The adhesion mechanism of barnacle and its cement proteins: a review / 生物工程学报

The adhesive protein secreted by marine sessile animals can resist the resistance of water and exert stickiness under the humid environment. It has become a candidate for the development of high-performance materials in the field of biomedicine and bionics. Barnacles are as one of the marine macrofoulers that can be firmly attached to the underwater substrate materials with different surface characteristics through its cement proteins. To date, the adhesion process of barnacle has been understood in-depth, but the specific underwater adhesion mechanism has not been elucidated and needs further exploration. This review first presented an overview of barnacle and its adhesion process, followed by summarizing the advances of barnacle adhesive protein, its production methods, and applications. Moreover, challenges and future perspectives were prospected.

Advances of enzymes related to microbial cement / 生物工程学报

Microbial induced calcium carbonate precipitation (MICP) refers to the natural biological process of calcium carbonate precipitation induced by microbial metabolism in its surrounding environment. Based on the principles of MICP, microbial cement has been developed and has received widespread attention in the field of biology, civil engineering, and environment owing to the merits of environmental friendliness and economic competence. Urease and carbonic anhydrase are the key enzymes closely related to microbial cement. This review summarizes the genes, protein structures, regulatory mechanisms, engineering strains and mutual synergistic relationship of these two enzymes. The application of bioinformatics and synthetic biology is expected to develop biocement with a wide range of environmental adaptability and high performance, and will bring the MICP research to a new height.

Recent advances in enzyme immobilization / 生物工程学报

Enzyme is an efficient and green biocatalyst, and widely used in many areas. Immobilized enzyme is superior to its free form in a variety of properties. Enzyme immobilization studies started in the 1970s in China. Till now, immobilized enzymes are widely applied in the fields of food, medical, energy, environmental management, among others. However, there are still some defects such as no universal method and the high cost. Therefore, based on the relatively mature traditional immobilization technologies, efforts have been made to innovate immobilization technologies. As a result, many new immobilization technologies focusing on new carriers and methods are continuously generated. Coupling with more than ten years' study on enzyme immobilization, we present here recent development and application of new immobilization technologies, as well as suggestions to future development of immobilization technology.

Advances in platform compounds and polymers from vegetable oils / 生物工程学报

Vegetable oil is one of the most potential alternatives of petroleum and has become a hot issue in recent years. This review focuses on the influence of vegetable oil structure on platform compounds and polymers properties, and further systematically introduces their developments and the latest progress. Meanwhile, we also summarized the main confronting problems and the future development directions in the research of oil-based platform compounds and polymers. The review provides useful information for readers to fully understand biochemical engineering of vegetable oils and their prospects.

Enhanced biohydrogen production by homologous over-expression of fnr, pncB, fdhF in Klebsiella sp. HQ-3 / 生物工程学报

To enhance biohydrogen production of Klebsiella sp. HQ-3, the global transcriptional factor (Fnr), formate dehydrogenase H (FDH1) and the pncB gene encoding the nicotinic acid phosphoribosyltransferase (NAPRTase) were for the first time over-expressed in Klebsiella sp. HQ-3. The fnr, fdhF, pncB genes were cloned from the genomic DNA of Klebsiella sp. HQ-3 by 3 pairs of universal primers, and introduced into the corresponding sites of the modified pET28a-Pkan, resulting in the plasmids pET28a-Pkan-fnr, pET28a-Pkan-fdhF and pET28a-Pkan-pncB. The 4 plasmids were then electroported into wild Klebsiella sp. HQ-3 to create HQ-3-fnr, HQ-3-fdhF, HQ-3-pncB and HQ-3-C, respectively. Hydrogen production was measured using a gas chromatograph and the metabolites were analyzed with a high-performance liquid chromatograph (HPLC). The results indicate that over-expression of fnr, fdhF and pncB significantly enhanced hydrogen production in the three recombinant strains. Hydrogen production per mol glucose for HQ-3 fnr, HQ-3 pncB, HQ-3 fdhF was 1.113, 1.106 and 1.063 mol of hydrogen/mol glucose, which was respectively increased by 12.26%, 11.62% and 7.28% compared with that of the control strain HQ-3-C (0.991 mol of hydrogen/mol glucose). Moreover, the analysis of HPLC showed that the concentrations of formate and lactate were markedly decreased, but succinate remained unchanged in culture media compared with those of the control strain HQ-3-C.

Expression of Vitreoscilla hemoglobin improves recombinant lipase production in Pichia pastoris / 生物工程学报

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.

Two-step synthesis of the full length Aspergillus niger lipase gene lipA leads to high-level expression in Pichia pastoris / 生物工程学报

Aspergillus niger lipases are important biocatalysis widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Full length gene synthesis is an efficient measure to enhance the expression level of the gene. In order to reduce the non-specific binding between oligonucleotides and bases mutation caused by the complicate secondary structure of DNA and excessive PCR amplification, a frequently phenomenon in one-step gene synthesis, we used a two-step method including assembly PCR (A-PCR) and digestion-ligation step to synthesis Aspergillus niger lipase gene lipA. Assisted by DNA2.0 and Gene2Oliga software, we optimized the codon usage and secondary structure of RNA and induced enzyme sites Cla I (237 site) and Pst I (475 site) into the gene. In the first step, fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) were separately synthesized by assembly PCR. In the second step, fragments F1, F2 and F3 were separately digested by Cla I and Pst I, and then ligated into a full length lipA gene. Two-step method efficiently enhanced successful ratio for full-length gene synthesis and dispersed the risk for gene redesign. The synthesized gene was cloned into pPIC9K vector and transferred into Pichia pastoris. After methanol inducement, the expression level of the codon optimized lipA-syn gene reached 176.0 U/mL, 10.8-fold of the original lipA gene (16.3 U/mL) in Pichia pastoris GS1115. The recombinant offers the possibility for lipase large-scale production.

Homologous expression of Burkholderia cepacia G63 lipase gene based on T7 RNA polymerase expression system / 生物工程学报

In order to realize over-expression of Burkholderia cepacia (B. cepacia) lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene (T7 RNAP) from the BL21 (DE3) and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene (lipA) promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMdeltalipAlipB, pUCPCMdeltalipA, pUCPCMdeltalipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/mg, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMdeltalipB were enhanced to some extent. The specific activities of wild type B. cepacia and B. cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain.

Directed evolution of lipase of Bacillus pumilus YZ02 by error-prone PCR / 生物工程学报

Random mutagenesis on Bacillus pumilus lipase YZ02 gene was conducted by using error-prone PCR strategy. Through two cycles of directed evolution, two optimum mutants BpL1-7 and BpL2-1369 with lipase activity improved 2 folds and 6 folds respectively were screened. The sequence of BpL2-1369 lipase gene showed that four nucleotides substitution, T61C, C147T, A334G and T371A have occurred, and three of them caused amino acid changes. Thus, amine acid Ser21 was changed into Pro21, Arg112 to Gly112, and Leu124 to His124. According to the 3D structure of Bacillus pumilus lipase mimicked by SWISS-MODEL Repository, three mutated amino acids were located at the third amino acid of the first alpha-helix, the turn between the fourth and fifth beta fold, and the first amino acid of the fifth beta fold, respectively. The BpL and BpL2-1369 genes were ligated into pET28a vector, and transferred into E. coli BL21 (DE3). After induced by IPTG the lipases were purified and characterized. The results showed that the specific activity of the evolved lipase was 1.31-fold than that of the wild lipase, and the Km decreased from 8.24 mmol/L to 7.17 mmol/L. The pH stability of the evolved lipase was better than wild lipase when pH>8.0.

Technologies for Surface Display of Lipase / 中国生物工程杂志

Lipase is a kind of widely used hydrolase.Surface display is an efficient method of highthroughput screening for protein engineering of lipase.Besides,lipase displaying on surface of microorganism has many advantages,such as higher stability against high temperature and organic solvent,compared with free lipase,so the host strain displaying lipase can be used as wholecell biocatalyst,which has some advantages compared with traditional immobilization of lipase.There are three kinds of host strain for displaying lipase:phage,bacteria and yeast.The surface display of lipase in the three display systems were systematically discribed,and their current uses and possible trends in the future were discussed also.