Isolation Of Molds With High-activity Of Cellulase And The Purification Of Cellulase System

The cellulose resources in nature are extremely rich.In view of social development and human needs,the exploitation and utilization of natural resources can not only alleviate the problem of resource shortage,but also play a positive role in environment protection.Therefore,the development and utilization of cellulase has always been a research hotspot.Cellulase is the only effective catalytic enzyme for degrading cellulose.It plays an important role in juice processing,feed production(as addition of enzyme preparations in feed),weaving as well as denim processing and dyeing,papermaking(such as deinking and recycling of waste paper),besides it can also convert biomass into bioenergy(for example bioethanol).It has become one of the important methods for industrial production of cellulose by fermentation of microorganisms.The research of new technology of cellulase production is significant for the development and industrial application of cellulase,for example,screening the cellulase or enzyme-producing microorganisms with new characteristics.In this study,a fungus,Penicillium W-B23,with the ability to efficiently degrade cellulose was selected from the humus pulp.We used morphological and molecular biology methods to identify the Penicillium;The enzyme activity of the produced cellulase is improved by optimizing the culture medium and culture conditions the enzyme with higher purity component is obtained after separation and purification.This research provides a new strain for cellulase research,which has guiding significance for the technology of industrial enzyme production,and also has a positive impact on improving the purity of purified cellulase.1.In this experiment,we separated and screened the fungi with cellulose degradation ability from the pine root soil,dead leaf pile,and humus pulp,and finally selected a cellulase high-yield strain derived from humus pulp.Which numbered as WB23.It was identified as Penicillium by morphology and molecular biology methods.In the CMC-Na fermentation medium,the filter paper enzyme activity was 3.15 � 0.12 U/mL,and the CMC enzyme activity was 2.23 � 0.07 U/mL.2.In order to further improve the enzymatic activity in liquid fermentation of Penicillium W-B23,we optimized the nutrient factors in the fermentation medium,including carbon source,nitrogen source,and fermentation conditions,as well as inoculation amount of seed liquid in shake flask,and initial pH,temperature during fermentation,in liquid medium.Based on the determination of CMC-Na and yeast powder as the best carbon and nitrogen sources,the best fermentation conditions were obtained through Single Factor Test and Response Surface Test: It is most suitable for W-B23 to grow and product enzyme at an inoculation amount of 5%,with the initial pH value of 7.0,and at fermentation temperature of 28 ?.Under this optimal culture conditions,after 6 days of cultivation,the CMC enzyme activity of the strain increased from 2.23 � 0.07 U/mL to 3.79 � 0.11/mL,and the FPA enzyme activity increased from 3.15 � 0.12 U/mL to 4.76 � 0.06 U /mL.The enzyme activity was improved by 69% and 51% compared with the samples before optimization.3.In order to obtain higher purity cellulase components,this experiment separately used cation exchange chromatography to separate and purify cellulase from Penicillium strain W-B23 fermentation broth,such as salting out(ammonium sulfate precipitation),Sephadex G-50 gel filtration chromatography,DEAE-Sepharose FF anion exchange chromatography,and CM-Sepharose FF Separation and purification techniques.The cellulase consists of three components,including endoglucanase,exoglucanase and ?-glucosidase.It has been determined that specific activity of endoglucanase was increased from 1.15 U/mg to 23.41 U/mg,and the specific activity of exonuclease was increased from 1.21 U/mg to 8.07 U/mg.The specific activity of ?-glucosidase increased from 1.28 U/mg to 8.63 U/mg.In this study,a new strain of cellulase-producing mold,numbered Penicillium Penicillium W-B23 was selected from nature.Compared with the commercial strain 40108(Trichoderma koningii)purchased from the market,the strain obtained from the experiment exhibited 43.07% and 73.35% higher separately in a filter paper enzyme activity and a CMC enzyme activity under the same conditions.This study has laid a foundation for the in-depth study of cellulase,which has a good application prospect,and it has a guiding significance for the research of engineering bacteria based on the mold of Penicillium.