /
• pp.587-601
• /
• 2013

During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.