| Galactooligosaccharides?GOS?are a kind of oligosaccharides which were first found in milk as a natural probiotic oligosaccharide.They have high stability and good beneficial effect,and are widely used in food and health products industry.In industry,GOS are usually obtained from lactose catalysised by?-galactosidase.At present,Japan has relatively mature industrial production technology,and its annual output ranks first in the world.However,due to the lack of?-galactosidase with better performance and backward production technology,the production of GOS in China is still at a low level.In this study,?-galactosidase from A.oryzae RIB40 was linked to the expression vector pPIC9K,and the recombinant plasmid was transferred into Pichia pastoris KM71 to obtain recombinant strain which is capable of expressing?-galactosidase.Three mutants with enhanced transglycoside ability were obtained by site-directed mutagenesis.The reaction conditions of GOS production by mutant and wild-type enzyme were optimized.The mutant N140C/W806 with the highest transglycoside ability was fermented in 3 L fermentor.Finally,the reasons for the mutant's effect were analyzed and verified.The main results are as follows:?1?The gene of?-galactosidase from A.oryzae RIB40 was linked to the expression vector pPIC9K,and the recombinant plasmid was transferred into Pichia pastoris KM71 to obtain recombinant strain which is capable of expressing?-galactosidase.The activity of enzyme towards o-nitrophenyl-beta-glucoside?oNPG?reached 198.7 U·mL-1.Site-directed mutagenesis of the wild-type gene was carried out,and several mutants with enhanced transglycosidase ability were obtained.The kinetic parameters of the mutants were analyzed.Among them,the Km value of the mutants N140C and N140C/W806F increased greatly,which indicated that the affinity of the two mutants to lactose decreased and the hydrolysis activity decreased,which met the expectation of the experiment.?2?The conditions of GOS production by mutant and wild type were optimized.The productivity of GOS of W806F mutant was 49.3%under the optimum conditions,50.7%under the optimum conditions of N140C mutant,35.2%and 31.5%higher than that of wild type enzyme,respectively.The conditions of GOS production by double mutants were also optimized.The optimal reaction conditions of double mutant N140C/W806F were as follows:lactose concentration 600 g·L-1,enzyme dosage 2.5 U·mL-1,reaction temperature 40?,reaction pH 4.5 and reaction time 7 h.The final yield was 59.8%,which was 59.2%higher than that of wild type.The transglycoside ability of?-galactosidas was successfully improved.The double mutant N140C/W806F was fermented in 3 L fermentor.Although the enzyme activity reached 141.6 U.mL-1 at the induction temperature of 20?,it was difficult to achieve in industry.Therefore,the optimum induction temperature was 28?.At this time,the enzyme activity reached 65.3 U.mL-1,which was 4.44 times higher than shaking flask.?3?The reason for the mutant's effect was analyzed.It was considered that the reason for the effect of N140C mutation was related to the destruction of hydrogen bond between the amino acid at this position and galactosyl.Although the destruction of hydrogen bond had an effect on both hydrolysis and transglycoside reaction,it had a greater impact on hydrolysis reaction,thus transglycoside reaction was relatively dominant and the final yield was increased.The reason for the effect of W806F mutation is related to the increase of hydrophobicity at this site.Increased hydrophobicity reduces the opportunity for water to participate in the reaction as a receptor,and the corresponding glycosyl reactions as a receptor are enhanced,thus enhancing the transglycoside ability.The positive mutation N140A and the reverse mutation W806H were designed.After the mutation of N140A,the yield of GOS was 44.9%,19.7%higher than that of wild type,and the yield of W806H was 22.7%lower than that of wild type which successfully verified our analysis. |
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