Heterologous Expression Of Sulfolobus Solfataricus β-galactosidase F441Y In Pichia Pastoris And Its Application

β-galactosidase （EC3.2.1.23）, usually known as lactase, is a kind of multifunctionalenzyme widely exists in animals, plants and microorganism. It can not only hydrolyze lactose,but transfer the galactosyl group. When using lactose as the substrate, it can cut the β-1,4glycosidic bond, and connect the free galactosyl group with other glycosidic receptors byglycosidic bond like β-1,3, β-1,4or β-1,6to form non-digestible oligosaccharides. This kindof oligosaccharides were called galacto-oligosaccharides （GOS）. Compared with mesophilicenzymes, thermostable β-galactosidase has good thermostability and high enzyme activityunder higher temperature, these advantages make it become an issue in research now.As reported, β-galactosidase from extreme thermophilic archaea Sulfolobus solfataricusP2has high transgalactosylation activity and the reaction system can avoid bacterialcontamination. In the early stage of work, a mutant of this enzyme having highertransgalactosylation activity called F441Y （the mutation of Phe441to tyrosine） was achievedby molecular modification. And it was susccessfully expressed in E. coli BL21（DE3）. In thisstudy, the enzyme mutant was expressed in Pichia pastoris KM71. Fermentation processoptimization and enzymatic properties were investigated, the reaction conditions for GOSproduction were optimized. Some factors affect the yield of GOS during reaction wasanalysed, they are initial pH, temperature, lactose concentration, enzyme concentration andtime. The yield of GOS under optimized conditions was61.0%. These results lay thefoundation for its large-scale industrial application.（1） A gene of β-galactosidase called F441Y was achieved by PCR. The target gene waslinked with expression vector pPIC3.5K to form the recombinant plasmid pPIC3.5K-F441Y,the linearized plamid was mixed with competent cells and was successfully electrotransportedinto the cells. The recombinant strain P. pastoris KM71/pPIC3.5K-F441Y with oNPGhydrolysis activity was selected. After induction by methanol for120h, the enzyme activitytoward oNPG reached27U mL^-1.（2） Flask-shaking optimization of the recombinant strain P. pastorisKM71/pPIC3.5K-F441Y was completed. The optimized conditions were as follows: initialcell density （DCW） was11g L^-1, temperature was26°C,10g L^-1of methanol was added every24hours. Induction was maintained for about120h, total enzyme activity reached44.6U mL^-1.（3） Conditions for fed-batch cultivation of the recombinant strain in a3L fermentor wasoptimized. Initial cell density and maintained methanol concentration on β-galactosidaseproduction was53.9g L^-1and10g L^-1, after the induction was maintained for about84h, theactivity of β-galactosidase reached204.9U mL^-1, and it was18.8%higher than that of E.coli BL21（DE3）and was about3.0-fold higher than that of flask.（4） The enzymatic properties of the pure enzyme were investigated, the results showedthat the optimum temperature and optimum pH was85°C and5.0, and it has goodthermostability at75°C, pH5.0and its half-time was30h. Using lactose as the substrate, theKm, Vmaxand kcatvalue was26.3mmol L^-1,8.41mmol L^-1 s^-1and160.1min-1at30°C, pH6.0.（5） The reaction conditions for GOS production using whole cell enzyme and free enzyme were optimized. For whole cell enzyme, the good conditions were as follows: initialpH, lactose concentration, enzyme concentration and temperature was5.0,700g L^-1,5U mL^-1,75°C. After30h, the yield of GOS reached59.9%.For free enzyme, the good conditions were as follows: initial pH, lactose concentration,enzyme concentration and temperature was5.0,700g L^-1,9U mL^-1,75°C. After44h, theyield of GOS reached61.0%. This research lay the foundation for the application ofβ-galactosidase in GOS production.