Richard D. Tambalo

Over-expression of the endoglucanase protein from the transformant Escherichia coli BL21 clones and confirmation of the endocellulase activity of the protein through hydrolysis of lignocellulolytic materials at high temperature and low pH conditions

· Glycerol and lyophilized stocks of the culture will always be maintained in the Microbial Culture Collection of BIOTECH under the classified and secured category.

· Revived stock culture tubes will be secured in a plastic container kept in a plastic box to prevent breakage, spillage and contamination.

· In case of flooding or typhoon the working culture tubes will be transferred to the plastic box in the refrigerator for cold storage.

· In case of flooding or typhoon, content of shake flasks and culture broths will be transferred to sealable containers and then placed in the refrigerator for cold storage.

· In case of brownouts, BIOTECH has an electric generator that can supply electricity to the laboratories.

· In case of extended electricity outages, content of ongoing shake flasks experiments and culture broths will be transferred to sealable containers and then placed in the refrigerator for cold storage or in the autoclave for decontamination.

· All activities shall be conducted at the Microbial Genetics Laboratory and Enzyme Laboratory of BIOTECH, UP Los Banos, College, Laguna

· The proponent shall adhere as closely as possible to the schedule of activities reflected in the submitted Gantt chart.

· Any modifications in the schedule of activities shall be made with the concurrence of the UPLB-IBC and the DOST-BC.

· A biosafety contingency plan shall be submitted before the conduct of the experiment

· The proponent shall be informed which activities would require the presence of the UPLB-IBC, DOST-BC and PEQS-BPI personnel

· The proponent shall ensure that only the DOST-BC authorized personnel are allowed inside the experimental facilities.

· The DOST-BC and PEQS-BPI personnel should be informed in advance of any visitations by unauthorized persons

· The DOST-BC and the PEQS-BPI personnel shall be informed immediately of any intrusion by unauthorized persons

· The proponent shall ensure that stray animals are excluded from the experimental facilities while tests are being conducted

· In case of undue destruction of experimental facilities, the proponent shall implement measures to prevent the inadvertent escape of any viable material within the facility

· The proponent and the supervising IBC shall be held accountable for the undue destruction of the experimental materials and the consequences that their inadvertent escape may cause to the surrounding environment

· All viable materials within the experiment area shall be accounted for

· The proponent shall strictly observe proper disposal procedures for all materials

· Movement of all materials will be done in compliance with all relevant biosafety and phytosanitary requirements of the Philippines

· Any additional requirement that the DOST-BC may impose, as necessary, for the duration of the experiment shall be complied with

· The proponent shall submit through the IBC a completion report within 90 days after completion of the experiment

· The IBC shall submit to the DOST-BC a report on all its monitoring activities, upon completion of this project, in the prescribed format

The endoglucanase gene was obtained by polymerase chain amplification reactions using genomic DNA from cellulose enriched mixed culture originally obtained from Mudspring of Mt. Makiling, Laguna, Philippines as template. The transformant Escherichia coli BL21 contains a pET21 plasmid with an endoglucanase gene identified to be from Sulfolobus solfataricus inserted within the MCS region.

The study will be undertaken to conduct initial studies on the over-expression of an endoglucanase gene contained within a recombinant plasmid into a functional protein. The endoglucanase gene will be expressed into the protein molecule for partial purification and/or concentration. Initial application trials for lignocellulose hydrolysis will be conducted to determine the efficacy of the expressed endoglucanase protein for the hydrolysis of complex bio-materials/agricultural by-products at high temperature and acidic conditions.

The specific objectives of the study are: 1) to express the endoglucanase gene into a functional protein, 2) to conduct enzyme activity assay on the endoglucanase protein at high temperature (70-90°C)-low pH (pH 1-4) conditions using carboxymethylcellulose as substrate, 3) conduct hydrolysis experiments using the expressed endoglucanase, high temperature (90, 100, >100°C) and low pH (1.0, 2.0, 3.0 and 4.0) conditions and different lignocellulosic materials as substrate, and 4) determine the hydrolysis products and efficiency of hydrolysis using the endoglucanase protein and different lignocellulosic materials at high temperature-low pH conditions.

The experiments will be conducted at the Microbial Genetics Laboratory and Enzyme Laboratory of BIOTECH, U.P. Los Banos, College, Laguna from August to October, 2013.