Tunçer H. Özdamar

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.

Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and α-amylase

The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and alpha-amylase (AMY) on five carbon sources that have different reduction degrees (gamma), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design. Glucose (gamma=4.0), acetate (gamma=4.0), and the TCA cycle organic-acids succinate (gamma=3.5), malate (gamma=3.0), and citrate (gamma=3.0) were selected for the theoretical analyses and comparisons. A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed. Highest and lowest specific cell growth rates (&mgr;) were calculated as 1.142 and 0.766h(-1), respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources. Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used. Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191mmolg(-1)DWh(-1) when glucose uptake rate was 10mmolg(-1)DWh(-1) and specific growth rate was zero. The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity. For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points. Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high. The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY.

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.

Mass flux balance-based model and metabolic pathway engineering analysis for serine alkaline protease synthesis by Bacillus licheniformis

A mass flux balance-based stoichiometric model of Bacillus licheniformis for the serine alkaline protease (SAP) fermentation process has been established. The model considers 147 reaction fluxes, and there are 105 metabolites that are assumed to be in pseudo-steady state. Metabolic flux distributions were obtained from the solution of the model based on the minimum SAP accumulation rate assumption in B. licheniformis in combination with the off-line extracellular analyses of the metabolites that were the sole carbon source citrate, dry cell, organic acids, amino acids, and SAP; variations in the intracellular fluxes were demonstrated for the three periods of the batch bioprocess. The flux distribution maps showed that the cells completed the TCA cycle and utilized the gluconeogenesis pathway, pentose phosphate pathway, and anaplerotic reactions throughout the fermentation; however, the glycolysis pathway was inactive in all the periods of the fermentation. The flux values toward SAP increased throughout the bioprocess and slightly decreased in the last period; however, SAP selectivity values were almost the same in Periods II and III and higher than Period I. The diversions in the pathways and certain metabolic reactions depending on the bioprocess periods are also presented and the results indicated that the intracellular amino acid fluxes played an important role in the SAP fermentation process.

The influence of carbon sources on recombinant‐human‐ growth‐hormone production by Pichia pastoris is dependent on phenotype: a comparison of Muts and Mut+ strains

The influence of carbon sources on rhGH (recombinant human growth hormone) production by two Pichia pastoris strains having different methanol utilization phenotypes (P. pastoris‐hGH‐Mut+ and P. pastoris‐hGH‐Muts) was investigated using batch bioreactors. The effect of methanol concentration (CMeOH) in defined and complex media, and further glycerol/methanol mixed defined media, was analysed systematically over a wide range. With methanol as the sole carbon source, strain Muts grew only slightly, whereas with Mut+, a cell concentration (CX) of 6.0 g of dry cells/dm3 was obtained and an rhGH concentration (CrhGH) of 0.032 g/dm3 was produced. In complex medium without glycerol at a CMeOH of 2% (v/v), a CrhGH of 0.16 g of rhGH/dm3 was produced by Muts, a value 3‐fold higher than that produced by Mut+, despite the fact that the CX of Mut+ (6.1 g/dm3) was 2‐fold higher than that of Muts (3.0 g/dm3). In a glycerol/methanol mixed defined medium, methanol consumption began when glycerol was totally depleted, indicating that glycerol is a repressor of the AOX1 (alcohol oxidase‐1 gene) promoter. With strain Muts at a glycerol concentration (CGly) of 30 g/dm3 and a CMeOH of 1% (v/v), the CrhGH produced was 0.11 g/dm3, whereas, with the Mut+ strain, a CrhGH of 0.06 g/dm3 was obtained at a CGly of 30 g/dm3 and a CMeOH of 4%. As methanol is not consumed by Muts strain effectively and the presence of methanol in the fermentation broth triggers induction of the AOX1 promoter, our results encourage the use of the Muts strain for rhGH production. In addition to rhGH production, the specific cell growth rates, specific methanol and/or glycerol utilization rates and maintenance coefficients in methanol‐ and glycerol‐based defined media were determined. With a methanol‐based defined medium and using the Mut+ strain, a higher specific growth rate (μ) of approx. 0.14 h−1 was observed during the exponential cell growth phase at a CMeOH of ≤2.0%. When glycerol was used as a sole carbon source, both phenotypes showed similar cell‐growth and glycerol‐utilization rates. The results of the present study should enable one to optimize the expression of other therapeutic proteins by P. pastoris.

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

Bioconversion of trans-cinnamic acid to L-phenylalanine by L-phenylalanine ammonia-lyase of Rhodotorula glutinis : parameters and kinetics

The kinetics of the bioconversion of trans-cinnamic acid to l-phenylalanine by the l-phenylalanine ammonialyase (PAL) enzyme of resting cells of Rhodotorula glutinis have been studied under the optimum bioreaction conditions determined. The optimum pH, temperature, ammonia concentration, and biocatalyst loading were 10.5, 30°C, 8 m, and ca. 2 (w/w), respectively. Among various chemicals, sodium glutamate and penicillin were found to increase the PAL activity of the cells. A maximum concentration of 76.18 mm (12.57 g dm−3) l-phenylalanine was maintained from 100 mm (14.8 g dm−3) trans-cinnamic acid after 104 h of residence time in the fed-batch operation of the bioreactor. trans-Cinnamic acid concentrations higher than 30–50 mm were shown to cause the substrate inhibition, besides the mixed-type inhibition effect of the chloride ions. Mechanistic bioreaction rate equations that incorporate separately the inhibition effects of the trans-cinnamic acid and chloride ions, and the activation effect of sodium glutamate, were proposed and the kinetic parameters of the models were calculated.

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.