High - Level Expression Of Very Heat - Resistant β - Glucosidase And Its Application In The Production Of Gardenia Blue Pigment

The transformation of blue pigment from Gardenia jasminoides Ellis is based on the hydrolysis of Geniposide by β-glucosidase to generate Genipin, and then polymerize with Amino Acid. The function of β-glucosidase is necessary to the process.In our study, a β-glucosidase gene A was cloned which comes from thermotoga maritima MSB8 (ATCC43589), using a heat shock induced expression vector pHsh constructed by ourselves. Thermotoga maritima MSB8 is an anaerobic thermostable bacteria found near the volcanic vent. The recombinant plasmid pHsh-bglA-WT was transformed into E.coli BL21 CodonPlus(DE3)-RIL and DH10B.The specific activity of the recombinant protein in E.coli DH10B was 6.4U/mg while in E.coli BL21 CodonPlus(DE3)-RIL was 25.7U/mg. The expression was improved about 4 times without inclusion bodies. In order to further improve the expression level, we changed the partial vector sequence and gene sequence using reverse PCR so as to optimize the secondary structure of the mRNA. The decreased of the mRNA freedom energy can lessen the spacial obstacles when the ribosome combined with the SD sequence during the translation period. The reconstructed plasmid was called pHsh-bglA-M. The plasmid was transformed into E.coli BL21 CodonPlus(DE3)-RIL and DH10B, the same as pHsh-bglA-WT. The specific activity in E.coli BL21 Codon Plus(DE3)-RIL was 107.1U/mg while in E.coli DH10B was 88.5U/mg. Above all, the recombinant protein was highly expressed in the E.coli BL21 CodonPlus(DE3)-RIL and the specific activity was 107.1U/mg.Taking the recombinant E.coli BL21 CodonPlus(DE3)-RIL/pHsh-bglA-M as seed fermentation broth, the recombinant protein was fermented in the 25 L doubled-fermentation tank using TB medium. The method of the induction was based on our patent. The production was 17834U/L.The recombinant cells was suspended with PBS buffer and broken by French press. The supernatant was obtained after centrifugation called crude enzyme. It was heat treated by 70℃,25min and then went through the ion-exchange column DEAE-Sepharose Fast Flow obtained pure recombinant protein assayed by SDS-PAGE. The size was in accordance with the theoretical value and the specific activity, the purification fold, the yield was 1746.2U/mg,16.2 and 17.5%, respectively.Geniposide and Amino Acid was the raw material, then the β-glucosidase was added into the mixed, keeping the mixed in 80℃ for 3h to produce blue pigment. Different conditions can generate natural blue pigment or brownish-violet pigment.