| Cold-adapted esterases have high catalytic activity at low temperature and potential application in food, detergent, environmental protection and other industrial fields. Medium and short carbon chain fatty acids with special milk aroma have been produced by esterase hydrolysis, which could be used in the development of dairy essence bases. Glacier environment is the openness system with less influence by human activity and contain the abundant resources of cold-adapted microorganism and enzyme. Resent years, Glacier continue to melt and retreat because of global warming. Thus, it is very urgent to develop its resources, which become one of the hot spots of extremophiles research currently. In this study, based on the resources of glacier No.1, Tianshan, Xinjiang, cold-adapted esterase producing bacteria were screened, which could hydrolyze single cream to produce medium and short carbon chain fatty acids. Esterase production were improved by modification technologies such as atmospheric room temperature plasma(ARTP) mutagenesis, genetic engineering, etc. The process and parameters of esterase hydrolysis were optimized Meanwhile, the volatile compounds of milk-based products, cold-adapted esterase hydrolysates, homemade milk flavor essence and commercially available milk essence were compared, analyzed and identified by modern food flavor analysis methods. These studies would provide important theoretical basis for the development of new type low temperature esterase. The main contents and results are as follows:(1) 377 strains were screened out from permafrost and the glacier melt water samples of glacier No.1, Tianshan. Through technical identification of rep-PCR, different colony morphology of 61 strains were selected and their morphological characteristics and cells’ shapes, etc. were studied. 10 strains were screened out from 61 strains by three selective mediums and larger transparent circles could be observed on tributyrin agar plate, and then, 5bacteria strains with higher production of esterase were selected based on their enzyme activity. The enzyme activity of strain T1-39 was highest(8.96 U/mL) by pNPB. The colonial morphology, physiological and biochemical character, molecular biology were analyzed and identified. Strain T1-39 was identified as Pseudomonas sp. and named Pseudomonas sp.T1-39, the GenBank accession number of 16 S rRNA gene sequence was KP780205.(2) Pseudomonas sp.T1-39 was mutated by ARTP mutagenesis, the three mutant strains(TB1, TB11 and D3) were obtained by tributyrin agar plate and determination method of pNPB. Pseudomonas sp.TB11 was found as good genetic stability, fast proliferation rate and its enzyme activity was 3.45 times higher than the original strain and strain TB11 was subjected to optimize the fermentation process for esterase prduction. The results were found single cream as inducers, typtone and ammonium sulfate as nitrogen source, addition of 2%PVA, intial pH 9.0, fermentation temperature 15 ℃, inoculation quantity of 2%, loadingvolume was 20 mL/250 mL, agitation speed of 200 rpm and the fermentation time of 32 h. In optimization conditions, the esterase was produced and named EstTB11 with enzyme activity of 58.92 U/mL, which was 1.48 times before optimize and 6.58 times of original strain esterase.(3) EstTB11 was purified by ammonium sulfate precipitation, Q Sepharose FF ion chromatography and Superdex G75 gel filtration chromatography. Then on this basis,molecular weight of esterase, peptied fingerprint and N-terminal amino acid sequences were analyzed, the molecular weight of esterase was 64.601 kDa by 5800MALDI-TOF/TOF, and ten amino acid sequences of N-terminal was obtained as GVYDYKNLTT. Enzyme characteristics of purified esterase were studied. EstTB11 had optimal pH of 8.5, temperature25℃, pH and thermal stability in the scope of 7.0~10.0 and 15℃ to 35℃(at around 90% at15℃~25℃ and about 80% at 35℃ for 720 min) respectively, however, EstTB11 activity at45℃ was remained above 65% for 60 min, thus it had good stability. The esterase showed higher activity towards p-NPB and p-NPC(C4~C5). Therefore, EstTB11 had good catalytic action to medium and short carbon chain esters, indicated that, the esterase has potential application value in the food industry and in increasing the aroma of dairy products.(4) Cloning and expression of the cold-adapted esterase gene, purification and properties of the recombinant protein were investigated. An ORF reading frame with a long for 1848 bp,encoding 615 aa, were obtained by genome walking and N-terminal sequences of EstTB11,then the secondary and tertiary structures of esterase were predicted. The recombinant vector was constructed and expressed successfully in E. coli Rosetta(DE3). The activity of expression protein was 10.67 U/mL, which was 1.19 times of the original strain esterase.However, compared with EstTB11 activity was lower, as most expression protein existed in a form of inclusion body. The recombinant protein was purified by Ni-affinity chromatography,and dialysis for protein renaturation. The purified enzyme named rest T, which also showed higher hydrolysis ability towards medium and short carbon chain esters, specific activity was195.03 U/mg by pNPB. Further research on the characteristics of recombinant esterase, the result showed that the optimum temperature and pH of rest T were 20℃ and 8.5 respectively,the presence of K+, Ba2+ and Na+ ions with a concentration of 5 mmol/L activated on rest T.(5) The volatile compounds of single cream, restT hydrolysates, Novozyme Palatase20000 L hydrolysates and two kinds of commercially available milk essences were analyzed by SPME-GC/MS and electronic nose detection & their aroma similarity was compared. The result showed that cold-adapted rest T performed well in hydrolyzing single cream at lower temperature and even at 4℃, which could keep aroma components of enzymolysis products to a great extent and their flavor were more close to the milk fragrance. Eight different samples were detected on the analysis of radar fingerprint, PCA and DFA by electronic nose technique,the results suggested that homemade natural milk fragrance was close to the flavor of singlecream and raw milk and significant differences from synthetic fragrances. Meanwhile, it is further showed that electronic nose technology will assist SPME-GC/MS in analysis and detection, and it is able to quickly assess the aroma quality of the sample. |
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