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히알루론산 생산성 향상을 위한 Streptococcus zooepidemicus 균주 개량 및 발효조 배양공정 최적화

Strain Improvement and Bioprocess Optimization for Enhanced Production of Haluronic Acid(HA) in Bioreactor Cultures of Streptococcus zooepidemicus

  • 김수연 (강원대학교 의생명과학대학) ;
  • 전계택 (강원대학교 의생명과학대학)
  • Kim, Soo Yeon (College of Biomedical Science, Kangwon National University) ;
  • Chun, Gie-Taek (College of Biomedical Science, Kangwon National University)
  • 투고 : 2020.08.19
  • 심사 : 2020.09.02
  • 발행 : 2020.09.28

초록

Streptococcus zooepidemicus 유래의 세포외 고분자물질인 히알루론산(hyaluronic acid) (HA)을 대량 생산하기 위해, 균주 개량, 생산배지 및 배양공정 개발에 관한 연구를 수행하였다. HA 고생산성 변이주를 선별하기 위해 약 99%의 사멸률을 보이는 ethylmethane sulfonate (EMS) 처리조건을 적용해서, 지속적인 random screening 방법으로 고생산성, 고안정성의 변이주들을 선별할 수 있었다. HA를 고농도로 생산하기 위해서는, 이 균주의 생화학 및 배양생리적 특성에 기반한 최적 배지개발이 필수적이라고 판단하여, one-factor-at-a-time (OFAT), full factorial design (FFD), steepest ascent method (SAM) 및 response surface method (RSM) (반응표면분석법)을 순차적으로 적용하여 통계적 배지 최적화 실험을 수행하였다. 최적 배지조성에서 플라스크 배양에 의한 HA 생산성은 5.38 g/l로서, 이전 배지(3.54 g/l)에 비해 약 52% 향상된 생산량을 얻을 수 있었다. 또한 선별된 우량균주와 최적화된 생산배지를 이용하여 5 L 발효조에서 배양공정 최적화 연구를 수행하였다. 이 균주의 생리학적 특성을 고려할 때, HA 생산성을 높이기 위해서는 (배양 중 HA 축적으로 인해 고점도를 띠는) 배양액으로의 충분한 용존산소 공급이 매우 중요한 요인인 것으로 판단되었다. 따라서 용존산소 공급과 밀접하게 관련있는 발효조의 교반시스템(교반 날개 종류, 크기 및 배치 등) 및 교반속도에 대한 최적화 연구를 수행하였다. 그 결과, 교반축 하부에는 Rushton turbine-type, 상부에는 marine-type의 확장된 교반날개(기존 대비 직경 1.3배 확장)가 설치된 경우, 450 rpm에서 강화된 혼합력과 충분한 용존산소 공급으로 인해 HA 생산성이 기존 플라스크 배양 대비 약 1.8배(9.79 vs. 5.38 g/l) 더 높은 것으로 확인되었다. 최종적으로 HA 배양공정의 scale-up 가능성을 확인하기 위해, pilot 규모의 50 L 발효조 배양을 최대 300 rpm의 교반속도에서 수행하였다. 처음으로 시도한 50 L 배양임에도 불구하고, HA 최대 생산성 면에서 볼 때, 5 L 발효조 결과와 거의 동일한 수준(98.5%) (9.11 vs 9.25 g/l)의 생산량을 얻을 수 있었다. 반면 지수기 성장단계인 배양 15시간까지의 50 L 배양의 HA 평균생산속도(rp)는 0.46 g/l/hr로서 0.62 g/l/hr인 5 L 배양 대비 약 74% 정도에 머무는 것으로 나타났다. 따라서 생산 발효조의 scale-up 시, 생산균주의 전단응력 민감성(shear damage)을 함께 고려하면서, 산소전달계수(kLa)를 기반으로 하는 교반시스템에 대한 체계적인 연구가 진행된다면, HA 생산속도도 증가될 수 있는 긍정적인 결과를 얻을 수 있을 것으로 기대된다.

Strain improvement and bioprocess development were undertaken to enhance hyaluronic acid(HA) production by Streptococcus zooepidemicus cells. Using a high-yielding mutant strain, statistical medium optimization was carried out in shake flask cultures, resulting in 52% increase in HA production (5.38 g/l) at the optimal medium composition relative to the parallel control cultures. For sufficient supply of dissolved oxygen (DO), which turned out to be crucial for enhanced production of HA, agitation system and speed were intensively investigated in 5 L bioreactor cultures. Increase in oxygen mass transfer coefficient (kLa) through increment of agitation speed (rpm) and 35% expansion of diameter of the newly-designed impellers showed significantly positive effects on HA production. By installing an expanded Rushton-turbine impeller for efficient break-down of sparged air, and an extended marine impeller above the Rushton-turbine impeller for efficient mixing of the air-born viscous fermentation broth, maximum amount of HA (9.79 g/l) was obtained at 450 rpm, 1.8 times higher level than that of the corresponding flask culture. Subsequently, the possibility of bioprocess scale-up to a 50 L bioreactor was investigated. Despite almost identical maximum HA production (9.11 vs 9.25 g/l), the average HA volumetric productivity (rp) of the 50 L culture turned out only 74% compared to the corresponding 5 L culture during the exponential phase, possibly caused by shear damages imposed on the producing cells at the high stirring in the 50 L culture. The scale-up process could be successfully achieved if a scale-up criterion of constant oxygen mass transfer coefficient (kLa) is applied to the 50 L pilot-scale bioreactor system.

키워드

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