Beyond HPLC. Additional techniques for the analysis of your starch-derived products and transformation processes

Transcription

1 Beyond PLC. Additional techniques for the analysis of your starch-derived products and transformation processes 07 June 2011, Rafael F. Sala, Ph.D., Scientist II Applications Research Genencor (A Danisco Division), Palo Alto, USA

2 utline Introduction What other techniques besides PLC? NMR MALDI- MS Capillary Electrophoresis Follow up of saccharification End of Fermentation analysis igh Dextrose syrup analysis Conclusion Questions

3 ow to monitor starch processing enzymes?. Production of Glucose from starch: Alpha-Amylase and Glucosidase Glucoamylase: degrades starch by breaking the 1,4 and, to a lesser extend, 1,6 bonds from the non reducing ends of poly/oligosaccharides (i.e. exo-glucosidase)

4 Introduction Use of PLC is ubiquitous in the industry for analysis of starch derived products and follow up of processes. (SEC, IEX, ion exclusion, RP, NP, IC, ILIC, etc) ther analytical techniques are used, some of them very often, some rarely Calorimetry, Specific Gravity, Viscosity, %humidity, DE, Iodine, Brix, etc Gas Chromatography (GC), GC-Mass Spectroscopy (GC-MS) Near Infra Red Spectroscopy (NIR) Nuclear Magnetic Resonance (NMR) Matrix Assisted Laser Desorption Ionization MS (MALDI-MS) Capillary Electrophoresis (CE) Ultrasonic Spectroscopy Quartz Crystal Microbalance (QCM) ther

5 The eyes of the chemist : 1 NMR spectroscopy. too much physics for a chemist Based on behavior of nuclei with non-zero magnetic moment ( 1, 13 C, 15 N, 31 P) on a high magnetic field Information rich technique, signals characterized by their frequency (chemical shift), intensity, fine structure and magnetic relaxation properties. Most used 1 NMR, allow the mapping of each proton in a molecule based on its different magnetic microenvironment. Used in metabolomics and also amenable for in situ monitoring of transformations. Non destructive.

6 1 NMR analysis: initial soluble liquefact 1 NMR allows the direct observation of the components of the saccharification mixture. The anomeric region is particularly useful to evaluate the presence of fermentable sugars and reduced sugars. Can follow up progress (kinetics) of saccharification in situ, but can not follow up fermentations (C 2 evolution) Anomeric Region Non-reducing sugars Reducing sugars 1-r 1-r -1-4,nr -1-6,nr -1,r -1,r -1-4,nr

7 Use of 1-NMR in the analysis of starch processing Expected 1 NMR signals on Liquefact Reducing ends: Anomeric : 1 (5.2 ppm) and 1(4.6 ppm) 2 (3.26 ppm) ; 4 (3.41 ppm)? ther protons: undefined (3.5 to 4.0) ppm Non-reducing ends: Anomeric : 1-4 (5.40 ppm), no in branching (hump ppm) 1-6 (4.96 ppm) Reducing anomeric protons 1-r 1-r Non-reducing anomeric protons 1-4-nr 1-6-nr

8 1 NMR analysis Soluble Liquefact rganic Acid PLC analysis

9 End of Saccharification almost all glucose 1 1

10 1 NMR follow up of GA effect on soluble part of Liquefact 205 min 50 min 15 min Start

11 1 NMR follow up Pullulanase on soluble part of Liquefact 24.5 h 1 h 1-4nr 10 min 1r 1r 1-4nr Close to nr 2 min

12 ow sensitive the 1 NMR is? Standard igh resolution NMR can be sensitive enough to detect micrograms levels (in the NMR tube) A rough approximation of the detection limit of 1 NMR is 1-10 mg/ml for small molecules (e.g. for glucose aprox 5 mm or 0.1%). 1 NMR of an incomplete saccharification with 0.5% internal standard DSS.looks like pure glucose, however upon a closer look (expansion). 0.5%

13 same spectrum zoomed (20x) I can see that the saccharification is incomplete. DSS 1,4 nr unreacted?

14 Saccharification reaction follow up with 1 NMR An array experiment with quantification of each signal (integral value) allows the monitoring of the changes on liquefact after addition of glucoamylase or any other enzyme Array is made by changing the pre-acquisition delay to desired time intervals Monitor only the anomeric region gives plenty information

15 Saccharification reaction follow up with 1 NMR A detailed progress curve can be drawn using 1 NMR. Ratio of non-reducing / reducing sugars or extent of hydrolysis (reducing / total) can be monitored. Reducing sugars can be correlated to DE numbers. More information can be elicited as the evolution of particular protons that make up the reducing and non-reducing components can also be monitored. Amount of enzyme can be tuned up for transformation on a desired length of time

16 a more detailed view: monitoring the anomers 1 NMR allow observation of the hydrolytic cleavage for each anomer ( and ) The results show that the anomer is formed preferentially during the process and then equilibrate (mutarotate) to the standard mixture (36/64). This is consistent with an inverting mechanism known for glucoamylases.

17 ..and the branching number Changes on hydrolysis of 1,4 and 1,6 can be followed up Change on the degree of branching (defined as the ratio 1 nr total / 1,6 nr) can also be monitored. Comparison among enzymes or blends on the effect on branching points can be studied using this technique.

18 1 NMR studies on model substrate: Panose 26h follow up, only 3 anomeric protons 1,4nr, 1,6nr, 1r ( and Cp Bp Ap Panose k 1 k 2 Bm Am Maltose Glucose

19 Progress curve analysis Maltose by difference Simulation suggest k 2 =2.5 k Panose hyd. WT Glucose gen. WT Maltose rem. WT Time(min)

20 EF analysis By the time EF is collected (55h) there is negligible production of Ethanol from fermentable sugars. owever the DP4+ peak is still a major peak on the PLC trace EF was collected, filtered, extracted with organic solvent and lyophilized to facilitate its analysis Liophylized soluble part of EF was analyzed by PLC & NMR What is in the DP4+ peak? but also, what is on the DP2 / DP1 regions? DP2s DP1s No Ethanol Lyophilized EF residue PLC on PX-87 column of the EF residue

21 1 NMR of liophylized EF helps to identify metabolites... 1 NMR show unambiguously that all fermentable sugars have been depleted from the soluble part of liquefact. As observed on the PLC Glycerol and Lactic acid are shown in the 1 NMR analysis A signal in the -anomeric region of the 1 NMR suggest the presence of a nonfermentable sugar of unknown structure Start liquefact 4. 0 Glycerol Lactic acid? fermentable sugars Single anomeric signal?? Lactic acid?? Clear

22 Identifying Trehalose as the unknown sugar at EF The single anomeric nature of the sugar (no -anomer) hints at Trehalose as the undigestable disaccharide. 1 NMR confirms Trehalose as the unknown sugar residue at the EF Trehalose -1,1 Gluc EF

23 Identifying other metabolites. Glycerol Lactate Propylene glycol, 2,3 butanediol?? Trehalose Phytic acid or Phosphate Inositols? Trehalose Lactate Trehalose

24 Analysis of igh fermentable sugar syrup (igh Glucose syrup) igh Glucose syrup (96+%) needed for Sweeteners applications Problem with GS is formation of reversion products at high Glucose concentration: Isomaltose and Maltose. Structurally similar saccharides are difficult to separate and detect: They are polar but usually not charged (unless high p or has charged groups). Detection usually by Refractive index but poor sensitivity and not amenable to gradients. PLC separation of Maltose/Isomaltose not trivial, need a technique which could deliver a better resolution GC is an option but is there other options?.

25 PLC (rganic Acid column) of conventional process Isomaltose Maltose

26 Putting your PLC on steroids: Capillary electrophoresis CE = Gel Electrophoresis + PLC CE employs narrow-bore ( mm i.d.) capillaries to perform high efficiency separations of both large and small molecules. Separations are facilitated by the use of high voltages, which may generate electroosmotic and electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary. Analytes can be separated by mass, charge or isoelectric point Usually state of the art detection technology is used for registering the electropherogram Uses small amounts of reagents and sample. Resolution as good or better than capillary GC. Figures: research.org and arris, D.C., Quantitative Chemical Analysis, 7 th ed., W.. Freeman Co., NY, 2007.

27 FACE: Labeling reducing sugars for separation and detection

28 Detection with Laser-induce fluorescence allows high resolution Figure: Shea M.G., et al. Carbohyd. Res. (1998) 307, 1 12

29 CE analysis on high glucose syrup for conventional process 300 Standards Isomaltotriose Isomaltose Maltose Maltotriose Conventional process 50 % ratio: unknown: 2 isomaltose: 52 Isomaltose Maltose maltose: 46 Maltotriose Minutes

30 1 NMR confirms FACE analysis (anomeric region only) -Glucose + other -reducing ends -Glucose + other -reducing ends Maltose Maltotriose Isomaltose

31 Use of 1 NMR in Biomass analysis. Biomass treatments produces complex mixtures 1 NMR anomeric analysis can be used on analysis of hydrolysates. Ratios of different sugars on mixture is important for SSF processes.

32 1 NMR of pretreated Biomass -xylose 1 -reduced (Glucose) 1 - Glucose 1 mannose 2 -reduced (Glucose) 1 - Glucoronic acid 1 mannose -xylose + -galactose -L-arabinose DSS 1 galactose 1,6-? 1 -nonreduced region

33 Another tool. MALDI-MS Matrix Assisted Laser Desorption Ionization (MALDI) was one of the first MS techniques available for the analysis of proteins due to its gentle ionization. It is also useful for the analysis of polydisperse oligosacharides such as the ones found in starch processing An alternative to other high resolution chromatographic techniques such as FACE and PAEC-PAD. Time-of-flight (TF) Mass Analyzer

34 Follow up of SSF process: Start, 6h, 18h 18h 6h Start Lqfct

35 25,43, 55h (EF) 25h 43h 55h 55h

36 Conclusions PLC is the workhorse for analysis of saccharides in saccharification / fermentation processes but has limitations on the information it provides. ther analytical techniques such as NMR, CE and MALDI-MS can provide additional information to monitor a process or to detect, isolate and quantify a component. 1 NMR is particularly useful to monitor the branching point evolution during a saccharification reaction. MALDI and CE are useful to analysis of monomer distribution of polysaccharides. ther techniques are being developed that can assist in a better understanding of processes associated to the Grain / Biomass industry.

37 1 NMR sensitivity sample requirement