Cloning And Expression Analysis Of α-L-rhamnosidase Gene From Aspergillus Niger JMU-TS528

As widely used in biotechnology for the production of food ingredients, pharmaceuticalsand industrial enzymes, Aspergillus niger is the safety strain permitted by FDA.α-L-rhamnosidase （EC3.2.1.40）, an important industrial enzyme synthesized by A. niger, is quitevaluable in the commercial industry. However, the current production strain exists a largenumber of disadvantages, such as low enzyme production, high cost of fermentation, longfermentation period. The synthesis of α-L-rhamnosidase needs carbon inducer and is regulatedby carbon catabolite repression. Therefore, the productivity of α-L-rhamnosidase produced by A.niger is insufficient to meet the requirements of mass production. The purpose of this paper is toclone the gene of α-L-rhamnosidase from A. niger using PCR and RACE technique, explore theexpression level of glucose repression gene CreA and α-L-rhamnosidase gene L-rha when usingdifferent carbon. The L-rha cDNA was subcloned into vector pPIC9K, then transformed intoPichia pastoris GS115, and obtained the recombinant strain which can produceα-L-rhamnosidase without carbon inducer. The following procedures were done:1. The cDNA of α-L-rhamnosidase gene （access number: KC750908.1） was cloned from A.niger JMU-TS528. It is named L-rha. The obtained cDNA was2062bp and the longest openreading frame of this gene was1968bp which could encode655amino acid residues. Moreover,analysis of signal peptide cleavage site confirms the1-17amino acids are signal peptide. Whencomparing L-rha with α-L-rhamnosidase gene from other aspergilli, higher homology wasobserved with A. kawachii （AB374267.1） and A. niger513.88（XM₀01389049.1）, reached93%and91%respectively. Homology modeling analysis found that the α-L-rhamnosidase from A.niger JMU-TS528has the similar tertiary structure with other α-L-rhamnosidase from GH78,and it has a （α/α）6-barrel domain which is a typical structure of α-L-rhamnosidase from GH78.2. α-L-rhamnosidase can be induced when naringin, hesperidin, rutin and rhamnose wereused as the sole carbon source; while no activity of α-L-rhamnosidase was detected whenglucose was used as the sole carbon source. Furthermore, SYBR Green Real-time PCR assaysystem has been established in this part to examine the relative expression of the L-rha and CreA.The results show that a lower enzymatic activity was observed by adding of glucose, and theexpression of L-rha was decreased also. In the same time, it was found that the expression levelof CreA was increased greatly. 3. The L-rha gene was inserted into the expression vector pET32a and pPIC9K to constructthe recombinant plasmids pET-rha and pPIC9K-rha. The recombinant plasmid pET-rha wastransformed into Escherichia coli BL21（DE3）. The pPIC9K-rha plasmid was subcloned into thevector pPIC9K and then transformed into P. pastoris GS115cells by electroporation. Both ofprokaryotic and eukaryotic expression system can induce the recombinant protein. But noactivity of the recombinant α-L-rhamnosidase produced by E. coli BL21（DE3） was detected.However, when the engineering strain P. pastoris GS115induced after7d, recombiantα-L-rhamnosidase activity reached711.9U/mL.4. The optimum pH and temperature of recombiant α-L-rhamnosidase is6.0and60°Crespectively. Recombinant α-L-rhamnosidase enzyme showed derhamnosylation activity fornaringin, hesperidin, rutin, p-nitrophenyl-α-L-rhamnoside and myricetrin. When Mg2+, Zn2+,Ca2+, Cu2+, Co2+, Ni2+and K+were added, the effect on the enzymatic activity was positive at1、10and100mmol/L. Recombinant α-L-rhamnosidase was inhibited by Fe3+（10and100mmol/L）,Mn2+（1mmol/L）, Na+（1and10mmol/L） and β-mercaptoethanol （1%、0.1%）. With Fe2+, noα-L-rhamnosidase activity was measured at a concentration of100mmol/L. Fe3+at1mmol/Lhad slightly positive influence on α-L-rhamnosidase activity, but the recombinant enzymeactivity reduced quickly when the concentration of Fe3+was increased. Glucose, L-rhamnose,EDTA and DTT showed negative effects at concentrations of1and10mmol/L. For thehydrolysis of naringin at pH5.0and50°C, the recombinant α-L-rhamnosidase showed michaelisconstent （Km） of0.04mmol/L, maximum velocity （Vmax） of811.93U/mg.