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Protein is the main component of aquatic feed. Fish meal, as the main protein raw material, has seen a rapid increase in price in recent years. So the development of new protein ingredients has become a hot issue in aquaculture. However, the new protein ingredients contain anti-nutrition factors, which are not conducive to the digestion of aquatic animals. Therefore, it is necessary to degrade anti-nutrition factors, release nutrients, and improve the nutritional value through enzymatic hydrolysis. The paper discusses the status of enzymatic hydrolysis of different protein raw materials and their application as substitutes for fish meal in aquaculture, to provide a reference for the extensive application of enzymatic hydrolysis protein raw materials in aquatic feed. [ABSTRACT FROM AUTHOR]

Short-chain chlorinated paraffins are persistent organic pollutants, and chlorinated paraffins were widely used as sizing agent in the paper industry. In order to investigate the levels and risk assessment of short-chain and medium-chain chlorinated paraffins in the paper mill plant, the surface soil and soil of different depths were collected.The concentrations, congener group profiles of short-chain chlorinated paraffins (SCCPs) and medium-chain chlorinated paraffins (MCCPs) in soil were determined by two-dimensional gas chromatography coupled with electron capture-negative ion mass spectrometry. The SCCPs and MCCPs concentrations were 42-3853 ng·g -1 and 34-2091 ng·g -1 . The chlorine contents were 59.9%-61.9% and 48.7%-52.8%. The concentrations of SCCPs and MCCPs were different in the soil collected in different sampling site. The concentration of SCCPs and MCCPs were relatively higher in soil of sewage treatment area and coating area. The CP levels in soil from the paper mill plant were at a high level compared with those in other regions. C 10 Cl 6-7 and C 14-15 Cl 5 were the main congener groups in most soil samples. The results of principal component analysis showed that the CP52 commercial products may be sources of SCCPs and MCCPs in the soil. The risk quotient (RQ) for SCCPs and MCCPs were assessed in soil of paper mill plant. The results showed that the RQ values for SCCPs in soil ranged from 0.01 to 0.73 which are the medium risk, and the RQ values for MCCPs in soil ranged from 0 to 0.07, which are the low risk. The human exposure values of children and adults are lower than TDI[10 μg·(kg·d) -1 ] in both cases. The health risks caused by non-dietary exposure under paper mill area are low.

In this paper, a simple universal high-throughput method of constructing the vectors was developed. It could add proper adapter when the PCR primers were designed, and the purpose fragments were cloned by PCR, and the various complementary sticky ends were created by T4 DNA polymerase's 3' -exodeoxyribonuclease activity. If all these fragments were put together with DNA ligase, they would recombinate in an orientation. If they had been transformated, the tansformants would be identified. Let's take the Oryza sativa single-cross homologous recombination chloroplast expression vector pRSMGA which was constructed with seven fragments as an example, if the vector pRSMGA was constructed in using the method what had mentioned, only twice recombination and transformation would be done. Scores of experiments had proved that it is a simple universal high-throughput novel method to construct the complicated vectors, which has not appeared in the periodical.

According to the published DNA sequence, a serial of elements for constructing the tobacco chloroplast multicistron site integrating expression vectors have been cloned by PCR technique, which include Prrn (a modified plastid ribosomal RNA operon promoter), psbA3' (the 3' region of the plastid psbA gene), aadA gene (encoding aminoglycoside 3'-adenylytransferase), man gene (encoding mannase), gfp gene (encoding green fluorescence protein) and tobacco chloroplast high-frequency homologous recombination ctDNA fragment (psaA/psbC, 3463 bp) (Fig.2). A tobacco chloroplast multicistron expression vector pLM4 (Fig.1) (-psaA-Prrn-SD-man-SD-gfp-SD-aadA-psbA3'- psbC-) was constructed with these elements. Then the tobacco leaves were bombarded 5 times with gold particles coated with the vector pLM4. After growing on the screening medium, the function of aadA gene was identified (Fig.3), and the function of gfp gene was confirmed by laser scanner (Fig.4), the expression of man was identified by Western blot (Fig.5). All these genes man, gfp and aadA being integrated in the tobacco chloroplast genome DNA were confirmed by PCR (Fig.6). And the multicistron expression cassette integrating in tobacco chloroplast genome DNA was confirmed by RFLP (Fig.7). All these showed that the three genes in the tomato vector pLM4 were expressed in tobacco chloroplast genome DNA.

A strain containing alkaline mannanase gene was isolated from soil by functional plates and the genome library was constructed. From it a mannanase gene TM1 was acquired and was sequenced. The BLAST analysis showed a lower-than-60% similarity of the amino acid sequence to those in GenBank and proved TM1 to be a new mannanase gene (GenBank accession number AY623903). The new gene without signal peptide was cloned into the Pichia pastoris expression vector pHBM905C. The recombinant plasmid pHBM1201 was digested by Sal I and transformed into Pichia pastoris KM71, GS115, SMD1168, respectively. All of the recombinant Pichia pastroris strains containing pHBM1201 secreted functional beta-mannanase. Because of its high mass of expression, the recombinant Pichia pastoris SMD1168-3 containing pHBM1201 was induced at shake flasks. The optimal temperature and pH of the beta-mannanase produced by the recombinant strains were 55 degrees C and 7.5, respectively. The enzymatic activity for konjak powder reached 41.8 with a half life of one hour. After keeping at 80 degrees C for 5 min, the enzymatic activity declined from 77% to 11% and the enzymatic activity could recover up to more than 60% when the temperature descended to 55 degrees C.