Effect On β-D-GALACTOHYDROLASE Gene Of Kluyveromyces Fragilis Caused By UAS

Lactose is also known asβ-D-galactohydrolase . Product food-grade lactose enzyme strains are mainly Pichia, Kluyveromyces Fragilis, etc. Kluyveromyces Fragilis is from the natural fresh milk,and its lactose enzyme is a kind of neutral lactose enzymes,its optimal pH is 6.6-6.8 which is close to milk ,and at the same time ,Kluyveromyces Fragilis has a higher production and enzyme activity of lactose.In addition, the lactose enzyme from Kluyveromyces Fragilis still has good enzyme activity in low temperature,and the low temperature environment will avoid the contamination from process of miscellaneous bacteria which is apt to cause the milk contaminated.But the traditional industrial methods of enzyme production such as has a low volume, not easy to separate purpose protein, not accord with food-grade disadvantages express requirement have limited the fast development of lactose. Using genetic engineering means to improve the expression of lactose will promote the development of lactose .In this paper,we study on lactose gene of Kluyveromyces Fragilis, and from the transformation of promoters, design and construct the new Kluyveromyces Fragilis expression vector for lactose, and it is to efficiently express discusses the safety in conditions and methods, the content and the results are as follows:1. Construction of Kluyveromyces Fragilis resistant vector K.f.-kanAccording to the Genbank published lactose gene sequence of Kluyveromyces Fragilis and the sequence of plasmid pPic9k,we design double enzyme cutting sites to construct the plasmid pPkan. The plasmid contains the resistance gene Kan, and based on the gene of Kan, each side of the resistance gene has a homologous arm around 1kb to turn to Kluyveromyces Fragilis . Using the linearization of plasmids pPkan transformate into Kluyveromyces Fragilis to construct Kluyveromyces Fragilis-kan which with resistance of kan ,and meanwhile, we make it instead of Lac promoter integration in its position.Using the homologous recombination to knockout promoter region and part of lactose enzyme gene, to make the lactose gene inactivation.It is meant that the resistance gene kan is adding a mark. Kluyveromyces Fragilis-kan can be used as constructing promoter, try different transformation among carrier signal peptide.2. Construction of contains a repeat of upstream activation sequence vector K.f.-UASRAccording to the Genbank published lactose gene sequence of Kluyveromyces Fragilis , design a pair of primers, using the method of SOE-PCR, constructing the plasmid pTUASR of which contains a repeat of the upstream activation sequence, the plasmid contains the same homologous arm with pPkan.With Kluyveromyces Fragilis for receptor bacteria, using the method of electric shocks reforming process we transformate the linearization plasmid of pTUASR into Kluyveromyces Fragilis-kan, and get Kluyveromyces Fragilis-UASR. The Kluyveromyces Fragilis -UASR has a repeat of UAS instead of the original promoters, and restore the lactose enzyme gene knock put away, restored lactose . 3.Screening of transformation, and detection of enzyme activityUsing PCR to determate the positive K.f.-UASR ,and using the method of ONPG to measure the activity of lactose in fermentation. The results show that the activity of lactose of K.f.-UASR is 22.823 U/ml instead of 15.646 U/ml of Kluyveromyces fragilis.