March 4, 1970 h Status of Glucose Isomerase Project f I, Production of Cells ~ (On schedule for pilot run) i A, Total activity on hand as of 3/4/70 1s about 400 units - (Will have about f 800 U by 3/9/70, which will be enough for the pilot run. att B. The activity continues to be in the 700-800,range and fermentation can be scaled up. II, Release of Enzyme from Celle A. Engymatic Treatment 1. Lfsozyme is used at rate of 1/12,500 to loosen cell wall; other studies indicate that this level may be reduced (i.e. 1/20-25,000) - these results ust’ be repeated’ ~ (no problem in pilot run) 2, Cell concentration in lysis - presently using 5% cells ~ and checking up to 20% cells - (No problem for PR) 3. Optimum temperature and time for lysis still not known but a workable level is known - (60°C - 2 hours) - (No problem for PR) jrPs Complete Release of Enzyme and Viscosity Reduction 1. Sonification is used for laboratory studies - (No industrial equipment - hence problem area for PR) 2. Other Shear equipment ~ homogenizesy @mni Mixer and Cowles dissolveg - not successful for viscosity reduction 3. Enzymatic viscosity reduction - may be possible by adding back whole cells and incubating ~ (Possible for commercial production but may require too much time for pilot run) 4, Study precipitation of nucleic acids (viscous material?) without enzyme renoval. 5, Summary - This area is one of the main problems in preparing for the pilot run - can be done on lab scale but requires more information for pilot run. III, Binding of Enzyme to Inert Supports ] A. DEAE - Cellulose No difference in acetone fractions vs. OFX in binding characteristics 9590 E£80S2, If viscous material is not removed from CFX , the amount of binding and enzymatic activity is very poor. 3. The cellulose swells to 4-8 times ifdry weight depending on pre- treatment (Use 50 U/kg wet we cellulose) 4, Enzyme removed by salt solution and cellulose can be reused at least 2x. 5. Enzyme stable on wet DEAE for at least 10 days at refrigeration ' temperature and longer when frozen (presently investigating the effect of drying on stability) B) Other Supports 1, Resins - (Presently under investigation) 2, Glass Beads - (comments) C) No Major problems in this area for pilot run IV. Column Isomerization A) Initial Substrate - Deionized total sugar - problems with carbongrechecking flow rates and % conversion, B) Temperature - Optimum 60-65°C; at 70°C obtain a 8% increase in conversion but a 33% reduction in enzymatic activity Jf ©) pil control - Presently using a buffer system but should reduce of eliminate it for pilot run. pli control by addition of base, changes the iontcbtrength and could wash enzyme off the column - Experimenting with multiple columns with pH control between columns, D) Magnesiun Level - Any concentration over 0.05M - reduced substantially the enzymatic activity ~ rechecking minimum level for optinu conversion- yy ©) Flow Rates & Retention Time - These process variables present largest problems in scale-up for pilot run. Difficult to scale directly from very small to large columns - Possible use of small column at W/S, to recheck, Bh fick noe tom flows - (similar to batch process at W/S before pilot run in CR) dle Calculated flow rates 0,/5~ gals/nin/ft> elt Pla nuke. — Wines wt 2 tab, aS F) Number of Columns ~ Due to pH control, we may want to use a multiple yt=+ pal— column system (Presently under investigation) G) Isomerization will be followed by optical rotation (unit has been modified to measure up to 50% conversion) V. Syrup Cleanup A) Mixed Bed Resin - (IRC - 120 - strong acid cation & TRA-93 weak base anion) ~ when isomerized dextrose was used as substzate - the: thr&é put between regenerations was approximately 850 gals/ft? (calculated from small columns by PD). 6590 ceses