Biodiesel, a nontoxic,cleaning, renewable and biodegradable fuel, is expected as a substitute for conventional fossil diesel. There are three main approaches to produce biodiesel, alkali-catalysis processing, enzymatic-catalysis processing and supercritical processing. With the unique property of energy-saving and environment-friendly, enzymatic-catalysis appears a great potential for industrial application. The main bottleneck of this technology is high cost and low stability of the lipase, as well as the inactivation of lipase by methanol and so on. To settle the problem, several methods have been used including the fixed-bed bioreactor, enzyme immobilized processing, whole-cell biocatalyst, changing addition method of methanol, developing of novel acyl acceptor, enhancing methanol resistance of lipase. The main problems and the relative strategy research of the enzymatic-catalysis technology were sum up.

Lipase gene from Penicillium expansum(lip07) was cloned and over-expressed in Pichia pastoris.a random mutant named ep8,which contained a single amino acid substitution,was obtained by using the lip07 as an error-prone PCR template in previous study.ep8 shows higher thermostability than that of lip07,To further improve the thermostability of the lipase,the Lys of wild-type(lip07) and mutant(ep8) in 202 were substituted by Ala using the Overlap extension PCR technique respectively.The mutant genes(lip07-K202A and ep8-K202A) were subcloned into pAO815,and then transformed into the Pichia pastoris GS115 for extracelluar expression,respectively.15% SDS-PAGE analysis indicated that the molecular mass of PEL-ep8-K202A and PEL-lip07-K202A are both about 28kDa,which is same with the wild-type lipase.The Tm of PEL-ep8-K202A is 41.66℃,2.63℃ higher than that of the wild-type(39.03℃) and 1.21℃ higher than the random mutant(PEL-ep8:40.45℃);the Tm of single mutant(PEL-lip07-K202A) is 37.08℃,2℃ lower than that of the wild-type lipase.

Background:Clinical outcomes of undifferentiated arthritis (UA) are diverse,and only 40 ％ of patients with UA develop rheumatoid arthritis (RA) after 3 years.Discovering predictive markers at disease onset for further intervention is critical.Therefore,our objective was to analyze the clinical outcomes of UA and ascertain the predictors for RA development.Methods:We performed a prospective,multi-center study from January 2013 to October 2016 among Chinese patients diagnosed with UA in 22 tertiary-care hospitals.Clinical and serological parameters were obtained at recruitment.Follow-up was undertaken in all patients every 12 weeks for 2 years.Predictive factors of disease progression were identified using multivariate Cox proportional hazards regression.Results:A total of 234 patients were recruited in this study,and 17 (7.3％) patients failed to follow up during the study.Among the 217 patients who completed the study,83 (38.2％) patients went into remission.UA patients who developed RA had a higher rheumatoid factor (RF)-positivity (42.9％ vs.16.8％,x2=8.228,P=0.008),anti-cyclic citrullinated peptide (CCP) antibodypositivity (66.7％ vs.10.7％,x2 =43.897,P ＜ 0.001),and double-positivity rate of RF and anti-CCP antibody (38.1％ vs.4.1％,x2 =32.131,P ＜ 0.001) than those who did not.Anti-CCP antibody but not RF was an independent predictor for RA development (hazard ratio 18.017,95％ confidence interval:5.803-55.938;P ＜ 0.001).Conclusion:As an independent predictor of RA,anti-CCP antibody should be tested at disease onset in all patients with UA.