| κ-Carrageenan is liner sulfated galactans extracted from the extracellular matrixof red marine algae. Sulfated oligosaccharides and their derivates prepared fromκ-carrageenan have diverse biological and physiological activities, includingantitumor, anti-HIV, immunoregulation, and viral inactivation activities. Thus,κ-carrageenan oligosaccharides show potential use in functional foods andpharmaceutical areas. Undoubtedly, degrading carrageen with a specific enzymecalled carrageenase, which features high substrate specificity and mild reactionconditions, is thus a promising alternative to chemical degradation. However, theproductivity of κ-carrageenase from natural microbial cells can hardly meet thepractical requirement. Consequently, boosting the productivity of κ-carrageenase is aprecondition for achieving its application value. The advent of recombinant DNAtechnology has allowed the enhancement of κ-carrageenase production throughheterologous expression in E. coli. In this paper, the κ-carrageenase gene was clonedand heterogeneously expressed in E. coli, in order to boost the production ofκ-carrageenase and promote the application of κ-carrageenase. The results were asfollwes:(1)Accroding to the16s rDNA identification of wild-strain, the κ-carrageenaseproduction strain was assigned to the genus Zobellia and named Zobellia sp. ZM-2.Three pairs of primers were designed by Primer-Blast and used to amplify the nucleicacid fragment containing κ-carrageenase gene. Results showed that primer pairFP2/RP2could be used to amplify the target fragment under optimal annealingtemperature (58°C). The gene encoding κ-carrageenase (named cgkZ) contained asingle ORF1638bp long and displayed87%similarity with existing κ-carrageenaseencoding genes (GenBank accession nos. FP476052and AF007559). The deducedamino acid sequence of κ-carrageenase was composed of545aa, with a signal peptide of29aa. The deduced protein has the characteristic motifE(I/L/V)D(I/V/A/F)(V/I/L/M/F)(0,1)E，which features two glutamic acid residuesseparated either by three amino acids or by four residues. Therefore, theκ-carrageenase encoded by gene cgkZ also belongs to family16of the glycosidehydrolases (GH16).(2) Based on the ORF of κ-carrageenase gene, two pairs of primers with restrictionsites were designed. The κ-carrageenase gene with a signal peptide sequence (cgkZ)was amplified with the primer pair KC-PF3/KC-PR(2/3);the primer pairKC-PF2/KC-PR(2/3)was used to amplify the κ-carrageenase gene without a signalpeptide sequence (cgkZ). The target genes, cgkZ and cgkZ, were ligated into theexpression plasmid pProEX-HTa and then transferred to BL21(DE3) competent cells.Both the recombinant strain BL21-HTa-cgkZ and BL21-HTa-cgkZ showed apparentand almost-equivalent intracellular enzyme activities. However, compared withBL21-HTa-cgkZ, BL21-HTa-cgkZ displayed significant extracellular enzyme activity.The culture conditions of BL21-HTa-cgkZ were further optimized to enhance theenzyme production. The optimal process conditions for recombinant κ-carrageenaseproduction were: induction temperature23°C, inducer concentration0.9mM,pre-incubation2.5h, inoculation volume1%, initial medium pH5.5. Moreover, theextracellular enzyme was further promoted by adding0.5%lactose or0.1%TritonX-100or0.5%Gly into the medium at proper times. When cultured in LBmedium, BL21-HTa-cgkZ gave a maximum extracellular enzyme activity of9.37U/mL, and periplasmic enzyme activity of6.06U/mL. It proved that around51%κ-carrageenase was secreted into the culture medium, and33%of the total enzymeaccumulated in periplasmic space. The extracellular enzyme activity ofBL21-HTa-cgkZ was much higher than that of the natural strain Zobellia sp. ZM-2(0.33U/mL).(3) According to the SDS-PAGE analysis, the molecular weight of recombinantenzyme was45kDa, which was lower than the deduced molecular weight61.9kDa.This experimental phenomenon indicates the posttranslational removal of a largeportion of the C-terminal end of the protein, a process that is likely required for crossing the outer membrane. The existence of His-tag was beneficial to thepurification of the recombinant enzyme, a single band was obtained by loading into aNi sepharose6FF column.The optimal temperature of the recombinant κ-carrageenasewas39°C.This result is nearly consistent with that of the natural enzyme fromZobellia sp. ZM-2Thermal stability analysis of enzyme showed that it was stableafter incubation at35°C for3h with retention of its maximum activity of95%.Whenincubated at40°C for60min, approximately50%of the enzyme activity remained.The Maximum enzyme activity was observed at pH6.0in sodium phosphate buffer.The enzyme was stable from pH6.0–8.0; over85%of the total activity remainedafter incubation at pH6.0–8.0and20°C for120min. Partial activation (12.3–18.3%enhancement) was observed in the presence of TritonX-100,5mM Na+, and150mMNa+. More apparent stimulation (32.9–167.3%) was induced by100mM Na+,Tween-80, and DTT. In contrast, enzyme activity was strongly inhibited by Cu2+, Pb2+,EDTA, and SDS. According to ESI-MS spectra, carrageenan oligosaccharides,including tetrasaccharides[A-G4s]2, hexasaccharides[A-G4s]3, andoctasaccharides[A-G4s]4were released after hydrolysis by the recombinant enzyme.The IR spectrum of collected oligosaccharides displied the characteristic absorptionpeaks (848cm-1,929cm-1) of the basic units of κ-carrageenan, this further proved thatthe prepared oligosaccharides were kappa-type.(4) This experiment aimed to develop a method for preparing κ-carrageenanoligosaccharides by degrading Eucheuma cottonii with using cellulase andκ-carrageenase. The optimum hydrolysis conditions were as follows:3-4%（w/v）Eucheuma cottonii, addition volume of cellulase21000U/g, duration2.5h; additionvolume of κ-carrageenase5.5U/g, pH6.5, temperature35℃, duration6h.Underoptimized conditions, the yield of κ-carrageenan-derived oligosaccharides was higherthan50%. This technology peomotes the application of carrageenan oligosaccharidesin food, pharmaceutical and health care products. |
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