Fermentative Production Of Alkaline Pectinase By Bacillus Sp. WSHB04-02

Pectinases are the pectin depolymerizing enzymes that cleaveα-1,4-galacturonosidic linkages of pectic substances which exist in higher plants cellwalls. Alkaline pectinases are those which can cleave α-1,4-galacturonosidiclinkages of pectic substances at alkaline conditions. In this study, the alkalinepectinse is the polygalacturonate lyase (PGL) which cleave α-1,4-galacturonosidiclinkages of polygalacturonic acid (PGA) by a trans-elimination mechanism andrequires Ca2+ ions for its activity. Nowadays the acidophilic pectinases haveextensive applications in the extraction and clarification of fruit juices and wine.Besides alkaline pectinases have been used in the purification of plant virus andbleaching of paper pulp. They are being widely used for processing and degummingof plant fibers such as ramie, buel, flax, jute, as well as depolymerizing anddebarking, and are becoming the vital bioenzymes during the process of cottonpretreatment. The application of alkaline pectinases will change the traditionalprocess of cotton pretreatment. Thus it not only decreases the environment pollutionbut also reduces the consumption of industry water.In this paper, a bacterial strain producing high-yield alkaline PGL was obtainedfrom the cutis of a rotten apple. The phylogenetic analysis based on 16S rDNAshowed that the bacterial strain was close to Bacillus subtilis with 99% sequenceidentity. The characteristic and the enzyme activity assay conditions of the PGL werestudied. The nutritional and environmental conditions were investigated and theeffects of volume oxygen transfer coefficient (KLa) on PGL in 7 L jar fermentor wereanalyzed. The main results were given as follows:1. A bacterial strain with high-yield alkaline PGL was obtained from the cutis of arotten apple. This bacterium was primarily identified as a strain belonging toBacillus, named as WSHB04-02. It was a Gram positive rod and soporiferousbacterium with round and white colony. The phylogenetic analysis based on 16SrDNA showed that strain WSHB04-02 was close to Bacillus subtilis with 99%sequence identity. The phylogenetic analysis of strain WSHB04-02 wasperformed by DNAMANV₄.0 and the homology tree of strain WSHB04-02 andthe neighbouring micrograms producing PGL was given.2. As for PGL produced by Bacillus sp. WSHB04-02, the suitable temperatureranged from 55℃⁶6℃, and the suitable pH ranged from 9.4⁹.8. It is showedthat the suitable temperature and pH of PGL were according with therequirement of cotton pretreatment, in which suitable temperature ranged from50℃⁶0℃ and suitable pH ranged from 9¹0. The highest PGL activity couldbe achieved at 60℃, and the optimal enzyme reaction temperature was 45℃.The optimum pH was 9.4.3. The assay conditions for PGL activity were determined as follows. The reactionmixture contained 2 mL 0.2% PGA in 0.2 mol/L Glycine-NaOH buffer at pH 9.4and 20 μL of enzyme solution with 0.44 mmol/L of CaCl2. The mixture wasincubated at 45℃ for 15 minutes. The reaction was stopped by adding 3 ml of0.03 mol/L H3PO4. One unit of enzyme activity was defined as the amount ofenzyme liberating 1 μ mol of unsaturated galacturonate per minute. Themolecular extinction coefficient of the unsaturated galacturonate at 235 nm was4600 L·mol-1cm-1.4. The suitable fermentation medium for PGL production by Bacillus sp.WSHB04-02 contained: 8 g/L cornstarch, 8 g/L peptone, 10 g/L yeast extract, 9.2g/L K2HPO4 and 3 g/L KH2PO4. NH4+ was a good nitrogen source for cellgrowth and PGL production by Bacillus sp. WSHB04-02. Production cost ofPGL could be reduced by using a combined nitrogen sources including peptone,yeast extract and NH4+ during its large scale production. It was also found thatthe addition of Mg2+ did not favor the production of PGL.5. The suitable environmental conditions for PGL production by Bacillus sp.WSHB04-02 were as follows. The seed age was 14 h with inoculation size of 8%.The initial pH, fermentation temperature, medium volume in 250-ml flasks andfermentation time were 7.0, 37℃, 25 ml and 25 h, respectively. The PGLproduction was greatly affected by medium volume and the synthesis of PGLwas not induced by the addition of pectin.6. The analysis of fermentation process showed that the synthesis of PGL wascoupled with cell growth of Bacillus sp. WSHB04-02. In the flask culture, PGLproduction could be improved by adding 8 g/L corn starch in the early stage andadding 8 g/L peptone, 10 g/L yeast extract in the exponential stage of cultivation.7. Effects of dissolving oxygen level on PGL production were investigated bychanging volume oxygen transfer coefficient (KLa). At lower KLa (<100h-1),Bacillus sp. WSHB04-02 neither grew normally nor produced high level of PGL.While at high KLa (>200h-1), the dissolving oxygen could meet the requirementof cell growth and enzyme production. With KLa at 350 h-1, the strainWSHB04-02 grew well and dry cell weight reached the highest value at 6 h.Meanwhile, PGL activity reached the maximum value of 40 U/ml at 8 h whichwas the best result reported in literatures.8. The results showed that the proposed two-stage oxygen supply mode could notbe used to improve PGL activity and reduce fermentation time at the same time.