Güzide Çalık

Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: use of citric acid as the carbon source

Abstract The effects of oxygen transfer on serine alkaline protease (SAP) production by Bacillus licheniformis on a defined medium with C c = 9.0 kg m −3 citric acid as sole carbon source were investigated in 3.5 dm 3 batch bioreactor systems. The concentrations of the product (SAP) and by-products, i.e., neutral protease, amylase, amino acids, and organic acids were determined in addition to SAP activities. At Q o /V = 1 vvm air flow rate, the effect of agitation rate on DO concentration, pH, product, and by-product concentrations and SAP activity were investigated at N = 150, 500, and 750 min −1 ; these are named as low-(LOT), medium-(MOT), and high oxygen transfer (HOT) conditions. LOT conditions favor biomass concentration; however, substrate consumption was highest at HOT conditions. MOT was optimum for maximum SAP activity which was 441 U cm −3 at t = 37 h. The total amino acid concentration was maximum in LOT and minimum in MOT conditions; lysine had the highest concentration under all oxygen transfer conditions. Among organic acids, acetic acid had the highest concentration and its concentration increased with oxygen transfer rate. The oxygen transfer coefficient increases with the agitation rate and the oxygen consumption rate increased almost linearly with the biomass concentration.

Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis.

The effects of oxygen transfer on the production and product distribution in serine alkaline protease (SAP) fermentation by Bacillus licheniformis and oxygen-transfer strategy in relation to the physiology of the bacilli were investigated on a defined medium with citric acid as sole carbon source in 3.5-dm(3) batch bioreactor systems. By forming a 3 x 3 matrix with the parameters air-inlet rates of Q(O)/V(R) = 0.2, 0.5, 1.0 vvm, and agitation rates of N = 150, 500, 750 min(-1), the effects of oxygen transfer were investigated at nine different conditions. The concentrations of the product SAP and by-products, i.e., neutral protease, alpha-amylase, amino acids, and organic acids, and SAP activities were determined throughout the bioprocess. Among the constant air-flow and agitation-rate fermentations, Q(O)/V(R) = 0.5 vvm, N = 750 min(-1) oxygen-transfer conditions produced maximum SAP activity that was 500 U cm(-3), at t = 37 h. With the increase in Q(O)/V(R) and/or N, Damköhler number that is the oxygen-transfer limitation decreases; and the process passes from oxygen-transfer limited conditions to biochemical-reaction limited conditions. Further increase in SAP activity, A = 680 U cm(-3) was achieved by applying an oxygen-transfer strategy based on the analysis of the data obtained with the constant oxygen-transfer condition experiments, with a step increase in air-inlet rate, from Q(O)/V(R) = 0.2 to Q(O)/V(R) = 0.5 vvm at N = 750 min(-1) constant agitation rate at t = 24 h. Organic acids and amino acids that were excreted to the fermentation medium varied depending on the oxygen-transfer conditions. With the increase in oxygen-transfer rate acetic acid concentration increased; contrarily, with the decrease in the oxygen-transfer rate the TCA-cycle organic acids alpha-ketoglutaric and succinic acids, and gluconic acid were excreted to the fermentation broth; nevertheless, the application of the oxygen-transfer strategy prevented the increase in acetic acid concentration between t = 35-38 h. Under all the oxygen-transfer conditions, the amino acid having the highest concentration and the amino acid that was not excreted to the fermentation broth were lysine and asparagine, respectively; both of which belong to the aspartic acid-group amino acids. Further, this result indicates the requirement of the genetic regulation directed to the aspartic acid-group enzymes for the progress in SAP production in B. licheniformis.

Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium: effects of the oxygen transfer rate.

The metabolic fluxes through the central carbon pathways in the bioprocess for serine alkaline protease (SAP) production by Bacillus licheniformis were calculated by the metabolic flux-based stoichiometric model based on the proposed metabolic network that contains 102 metabolites and 133 reaction fluxes using the time profiles of citrate, dry cell, organic acids, amino acids, and SAP as the constraints. The model was solved by minimizing the SAP accumulation rate in the cell. The effects of the oxygen-transfer rate (OTR) on the metabolic fluxes were investigated in a defined medium where citrate was used as the sole carbon source. The central pathways were active for the growth and the SAP synthesis in all the periods of the bioprocess at low (LOT), medium (MOT), and high (HOT) oxygen-transfer conditions. The flux partitioning in the TCA cycle at α-ketoglutarate towards glutamate group and at oxalacetate (OA) toward aspartic acid group amino acids were dependent on the OTR. The flux of the anaplerotic reaction that connects the TCA cycle either from malate or OA to the gluconeogenesis pathway via the main branch point pyruvate (Pyr) was also influenced by the OTR. With the decrease in the OTR, the intracellular flux values after glycerate 3-phosphate (PG3) in the gluconeogenesis pathway and the specific growth rate decreased. The total ATP-generation rate increased with the increase in OTR. The pathway towards the aspartic acid family amino acids which is important for sporulation that precedes the SAP synthesis were all active throughout the bioprocess. Metabolic flux analysis results at LOT, MOT, and HOT conditions encourage the design of an oxygen-transfer strategy in the bioreactor; moreover, asparagine synthetase or aspartate kinase could be the potential metabolic engineering sites due to the low value of the flux from the branch point aspartate toward asparagine.

Bioreactor operation parameters as tools for metabolic regulations in fermentation processes: influence of pH conditions

The influence of controlled- and uncontrolled-pH conditions together with the initial pH on the product and by-product distributions and oxygen transfer characteristics, whereupon the process rate limitations in relation to the intracellular reaction rates were investigated in serine alkaline protease (SAP) fermentation process by recombinant Bacillus licheniformis carrying pHV1431::subC on a defined medium with the sole carbon source glucose in the pH range of 6.80-7.25 in batch bioreactors. Although the same amount of cell was produced at all the conditions in each type of operation, with the increase of initial pH the cell formation rates increased; moreover, uncontrolled-pH operation was favourable for the cell formation. The SAP synthesis rates and concentrations were higher at uncontrolled-pH operations. Among the fermentations, pH 0 = 7.10 uncontrolled-pH operation produced maximum SAP activity that was ca. 900 U cm -3 at t = 21 h. According to the biomass and SAP production profiles, bioprocess was divided into two periods; Period I(0 < t ≤ 10 h) covers the cell growth phase and Period II(10 < t ≤ 24 h) covers the SAP production phase. In Period I in both operations, while the oxygen uptake rate (OUR) increased with the increase in initial pH the oxygen transfer rate (OTR) decreased. In Period II, among uncontrolled-pH operations OUR was the lowest at pH = 7.10 while OTR decreased with the increase in initial pH. At the oxygen transfer condition applied, the bioprocess is biochemical reaction limited at all the conditions; nevertheless, with the decrease in initial pH, Damkohler number that is the oxygen transfer limitation decreases. Further, the perturbation effects of initial pH and the operation conditions on the intracellular reaction rates were calculated for Periods I and II by using the experimental data obtained and, the diversions in the pathways and certain metabolic reactions and potential strategies for improving SAP production are also discussed.

Metabolic flux distribution for the optimized production of l-glutamate

Abstract A comprehensive metabolic network was proposed for glutamic acid bacteria and used in a stoichiometrically based flux balance model for l -glutamate production. Theoretical metabolic pathways leading to optimized glutamate overproduction were determined for several specific cell growth rates; variation in the fluxes was obtained. The off-line extracellular analyses throughout the batch fermentation with Brevibacterium flavum showed the accumulation of arginine, aspartate, lysine, alanine, proline, lactate, α-ketoglutarate, succinate, pyruvate, and gluconate in the medium in addition to glutamate. Metabolic flux distributions in the cells throughout the fermentation were determined using the model in combination with the extracellular analyses of the metabolites. The flux distribution maps showed that the cells utilized the TCA cycle in part whereas the glyoxylate bypass was active throughout the fermentation. The results also indicated that the phosphate pentose shunt played an important role in the glutamate fermentation. These diversions in the pathways and certain metabolic reactions depending on the fermentation periods and conditions are also presented in this paper.

BIOPROCESS DEVELOPMENT FOR SERINE ALKALINE PROTEASE PRODUCTION: A REVIEW

This work is the result of the convergence of the research in biochemical reaction engineering and metabolic flux analysis for serine alkaline protease (SAP) production. The genus Bacillus includes a variety of industrially important species that are known to secrete a large number of extracellular proteases and are used among many species as producer of SAP enzyme. Therefore, in the first part of the present article an overview to serine alkaline protease and regulation of its synthesis and secretion in Bacillus is presented. In the second part, a detailed review of the published information on the bioprocess medium design and bioreactor operation parameters are discussed in relation to the concentrations of the byproducts, i.e. neutral protease, amylase, amino acids, organic acids and alcohols. In the third part, papers on metabolic flux analysis for SAP are reviewed with the emphasis on SAP overproduction potential of the Bacillus licheniformis, the effect of oxygen transfer on the bioreaction-network fluxes and the need for oxygen transfer strategies. The metabolic bottlenecks and strategies for increasing the yield and selectivity of SAP fermentation process are discussed. * To whom all correspondence should be addressed: Dr. Pinar Calik, Associate Professor, Department of Chemical Engineering, Middle East Technical University, 06531 Ankara, Turkey e-mail: pcalik@metu.edu.tr: Tel #: 00.90.312.210 43 85; Fax #: 00.90.312.210 12 64

Expression System for Synthesis and Purification of Recombinant Human Growth Hormone in Pichia pastoris and Structural Analysis by MALDI-ToF Mass Spectrometry

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZαA vector under the control of AOX1 promoter, which included a polyhistidine‐tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non‐native N‐and C‐termini. Analyses of the affinity‐purified rhGH product by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) showed a spectral peak at m/ z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu‐Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile‐Glu‐Gly‐Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N‐Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.

Expression system for recombinant human growth hormone production from Bacillus subtilis

We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid‐gene of two DNA fragments, i.e., signal (pre‐) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg‐promoter in B. subtilis. Recombinant‐hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N‐terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal‐peptidase. The highest rhGH concentration was obtained at t = 32 h as CrhGH = 70 mg L−1 with a product yield on substrate YrhGH/S = 9 g kg−1, in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic‐acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino‐acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species.

Serine alkaline protease overproduction capacity of Bacillus licheniformis

Abstract A comprehensive metabolic network that considers 147 reaction fluxes and 105 metabolites is used in a mass-flux-balance-based stoichiometric model for Bacillus licheniformis for serine alkaline protease (SAP) overproduction. The theoretical capacity analysis leading to optimized SAP overproduction was carried out by using a linear constrained optimization technique for several specific growth rates and the variation of the fluxes were calculated by fixing the sole carbon source citrate’s uptake rate at 10 mmol/gDW/h. The theoretical data-based capacity analysis was conducted by using the model in combination with the off-line extracellular analyses of the dry cell and the metabolites that were citrate, organic acids, amino acids, and SAP; and the variations in the intracellular fluxes were obtained for the three periods of the batch bioprocess. The flux distribution maps of the analyses showed that the tricarboxylic acid cycle was active and the cells utilized the gluconeogenesis pathway, the pentose phosphate pathway, and the anaplerotic reactions; nevertheless, the glyoxylate shunt and the glycolysis pathway were inactive. The theoretical capacity analysis showed that SAP synthesis flux increased with the decrease in the specific growth rate, and was the highest at μ = 0 h −1 as 0.0260 mmol/gDW/h. Both in the theoretical capacity and the theoretical data-based capacity analyses, among the fluxes towards the amino acid groups, aspartic acid group had the highest value and aromatic acid group had the lowest flux value; the flux distributions are similar. The flux values towards SAP was maximum in Period II, whereas it was minimum in Period I. In Period II of the theoretical data-based capacity analysis, the fluxes of alanine and valine are higher than the other amino acid fluxes; and the pyruvate branch point seems to be the potential metabolic engineering site. The results reveal that SAP production can theoretically be increased 1.09, 16.68, and 7.21 folds, respectively, in Periods I, II, and III. The diversions in the pathways and certain metabolic reactions depending on the bioprocess periods and potential strategies for improving SAP production are also discussed.

Metabolic flux analyses for serine alkaline protease production

The intracellular metabolic fluxes through the central carbon pathways in Bacillus licheniformis in serine alkaline protease (SAP) production were calculated to predict the potential strategies for increasing the performance of the bacilli, by using two optimization approaches, i.e. the theoretical data-based (TDA) and the theoretical data-based capacity (TDC) analyses based on respectively minimum in-vivo SAP accumulation rate and maximum SAP synthesis rate assumptions, at low-, medium-, and high-oxygen transfer conditions. At all periods of low-oxygen transfer condition, in lag and early exponential periods of medium-oxygen transfer (MOT) condition, and SAP synthesis period of high-oxygen transfer (HOT) condition, the TDA and TDC analyses revealed that SAP overproduction capacity is almost equal to the observed SAP production due to the regulation effect of the oxygen transfer. In the growth and early SAP synthesis period and in SAP synthesis period at MOT condition the calculated results of the two analyses reveal that SAP synthesis rate of the microorganism can be increased 7.2 and 16.7 folds, respectively; whereas, in the growth and early SAP synthesis period at HOT condition it can be increased 12.6 folds. The diversions in the biochemical reaction network and the influence of the oxygen transfer on the performance of the bacilli were also presented. The results encourage the application of metabolic engineering for lifting the rate limitations and for improving the genetic regulations in order to increase the SAP production.