Friday, September 20, 2019
Pectinase Production Techniques
Pectinase Production Techniques PECTINASE PRODUCTION ABSTRACT Pectin is found naturally in many plants. Pectinase enzyme has ability to break down pectin. Pectinase production has varying important needs industrially. Hence the production of pectin is important. Pectinase production has developed with the help of genetic engineering and novel strains. Production of alkaline pectinase is more important industrially than the normal pectinase. INTRODUCTION Pectin is structural heteropolysaccharide having esterification to galacturonic acid . It forms aÃ ±-1, 4 glycosidic bonds with arabinose, galactose, rhamnose and xylose . It is found in higher plants in the primary cell wall containing different composition of lignin, cellulose and hemicelluloses and proteins of microfibril that are cross linked which forms the hard tissue structure shape . Pectinase is breaking down of pectin substances by enzymes . It can be divided into different types like pectin esterases, pectin hydrolases, protopectinase and pectin lyases . It can also be divided into alkaline and acid pectinase . Pectin production has become industrially important because of its variety of applications like papermaking, extraction of natural products, textile degumming, juice extraction and clarification and so on . Pectin is observed in plants in middle lamella and primary cell wall. Pectin has a characteristic of gelation which can be divided into high and low methoxy gelation. In 1825 scientist Henri Braconnot first isolated pectin. PECTINASE PRODUCTION USING: GENETIC ENGINEERING The generation of alkaline pectinase through genetic engineering takes place as follows. The first step is the chromosomal DNA separation from a strain used for producing alkaline pectinase, primer designing, and use of PCR to obtain target gene recombinant plasmid construction, Bacillus Subtilis as means of expression, gene sequence measurement and comparative analysis [2, 7, and 8]. By using this method the enzyme activity of the engineered bacteria in the fermentation liquid increased 22 times compared to normal starting strain and the yield achieved was 330U/ml . After optimizing various culture conditions, the enzymatic activity of genetically engineered bacteria is 758.7825U/ml which is 50 times better than using original strain from Luria Bertani fermented media . NOVEL STRAIN Sometimes micro-organisms strain having unique characteristic are used for pectinase production for maximum growth example Aspergillus niger give rise to novel acid pectinase PEC 2 . Consider the strain Penicillium verruculosum TS63-9 have a gene sequence ITSrRNA which makes it useful in production . During fermentation crude enzyme liquid is obtained which is then applicable for production of tobacco . In another case novel strain like Paenibacillus is used for the production of alkaline pectinase . The production takes place in such a way that paenibacillus SJN-PL0602 is first inoculated in the fermentation broth to get initial system of which OD 600 is 0.05 -0.15 . The main fermentation shake cultivation takes place in two phase; in first at 30 -370C for 8 -12 hrs and in second phase at 22 Ã¢â¬â 260 C for 36- 40 hrs . Red algae or brown red algae were used for the production of ultra pectinase as clearing agents for wine and juices, sometimes ultrapectinase are also extracted from seaweed or waste liquor discharged from seaweed .Bacillus subtilis was used for alkaline pectinase production in paper making industry . Pichia pastoris GS115 is also used for alkaline production . Bacillus cereus is used for industrial production of pectinase . UNIQUE PROTEIN For the production of alkaline pectinase a protein Pel N having unique amino acid sequence was used . This protein has enzyme activity of 4590 Ã¢â¬â 4950 U/mg for degrading polygalacturonic acid, has better heat stability and heat preservation of 120 min at 450C gives 90% of relative heat stability . The optimum temperature is 650 C and p H value of 9.8 . ULTRA PECTINASE The production of ultra pectinase was done in order to replace pectinase and it is nonbiological . Since pectinase is used as clarifying agent it was necessary to replace pectin on industrial scale . The major advantage of ultra pectinase are the raw material are cheap, low investment, process takes place without heating, product output is higher, impurities are isolated, less moisture content is les and it takes place in less than 30 mins than the traditional method of 48 mins . ALKALINE PECTINASE The general steps that are followed during the production of alkaline pectinase the bacterial strain are selected for selective breeding, these strains are cultured, fermentation takes place through shaking and the enzyme liquid preparation takes place [2, 7, and 8]. The production of alkaline pectinase has optimized the culture condition method and they are; the fermentation medium is optimized by taking a single fact experiment, the target strain is optimized by using methods of response surface to get strain of target, the culture condition in the medium is optimized so that the fermentation condition for the target strain is optimized . In a same way nucleotide sequence of alkaline pectinase pel1685s is optimized, this is done by amplification using PCR, transformation and ligation of the pel168 gene . Industrial production of pectinase also takes place in same way as production of alkaline pectinase for high enzyme activity . ItÃ¢â¬â¢s an advantage in for having high enzyme activity for industrial pectinase production; fermentation period is short, cost is low, time taken is short and stable . APPLICATION OF PECTINASE PRODUCTION Pectin as an enzyme is used industrially for various application some of the specific areas are as follows: Papermaking pulping After pickling treatment the raw material is soaked with alkaline pectinase. The chemical extract prepared has favorable strength and property, after bleaching and chlorine treatment the plump has whiteness of 80-90%, there is 20-40% decrease in alkali consumption and the cost of production is also decreased 20% . Degumming When pectin is used for fiber blast degumming there is about 80% waste reduced, the ration of the finished is about 65%, the ratio of fiber dispersion is 100%there is almost no fiber strength destruction, also the defect in gum ratio is low, the time taken for degumming is also less . Juice making and clarification Pectin is normally found in most of the fruits. Pectin is used in juice clarification in the following way plant material to be provided, plant material need to be chopped and crushed into smaller pieces, taking this small piece and contacting it with pectinase with further clarifies the juice . Also sometimes low temperature pectinase are generated for the production of apple juice. Penicillium aculeatum is used for the generation of low temperature pectinase with the help of shaddock peel powder which acts as a carbon source for clarification of fresh apple juice. The medium used for fermentation is as follows anhydrous CaCl2 0.01%, potassium dihydrogen phosphate 0.2%, magnesium sulfate heptahydrate 0.2% , peptone 0.5-2% and shaddock peel powder 0.5-2% the p H of medium is maintained at 5.5. a clear apple juice is obtained with the help of fermentation condition like time taken is 75-147 hrs, the inoculums size is 2%, the temperature is maintained at 300 C, the speed of rotation at 3-30 rpm. In these conditions the pectinase of low temperature react with the apple juice at 100, 200 and 300 for 2 hrs to give clarified apple juice . Processing of soybean Pectinase produced by organism bacillus is used in soybean production. The processes takes place in such a way that first the soaking of soybean in water takes place which is further steamed, cooled and allowed to react with pectinase. This is held for some time period agitated further for enzyme treatment. Now slurry is generated from that single cell of soybean is taken and dispersed and the pectinase is inactivated. It is further dried to get soybean powder . CONCLUSION Different production methods of pectinase have been studied. The super or ultra pectinase production method for clearing agents in juices using seaweeds. The use of genetically engineered bacteria for the production of alkaline pectinase and the different optimization methods of culture conditions for the production. The nucleotide sequence used for the production of alkaline pectinase is also optimized. The applications of pectinase especially in the papermaking industry. The production of low temperature pectinase in juice clearing at low temperatures. Specific gene code, nucleotide and protein used for the production of pectinase are used for industrial production of alkaline pectinase.