Evaluation Of Hereditary Stability Of Engineered Pichia Pastoris And Isolation,purification And Application Of The Recombinant Thermomyces Lanuginosus Lipase From Fermentation

Lipases are a class of highly efficient biocatalysts,and play different roles in food processing.And the development of lipases by genetic and fermentation engineering has been one of the research hotspots in recent years.However,the expression of lipases is affected by many factors,and the severe loss of enzyme activity during separation and purification is common.Therefore,it is of great significance to study the production performance and hereditary stability of engineered strain,optimize the separation and purification process of lipase,and explore its application characteristics.An engineered Pichia patoris GS115-PT which could highly produce recombinant Thermomyces lanuginosus lipase(named PTL)was derived in our previous study.This study firstly evaluated the hereditary stability of GS115-PT and then separated and purified the PTL using the aqueous two-phase system,and finally explored the application characteristics of lipase in baking system.The main findings are as follows:(1)The hereditary stability of GS115-PT and its production performance were studied.GS115-PT were subcultured for 15 generations,and the 1^th 5^th,10^th,and 15^th generations were taken as the test samples.The results showed that the colony and cell morphology,the molecular weight of the target protein and the sequence of the target gene were consistent.The copy number was reduced from 64.99 to 17.80 and finally stabilized at around 7.00.At the same time,the enzyme activity of lipase increased by around 0.55 times,indicating that copy number of 7.00 is favorable for production,and GS115-PT has good hereditary stability and can be used for industrial production.(2)The conditions of aqueous two-phase system were optimized,and the enzymatic properties of lipases before and after separated and purified were determined and compared.The molecular weight and concentration of PEG,type and concentration of salt,pH and concentration of NaCl was confirmed based on purification fold and enzyme recovery.The optimal conditions were obtained:the concentration of PEG 6000 was 20%(w/w),concentration of K₂HPO₄was 10%(w/w),pH 7.0 and without NaCl.Under these conditions,the purification fold was 4.26,and the enzyme activity recovery was 94.6%.And the lipase after purified had higher thermal stability,the remaining activity of which was respectively103.4%and 99.4%at 60? and 70? after 5 h of treatment.Meanwhile,the purified lipase had higher pH tolerance in the range of pH 6.0 to 12.0.(3)To explore the application characteristics of PTL in bread making,optimal dosage of it based on the specific volume,crumb texture and lightness was determined.The results showed that the optimal dosage was 10?20 mg/kg.Furtherly baking quality of bread respectively with addition of PTL,that of commercial lipase FBG and chemical modifier DATEM was compared,and the gluten network and aroma were respectively analyzed by SEM and HS-SPME-GC-MS.It was found that PTL significantly reduced the hardness of bread,and also improved the cohesion and resilience to some extent.FBG performed better than PTL and DATEM on improving the specific volume,gluten strength and overall sensory acceptance of the bread.FBG and PTL could obviously increase the variety of flavor components,DATEM caused a large amount of acetic acid covering the aroma of bread,and the bread with PTL achieved the maximum score in smell and crumb texture.In conclusion,appropriate addition of PTL can effectively improve the baking quality of bread and has a unique advantages.