Preparation Of Antarctic Cold-Adapted Cellulase And Research On Kelp Cellulosic Ethanol

Cellulose is the most abundant biomass and most inexpensive renewableresources. Cellulases are inducible enzymes that can catalyze the hydrolysis ofcellulose, and can be widely used in the extraction of Chinese medicines, foodindustries, textile industries, feed processing, brewing industries, oil prospecting andmany other fields. Especially, cellulase plays an important role in the bio-energyindustry.Currently, cellulase adapting to different temperature is the further research trend.On the basis of this tendency cellulase produced by Antarctic bacteria at lowtemperature is being studied, and will be applied in the actual production industry.Industry waste residues from kelp processing plants become fuel alcohol byfermentation, which is kelp cellulosic ethanol. Using the waste fiber material toproduce cellulosic ethanol is not only a way of waste utilizatio n and pollutionabatement, but also provides a new, adequate and cheap supply of raw materials forthe preparation of cellulosic ethanol and means green concept of environmentalfriendly renewable energy.According to the literature, a cellulase-producing bacterium named NJ64wasisolated from82Antarctic bacteria strain and identified by16SrDNA sequenceanalysis. The phylogenetic tree was constructed by neighbor-joining, and wasconsistent with the results of16SrDNA sequence homology. The optimum fermentconditions of strains NJ64fermentation was to at15℃for72h with initial pH7.0.Cold-adapted cellulase was obtained preliminary purification by using the salt fractionation with ammonium sulphate at50%and90%saturation; the activityrecovery of the lyophilized cellulase following desalination was84%and thispurification processing concentrated the activity of the Antarctic cold-adaptedcellulase about4.6times with expected results. By SDS-PAGE analysis the molecularweight of this isolated cold-adapted cellulase was consistent with literature values; theoptimum pH value and temperature of this cold-active cellulase were9.0and40℃, which wastypical cold-active cellulase. The method of crosslinking-embedding by sodiumalginate was adopted to immobilize the cold-adapted cellulase, and studied the effectsof conditions on the activity recoveries. The orthogonal test was used to investigatethe optimal immobilization conditions: the best activity recovery rate was51.98%obtained when the sodium alginate concentration was3.0%, the calcium chlorideconcentration was0.5%, the glutaraldehyde volume fraction was0.3%and theimmobilization time was5hours. The rate of immobilized cellulose was （51.98±1.7）%after3verification experiments with desirable reproducibility; The optimumreaction temperature of immobilized enzyme was50℃, increasing10℃more thanfree cellulase; the optimum pH is still9.0and had expanded its range compared to thefree cold-adapted cellulase; after being recycled seven times, the remaining enzymeactivity of immobilized cellulase remains58.37%.Kelp cellulose can be hydrolyzed by cold-adapted cellulase for ethanolfermentation. In this experiment, the immobilized cold-active cellulase hydrolyzed theindustry waste residues from kelp processing plants for ethanol production and thefactors on the ethanol yield were studied: the ethanol yield reached37.37%when theconcentration of glucose in the substrate was70g/L, the volum of liquid was50%andfermented for48h.This study was mainly to make full use of Antarctic cold-adapted cellulase andprovided a new source of raw materials for the production of bio-ethanol fuel.