Hello Every one,
I am working on saccharification of biomass with crude enzyme. In this case I need to select certain concentration of enzyme (eg: 500 U/ ml), If suppose my enzyme source concentration is 2000 U/ ml . Can I simply dilute the crude source with sterile distilled water up to 500 U/ ml or something else? and what is the best concentration of crude enzyme for saccharification of biomass?
Thanking you all,
Dear Golla,
Yes you can dilute from 2000U/mL to 500U/mL by adding sterile water or any appropriate solvent.
Regarding the other part of question; attached please find two papers which fully cover the answer to your question. I have copied some important paragraphs for quick view.
2. Pretreatment of lignocelluloses
The samples were pretreated using conditions that previous research [4,5] proved to be optimal for this type of biomass: in NaOH 2% solution, and autoclaving at 2 bar for 30 minutes. The pretreated biomass was washed and neutralized. The amount of water used for washing was around 12 volumes of biomass volume. The reason for washing is to eliminate the inhibitors resulted after pretreatment (furfural, hydroxymethyl furfural, traces of acids, hydroxides, salts), and resulted glucose, due to its inhibiting effect for cellulases. All lignocellulosic materials which have undergone some aqueous pretreatment must never undergo any drying whatsoever prior to enzyme digestibility, since irreversible pore collapse
can occur in the micro-structure of the biomass leading to decreased enzymatic release of glucose from the cellulose. Additionally, all frozen lignocellulosic materials which are to be subjected to digestibility tests cannot have been frozen for more than one month prior to analysis, since, depending on the environment, sublimation could have occurred, leading to possible irreversible collapse of micropores in the biomass [6].
3. Evaluation of enzymatic activity of cellulases
The activity of several commercial cellulolytic enzymes and a crude extract of a local strain of Trichoderma viride have been evaluated. The enzymes are: Cellulase Onozuka, from Trichoderma viride (Merck 102321), Cellulase from A. niger (Fluka 22178), Zetalase and Hidrocell from a local provider, and the whole cellulolytic enzyme pool produced in laboratory by a culture of T. viride CMIT3.5 (other name: T. viride CMGB1, microorganism kindly donated by Dr. Săsărman Elena, from the University of Bucharest, Faculty of Biology). These enzymes were used individually or combined with cellobiase from Aspergillus niger – Novozyme 188 (Novo Industries) to hydrolyze cellulose Avicel. The load used was 15 U of cellulase/gram cellulose and 90 U of cellobiase/gram cellulose. The load of cellobiase was 6 units cellobiase/1 unit cellulase (after Lynd, 1996 [2]). Considering that the cellulases will be used in further experiments in simultaneous hydrolysis and fermentation
systems, besides cellulose, nutrients for yeast cultures were added to the hydrolysis probes: cellulose 1%; yeast extract 1%; peptone 2% [6]. The hydrolysis was carried out in sterile conditions at 50o C and pH 4,8 for 96 hours. Every 24 hours probes were harvested in aseptic conditions, centrifuged and stored in the freezer. After four days of hydrolysis the dry weight of the remained cellulose and the concentration of glucose in harvested probes were analyzed.
4. Enzymatic hydrolysis of Miscanthus
Using the results obtained in the previous part (evaluation of enzymatic activity of
cellulases), three enzymes were selected and used to hydrolyze Miscanthus. The reason of this study is to select one cellulase (from the cellulases tested previously to hydrolyze pure cellulose) to be applied to hydrolyze pretreated lignocellulosic biomass. We applied the same hydrolysis conditions described in the previous paragraph. The hydrolysis was carried out in sterile conditions, in a thermostatic water bath with shaker. The samples were harvested and stored as described previously.
5. Enzymatic hydrolysis of three types of lignocellulosic biomass
The enzyme giving the best yield of glucose in the previous study (enzymatic
hydrolysis of Miscanthus) is applied in this study to hydrolyze in the same conditions two more types of biomass from agriculture: wheat straw and corn stover. The reason of this study is to compare the sugar yields which can be obtained after pretreatment and enzymatic hydrolysis of Miscanthus (less studied) with yields obtained from wide-studied wheat straw and corn stover.
Hoping this will be helpful,
Rafik
