Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS-22 in batch, fed-batch, and continuous cultures

Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media.     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Application of factorial experimental designs for optimization of cyclosporin. A production by Tolypocladium inflatum in submerged culture

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.     

Application of factorial and Doehlert designs for optimization of protopectinase production by a Geotrichum klebahnii strain

Two successive factorial designs followed by two response surface methodology were applied to optimize protopectinase production by Geotrichum klebahnii. Factorial designs were used to study the effect of 11 variables (mineral pool and pH) on enzyme production. Only pH and Fe²⁺ had significant effect on the protopectinase production in the conditions of the assay, the interaction between them not being significant. According to this result pH and Fe²⁺ were multivariate according to a Doelhert design. The central points were pH 3.5 and Fe²⁺ 1.8 μmol L⁻¹. The results show a negative effect of pH and a positive (quadratic) effect of Fe²⁺ on enzyme production. The second Doelhert design was centered at pH 3.3. A relative maximum was obtained at pH 2.8 and Fe²⁺ 0.540 μmol L⁻¹, where the enzyme activity obtained was 236 U/mL. This value is two times higher than the value reported elsewhere.     

Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli

 The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2⁴ factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 g l^-1 to 1.9 g l^-1 (dry weight) and from 10 units ml^-1 to 210 units ml^-1 within 24 h at 30°C in shake flasks. Received: 26 July 1995/Received revision: 22 January 1996/Accepted: 29 January 1996     

Cyclodextrin production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrins (CD) are cyclic oligosaccharides with multiple applications in the food, pharmaceutical, cosmetic, agricultural and chemical industries. In this work, the conditions used to produce CD with cyclodextrin glycosyltransferase from Bacillus circulans DF 9R were optimized using experimental designs. The developed method allowed the partial purification and concentration of the enzyme from the cultural broth and, subsequently, the CD production, using the same cassava starch as enzyme adsorbent and as substrate. Heat-treatment of raw starch at 70 degrees C for 15 min in the presence of adsorbed cyclodextrin glycosyltransferase allowed the starch liquefaction without enzyme inactivation. The optimum conditions for CD production were: 5% (w/v) cassava starch, 15 U of enzyme per gram of substrate, reaction temperature of 56 degrees C and pH 6.4. After 4h, the proportion of starch converted to CD reached 66% (w/w) and the weight ratio of alpha-CD:beta-CD:gamma-CD was 1.00:0.70:0.16.     

Studies of the inhibition by malto-oligosaccharides of the cyclisation reaction catalysed by the cyclodextrin glycosyltransferase from Klebsiella pneumoniae M 5 al with glycogen

The substrate qualities of malto-oligosaccharides for the disproportionation reaction catalysed by the cyclodextrin glycosyltransferase [(1----4)-alpha-D-glucan:[(1----4)-alpha-D-glucopyranosyl]transferase (cyclising) EC 2.4.1.19] from Klebsiella pneumoniae M 5 al have been re-investigated. Maltose failed to be homologised with measurable velocity. The initial rates of disproportionation and the affinities of the enzyme increased with the chain lengths of the substrates. Maltopentaose was the smallest saccharide which, by disproportionation, yielded longer chains being cyclised initially. D-Glucose did not affect the initial cyclisation from glycogen, but served as acceptor for the "chain-shortening" reaction. Maltose inhibited the initial cyclisation reaction in a linearly competitive manner. Maltotriose and maltotetraose inhibited the cyclisation reaction competitively, the inhibition kinetics pointing to the binding of two effector-molecules to the enzyme. Competitive inhibition was also found with malto-pentaose, -hexaose, and -heptaose. The degrees of inhibition increased from maltose to maltotetraose, and decreased with the larger saccharides; maltotriose and maltotetraose were the most effective inhibitors of the initial cyclisation. Some possibilities for the subsite-mechanisms are discussed.     

Application of statistically-based experimental designs for the optimization of nisin production from whey

Statistically-based experimental designs were applied for the optimization of nisin production by Lactococcus lactis in a whey-based medium. Yeast extract, KH₂PO₄, and MgSO₄ were identified to have significant effects on nisin biosynthesis by a Plackett–Burman design. These three significant factors were subsequently optimized using central composite design, and the optimal conditions were determined to be 12.067 g l^–1 for yeast extract, 0.569 g l^–1 for KH₂PO₄, and 0.572 g l^–1 for MgSO₄. The validity of the optimal conditions was verified by a separate experiment.     

Chemical modification of lysine side chains of cyclodextrin glycosyltransferase from Thermoanaerobacter causes a shift from cyclodextrin glycosyltransferase to alpha-amylase specificity

Cyclodextrin glycosyltransferases and alpha-amylases are two groups of enzymes with related secondary structures. However, cyclodextrin glycosyltransferases display transferase activities not present in alpha-amylases, probably derived from the existence of two more domains and different amino acid sequences. The hydrolytic activity of cyclodextrin glycosyltransferases is generally quite low, except for two cyclodextrin glycosyltransferases from termophiles. In this work, we have carried out the chemical modification (with acetic anhydride) of the amino groups of cyclodextrin glycosyltransferase from Thermoanaerobacter to assess their contributions to protein function. The acetylated cyclodextrin glycosyltransferase showed a significant reduction of its cyclization, coupling and disproportionation activities. Surprisingly, the hydrolytic (saccharifying) activity was slightly enhanced. These results suggest the participation of one or more lysine side chains in the interactions contributing to the transferase activity, either in any of the S11 subsites or in the acceptor binding site.     

Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.     

Optimization of bio-hydrogen production from biodiesel wastes by Klebsiella pneumoniae

Biodiesel wastes containing glycerol were utilized by Klebsiella pneumoniae DSM 2026 to produce hydrogen. The optimization of medium components was performed using both Plackett-Burman and uniform design methods. Using the Plackett-Burman design, glycerol, yeast extract, NH(4)Cl, KCl and CaCl2 were found to be the most important components, which were further investigated by uniform design and second-order polynomial stepwise regression analysis. The optimized medium containing 20.4 g.L(-1) glycerol, 5.7 g.L(-1) KCl, 13.8 g.L(-1) NH(4)Cl, 1.5 g.L(-1) CaCl(2) and 3.0 g.L(-1) yeast extract resulted in 5.0-fold increased level of hydrogen (57.6 mL/50 mL medium) production compared to initial level (11.6 mL/50 mL medium) after 24 h of fermentation The optimization of fermentation condition (pH, temperature and inoculum) was also conducted. When the strain grew in the optimized medium under optimal fermentation condition in a 5-L stirred tank bioreactor for batch production, hydrogen yield and production reached 0.53 mol/mol and 117.8 mmol/L, respectively. The maximum hydrogen evolution rate was 17.8 mmol/(L.h). Furthermore, 1,3-propanediol (6.7 g.L(-1)) was also obtained from the liquid medium as a by-product.     

Improved process for exopolysaccharide production by Klebsiella pneumoniae sp. pneumoniae by a fed-batch strategy

Exopolysaccharide (EPS) was produced by Klebsiella pneumoniae K63 grown in fed-batch cultures using different procedures of the supply of carbon or nitrogen (N) source, or both. Cultures grown with excess of glucose and limitation or exhaustion of N produced 54.8 and 47.4 g(EPS) l(-1), respectively. These cultures also led to an accumulation of 'overflow' metabolites representing more than 16% of carbon conversion. The consistency indexes ( K ) obtained to the end of the cultures, characteristic of the rheological property of the biopolymer, were 16.4 Pa s(n) for N deficiency and 5.2 Pa s(n) for N limitation conditions. The simultaneous limitation of glucose and N decreased the excretion of co-metabolites (6.4% of carbon conversion) and the EPS production (18.1 g(EPS) l(-1)), while improving the quality of the polysaccharide, characterized by the highest K of 126.2 Pa s(n) and the highest pseudoplasticity degree (flow behaviour index, n=0.2).     

肺炎克雷伯杆菌疫苗中试生产中细菌培养工艺的改进及优化

目的中试生产中对肺炎克雷伯杆菌培养工艺进行改进及优化。方法采用液体综合培养基代替半综合培养基在10L和100L中国丽生物反应器中对肺炎克雷伯杆菌进行培养,在10L中国丽生物反应器探讨不同的培养基配方、pH值、培养温度、搅拌转速、溶氧,工艺参数稳定后,扩大培养到100L中国丽生物反应器,并探讨培养过程中补加葡萄糖的浓度及补加方式等对细菌浓度及荚膜多糖含量的影响。结果肺炎克雷伯杆菌液体综合培养基可代替半综合培养基用于该菌的培养,培养过程中维持pH值7．2、温度37℃、通气60L／h、搅拌转速250r／min、培养到2h时开始以恒速补加30mL／L40％葡萄糖溶液、培养时间为5h,细菌长势最好,收获的荚膜多糖含量最高。结论肺炎克雷伯杆菌的培养工艺放大到100L中国丽生物反应器中,经过多次试验初步建立了稳定的肺炎克雷伯杆菌中试培养工艺。     

Effect of various acceptors on the rates of the cyclization and chain-shortening of amylose catalyzed by the cyclodextrin glycosyltransferase from Klebsiella pneumoniae M 5 al. Improvement of new photometric assay methods

The effect of various acceptors on the cyclization and chain-shortening reaction of amylose catalyzed by cyclodextrin glycosyltransferase [(1 leads to 4)-alpha-D-glucan: [(1 leads to 4)-alpha-D-glycopyranosyl]transferase (cycling) EC 2.4.1.19] from Klebsiella pneumoniae M 5 al was studied by use of photometric-assay methods. The requirements for the acceptor were the same for both cyclization and chain-shortening, indicating the close relationship between both reactions. Maltose proved to be the most effective (2.48- and 5-fold acceleration of the cyclization and chain-shortening, respectively, in the presence of 584 micrometers maltose). The dependence of the chain-shortening reaction on the conformational state of the amylose molecules is discussed.     

Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme

During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases except that produced by Bacillus amyloliquefaciens. Received 06 January 1997/ Accepted in revised form 20 March 1997     

Sequence analysis of cyclodextrin glycosyltransferase from the alkaliphilic Bacillus agaradhaerens

The gene encoding an alkaline active cyclodextrin glycosyltransferase (CGTase) from the alkaliphilic B. agaradhaerens LS-3C was cloned and sequenced. It encodes a mature polypeptide of 679 amino acids with a molecular mass of 76488 Da. The deduced amino acid sequence of the mature CGTase revealed 99 and 95% identity to the CGTase sequences from the other B. agaradhaerens strains, DSM 8721^T and 9948, respectively. The next closest identity was of 59% with B. clarkii enzyme. CGTases from B. agaradhaerens, B. clarkii, and B. firmus/lentus formed a phylogenetically separated cluster from the other CGTases of Bacillus spp. origin. A number of usually conserved residues in the CGTases were found to be replaced in the sequence of B. agaradhaerens enzyme. The sequence analysis indicated the enzyme to be close to the so-called `intermediary enzymes' in the -amylase family.     

Application of fractional factorial designs to screen active factors for antibody production by chinese hamster ovary cells

With ever increasing need for cost-effective large-scale production of monoclonal antibodies, it is essential to develop highly productive and commercially viable processes. Previous research showed that growth and production capacity of the culture media can be improved by micronutrient supplements, such as insulin, vitamins, and growth factors. Since these micronutrients may not act independently of one another, factorial designs can expose critical interactions between nutrients as opposed to a serial approach of changing one factor at a time. In this study, fractional factorial designs were applied to observe the effect of several micronutrients on antibody production and culture longevity in shake flasks. Response surface designs were used to investigate the factors in depth and confirm the results of the fractional factorial study. The results demonstrate that fractional factorial design is an effective tool for rapid development of antibody-producing Chinese hamster ovary (CHO) cells.     

Development of a fermentation process for production of an alginate G-lyase from Klebsiella pneumoniae

A high-density-cell fermentation process for production of an exracellular alginat lyase from Klebseilla pneumoniae on a defined medium has been developed. The process employs a strategy using two carbon sources. One low-molecular-mass, low-viscosity carbon source (sucrose) with high water solubililty is used as the main carbons source for growth, while the high-molecular-mass and viscoous alginate in low concentration is used as an inducer for enzyme synthesis. The repression of algiante lyase production by sucrose and the growth inhibition that we observed at increased levels of ammonia were circumvented by a computer-assisted fed-batch addition of the carbon sources (succrose and alginate) and by supplying nitrogen source as ammonia in the pH control. No enzyme production was observed when dissolved oxygen limited growth at an oxygen uptake rate of 40%–50% of the maximum uptake rate. An optimal composition of the feeding solution (12.5 g alginate and 587.5 g sucrose 1^–1) was found both for the maximum final concentration of enzyme (1330 U 1^–1) and for the maximum volumetric rate of enzyme production (67 U 1^–1 h^–1). The enzyme production dependes of the growth rate in the linear growth phase, giving a maximum enzyme concentration at the highest growth rate tested. The final enzyme concentration shows a fiveflod increase compare with previously reproted daata where alginate was used as a carbon source. In addition, the ratio of alginate lyase by a factor of apporximately 15. A doubling in extracellular specific activity of the enzyme was observed, a property of significant interest, especially for purification of the enzyme. On the othr hand, the final dry cell weight concentration of the bacteria also increased by a factor of 15–20 thus giving a relatively lower specific productivity of 0.4 U (g cell dry weight)^–1 h^–1.     

Structure of cyclodextrin glycosyltransferase refined at 2.0 A resolution.

The previously reported structural model of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans has been improved. For this purpose the known sequence was built into an electron density map established by multiple isomorphous replacement and subsequent solvent-flattening at 2.5 A resolution. The resulting model was refined at 2.0 A resolution using a simulated annealing refinement method. Based on 70,171 independent reflections in the range 7.0 to 2.0 A resolution, a final R-factor of 17.6% was obtained with a model obeying standard geometry within 0.013 A in bond lengths and 2.7 degrees in bond angles. The final model consists of all 684 amino acid residues, two calcium ions and 588 solvent molecules.     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.