Screening And Cultural Condition Optimization Of Cold-adapted Lipase Producing Yeast And Its Enzymatic Properties

The cold-adapted lipase,which has become a good candidate for industrial low-temperature processes,plays an important role in food,leather,textile,detergent,biodiesel,cosmetics,medicine,wastewater treatment and other fields.In this study,a high-yielding strain was screened from 55 cold-adapted lipase-producing yeast strains preserved in our laboratory.The strain was identified,the fermentation conditions were optimized,and the properties of the cold-adapted lipase were studied after preliminary purification.This study not only provides important theoretical value for breeding cold-adapted lipase-producing strains,but also provides new strain resources for the development of cold-adapted lipase-producing yeasts,and provides important reference for the development,utilization and large-scale production of cold-adapted lipase resources in the industrial field.The results are as follows:1.Strain screening.Twelve strains with larger ratio of transparent circle to colony diameter were screened by glycerol tributyrate plate.Then a high-yield lipase-producing strain,NYNU 19160,was screened by shaking flask screening,and the enzyme activity reached 34.47 U/m L.2.Strain identification.Based on morphological and mhysiological characteristics,and phylogenetic analysis of ITS and 26S r DNA sequence,the strain NYNU 19160 was identified as Papiliotrema fonsecae.3.Optimization of fermentation conditions.The results of single factor screening for cold-adapted lipase production by strain NYNU 19160 showed that:the optimal carbon source was soluble starch,The optimal organic and inorganic nitrogen sources are yeast powder and ammonium tartrate,the optimal inorganic salt was calcium chloride,the optimal inducer was soybean oil,and the optimal surfactant was Tween20.On the basis of the above results,the optimal of fermentation medium composition was obtained by orthogonal experiment as follows:soluble starch 31 g/L,yeast powder 95 g/L,ammonium tartrate 8 g/L,calcium chloride 7.6 g/L,soybean oil2.5 g/L,and Tween 20 4 g/L.The optimum fermentation conditions as follows:temperature 15°C,p H 7.0 and fermentation time 7 d.Under the optimal fermentation conditions,the lipase activity of this strain could reach（81.44±0.8）U/ml,which was2.36 times higher than before optimization.4.Purification of lipase.The grading precipitation saturation was determined by the change curve of lipase activity in the supernatant of ammonium sulfate precipitation.Primary and secondary precipitation of ammonium sulfate with 30%saturation and 90%saturation respectively.After dialysis desalination and concentration,the purification multiple was 2.12,the specific activity of purified enzyme solution was 97.93 U/mg,and the active recovery rate reached 33.5%.5.Enzymatic property.The optimal reaction temperature of the lipase was 20℃,the best stability of enzyme activity at 20～30°C.The optimal reaction p H was 7.5,and the best stability of enzyme activity in p H 6.5～8.5.Cu²⁺significantly promoted the hydrolysis activity of the enzyme,while Na²⁺,Li⁺,K²⁺showed significant indigenous inhibition.Organic solvents,including acetonitrile,methanol and acetic acid,had a great promotion effect on the enzymatic activity,while benzene and n-hexane inhibited the enzymatic activity.The enzyme showed strong specificity for p-nitrophenol butyrate（pNPC4）substrate.