Introduction Introduction ABSTRACT Hydrolysis of cellulose by cellulase enzyme is a solid-liquid...
1
Introduction Introduction ABSTRACT ABSTRACT Hydrolysis of cellulose by cellulase enzyme is a solid-liquid heterogeneous reaction. As such the reaction is strongly affected by the physical resistances caused most notably by the crystalline stru cture. Recently we have invented a method to produce non-crystalline cellulose (NCC) from a commerci al α-cellulose or cotton by a relatively simple process. This material is drastically different from natural cellulose in that the crystalline structure is disrupted. We have used this material for the kinetic study of cellulose hydrolysis. Among the major findings is that a significant amount of oligomers was found to accumulate throughout the reaction. It appears that oligomers are inhibitory to cellulase enzyme, especially the endo-glucanase. We have also produced cello- oligosaccharides (beta 1-4 glucan) from alpha-cellulose and used as the substrate for cellulase. When treated separately, the oligomers can be easily hydrolyzed to glucose by sulfuric acid, but was not hydrolyzed significantly cellulase enzyme. Materials and Methods Materials and Methods • The crystallinity of cellulose is a major factor hindering the enzymatic reaction by cellulase. Under the influence of the crystallinity, it is difficult to obtain the intrinsic kinetic information. • We have produced non-crystalline cellulose (NCC) from alpha cellulose by treatment with concentrated sulfuric acid. • The main objective of this work is to investigate the kinetic behavior of enzymatic hydrolysis against NCC and seek additional information that may not be available from crystalline cellulose. Product distribution from the enzymatic hydrolysis of Avicel cellulose and NCC 1 FPU/g- glucan (6 hour) Glucos e Cellobios e oligomers 1 FPU/g- glucan (96 hours) Glucos e Cellobios e Gluco se Cellobios e Glucos e Cellobios e Avicel NCC Avicel oligomers NCC Profiles of Profiles of glucose, cellobiose, glucose, cellobiose, and and o o ligomers in hydrolysis of NCC ligomers in hydrolysis of NCC Summary Summary • NonCrystallineCellulose(NCC), and Cello- Oligosaccharides(COS) were prepared in our laboratory. • Cellulase enzyme : Spezyme CP, Genencor, .Lot No. 301- 00348-25 • The conditions for acid hydrolysis of cello- oligosaccharides were 121°C, 20 min., 4%H 2 SO 4 . • The enzymatic hydrolysis conditions: 1%(w/v) glucan concentration, pH 4.8, 50ºC and 150 rpm • Hydrolysis of NCC exhibits extremely high initial rate. The reaction then essentially ceases after 10 hours. • In the enzymatic hydrolysis of NCC, a significant amount of cellobiose and COS are formed as reaction intermediates. • Cellobiose gradually disappeared whereas COS remained constant throughout the enzymatic hydrolysis. • COS are easily hydrolyzed by sulfuric acid, but are not significantly hydrolyzed by cellulase. • Hydrolysis of COS by Spezyme CP was slower than that of Avicel. • COS appear to be inhibitory to endo-glucanase. • Acid hydrolysis of COS resulted in 93% glucose yield in 20 min • Enzymatic hydrolysis gave 17.7% of glucose yield Acid and Enzymatic Hydrolysis of Cello-oligosaccharides Acid Hydrolys is Enzymati c Hyd rolysis, 15FPU/g-glu c an Glucos e Cellobios e COS DP 3 DP 4 DP 5 DP 6 Glucos e Glucos e Cellobios e DP 3 DP 4 Glucos e Cellobios e Enzymatic Hydrolysis of COS and Avicel H ydrolysis ofC O S w ith differentenzym e loadings 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 0 20 40 60 80 100 Tim e in hours MolarYield CO S -1FPU/gm Glucan CO S -3FPU/gm Glucan CO S -15FPU/gm Glucan Avicel-1FPU/gm Glucan Avicel-3FPU/gm Glucan Avicel-15FPU/gm Glucan Cello-oligosaccharides are more difficult to hydrolyze than Avicel. Oligomers were not degraded throughout the reaction. Enzym e loading-1 FPU/gm Glucan 0% 10% 20% 30% 40% 50% 60% 70% 0 20 40 60 80 100 Tim e in hours MolarYield oligomers cellobiose glucose total Enzym e loading-3 FPU/gm Glucan 0% 10% 20% 30% 40% 50% 60% 70% 0 20 40 60 80 100 Tim e in hours MolarYield oligomers cellobiose glucose total
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Transcript of Introduction Introduction ABSTRACT Hydrolysis of cellulose by cellulase enzyme is a solid-liquid...
IntroductionIntroduction
ABSTRACTABSTRACTHydrolysis of cellulose by cellulase enzyme is a solid-liquid heterogeneous reaction. As such the reaction is strongly affected by the
physical resistances caused most notably by the crystalline structure. Recently we have invented a method to produce non-crystalline cellulose (NCC) from a commercial α-cellulose or cotton by a relatively simple process. This material is drastically different from natural cellulose in that the crystalline structure is disrupted. We have used this material for the kinetic study of cellulose hydrolysis. Among the major findings is that a significant amount of oligomers was found to accumulate throughout the reaction. It appears that oligomers are inhibitory to cellulase enzyme, especially the endo-glucanase. We have also produced cello-oligosaccharides (beta 1-4 glucan) from alpha-cellulose and used as the substrate for cellulase. When treated separately, the oligomers can be easily hydrolyzed to glucose by sulfuric acid, but was not hydrolyzed significantly cellulase enzyme.
Materials and MethodsMaterials and Methods
• The crystallinity of cellulose is a major factor hindering the enzymatic reaction by cellulase. Under the influence of the crystallinity, it is difficult to obtain the intrinsic kinetic information.
• We have produced non-crystalline cellulose (NCC) from alpha cellulose by treatment with concentrated sulfuric acid.
• The main objective of this work is to investigate the kinetic behavior of enzymatic hydrolysis against NCC and seek additional information that may not be available from crystalline cellulose.
Product distribution from the enzymatic hydrolysis of Avicel cellulose and NCC
1 FPU/g- glucan (6 hour)
Glucos
e
Cellobios
e
oligomers
1 FPU/g- glucan (96 hours)
Glucos
e
Cellobios
e
Glucos
e
Cellobios
e
Glucos
e Cellobios
e
Avicel
NCC
Avicel
oligomers
NCC
Profiles of Profiles of glucose, cellobiose, glucose, cellobiose, andand oligomers in hyoligomers in hydrolysis of NCCdrolysis of NCC
SummarySummary
• NonCrystallineCellulose(NCC), and Cello-Oligosaccharides(COS) were prepared in our laboratory.
• Cellulase enzyme : Spezyme CP, Genencor, .Lot No. 301-00348-25• The conditions for acid hydrolysis of cello-oligosaccharides were 121°C, 2
0 min., 4%H2SO4.• The enzymatic hydrolysis conditions: 1%(w/v) glucan concentration, pH
4.8, 50ºC and 150 rpm
• Hydrolysis of NCC exhibits extremely high initial rate. The reaction then essentially ceases after 10 hours.
• In the enzymatic hydrolysis of NCC, a significant amount of cellobiose and COS are formed as reaction intermediates.
• Cellobiose gradually disappeared whereas COS remained constant throughout the enzymatic hydrolysis.
• COS are easily hydrolyzed by sulfuric acid, but are not significantly hydrolyzed by cellulase.
• Hydrolysis of COS by Spezyme CP was slower than that of Avicel.• COS appear to be inhibitory to endo-glucanase.
• Acid hydrolysis of COS resulted in 93% glucose yield in 20 min• Enzymatic hydrolysis gave 17.7% of glucose yield
Acid and Enzymatic Hydrolysis of Cello-oligosaccharides
Acid Hyd
rolysis
Enzymatic Hydrolysis,
15FPU/g-glucan
Glucos
e
Cellobios
e
COS
DP
3DP
4DP5
DP
6
Glucos
e
Glucos
e
Cellobios
e
DP
3DP4
Glucos
e
Cellobios
e
Enzymatic Hydrolysis of COS and AvicelHydrolysis of COS with different enzyme loadings
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
0 20 40 60 80 100
Time in hours
Mol
ar Y
ield
COS-1FPU/gm GlucanCOS-3FPU/gm GlucanCOS-15FPU/gm GlucanAvicel-1FPU/gm GlucanAvicel-3FPU/gm GlucanAvicel-15FPU/gm Glucan
Cello-oligosaccharides are more difficult to hydrolyze than Avicel.
Oligomers were not degraded throughout the reaction.
Enzyme loading-1 FPU/gm Glucan
0%10%20%30%40%50%60%70%
0 20 40 60 80 100
Time in hours
Mol
ar Y
ield
oligomers
cellobiose
glucose
total
Enzyme loading-3 FPU/gm Glucan
0%10%20%30%40%50%60%70%
0 20 40 60 80 100
Time in hours
Mol
ar Y
ield
oligomers
cellobiose
glucose
total
