การโคลนยีนไฟเตสทนร้อนจากแอคติโนไมซีท Thermomonospora sp. RC7 เพื่อพัฒนาการผลิตเอนไซม์ไฟเตสทดแทนการนำเข้าจากต่างประเทศ

บทคัดย่อ

Phytase is commonly used as feed additive to improve phosphorus absorption which is not breakdown by enzyme in monogastric animal such as pig and poultry. The Antinutritional factors (ANFs) of phytic acid in animal feed is also reduced by phytase resulting in more efficiently absorption of other nutrients such as vitamin, mineral and protein, reducing the addition of such nutrients in animal feed and in turn lower the cost. Addition of phytase in animal feed also reduces pollution in surface and ground water created by phytate and phosphorus in animal manure in intensive livestock region. Therefore, phytase is an important enzyme in animal feed industry. However, phytase available for feed industry in Thailand is an imported enzyme. The most important source of phytase is microorganism. This study, therefore, is aimed to isolate microorganism capable of producing phytase. After that, phytase gene will be isolated, cloned and expressed in Pichia pastoris. The first microorganism, Thermomonospora sp. RC7 which has been reported to express thermostable phytase, was cultured and induced. However, the microoraganism lost the ability to produce active phytase probably due to long term storage. Therefore, isolation of phytase gene from Thermomonospora sp. RC7 was not possible. Thermotolerant microbial strains from several sources were screened. Six isolates were found to be thermotolerant strains. Three of of six (isolate 2, F and X) were found to exhibit phytase activity at 0.04, 0.49 and 0.48 U/ml, respectively. The 16S rDNA analysis revealed that they were Anoxybacillus sp. (accession no. FJ527831.1), Geobacillus sp. (accession no. JN866599.1) and Anoxybacillus sp. (accession no. FJ527830.1), respectively. Isolation of phytase gene from these strains using specific primers to Bacillus phytase was not successful. The isolation of phytase gene was successful using a theremotolerant Bacillus subtilis MR10 as a source. The nucleotide and amino acid sequence analysis revealed the similarity between B. subtilis MR10 and two other Bacillus strains (DS11 and MD2) with 4 amino acids different. The phytase was cloned into pUC19 for expression in Escherichia coli JM109. The recombinant E. coli expressed active phytase at 0.36 U/ml. However, the enzyme was not stable at high temperature. The amino acid sequence of this strain was then modified to match B. subtilis MD2 strain. The codon of the phytase was also optimized to match for Pichia pastoris expression before synthesis. The optimized sequence was cloned into expression vector pPICZA and transformed into P. pastoris. The integration of phytase gene into the genome was comfirmed by PCR technique using specific primers to optimized phytase gene. The randomly selected transformant was cultured and induced with methanol resulting in active phytase up to 0.103 U/ml.

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