title: Expression and characterization of camel chymosin in Pichia pastoris.
abstract: Chymosin efficiently coagulates milk and so is widely used in commercial cheese production. Traditional chymosin production requires the slaughter of a large numbers of unweaned calves. In the present study, a full-length camel prochymosin gene was synthesized and cloned into the pPIC9K vector, which was then inserted into the yeast strain, Pichia pastoris GS115. Expression of the chymosin gene in yeast was under the control of an AOX1 inducible promoter. The yeast system produced approximately 37mg/L of recombinant enzyme under lab conditions. SDS-PAGE of the raw supernatant revealed two molecular bands, which were approximately 42kDa and 45kDa in size. The 45kDa band disappeared after treatment of the supernatant with N-glycosidase F (PNGase F), indicating that the recombinant protein was partially glycosylated. When subjected to a low pH, recombinant prochymosin was converted into mature and active chymosin. The active chymosin was capable of specifically hydrolyzing κ-casein. A pH of 5.04, and temperature range of 45-50°C, was optimum for milk clotting activity. Maximum milk clotting activity was detected with the inclusion of 20-40mM CaCl2. The recombinant enzyme was highly active and stable over a wide pH range (from 2.5 to 6.5) at 20°C for 8h. Thermostability of the recombinant enzyme was also analyzed. Pilot-scale production (300mg/L) was attained using a 5L fermenter. We demonstrated that expression of the camel chymosin gene in P. pastoris could represent an excellent system for producing active camel chymosin for potential use in the commercial production of cheese.
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.affiliation: Department of Natural Sciences, Northeastern State University, Broken Arrow, OK 74014 USA.
authors.forename: Kevin Yueju
authors.initials: KY
authors.lastname: Wang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: GangQiang
authors.initials: G
authors.lastname: Li
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: WenFang
authors.initials: W
authors.lastname: Guo
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Electronic address: liudehu2006@126.com.
authors.forename: DeHu
authors.initials: D
authors.lastname: Liu
mesh_terms: D000818:Animals; D002162:Camelus; D012085:Chymosin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D012085:Chymosin
keywords: Camel chymosin; Milk-clotting; P. pastoris; Recombinant; Temperature; pH