This study presents a one-step encapsulation method for synthesizing X-shaped zeolitic imidazolate frameworks (ZIF-8) and immobilizing Rhizomucor miehei lipase (RML). We proved that the morphological structure of ZIF-8 had changed after immobilization with enhanced characterization using a field-emission scanning electron microscope, an energy-dispersive spectrometer, a transmission electron microscope, a Fourier transform infrared spectrometer, and powder X-ray diffraction. The surface area and pore size of the carrier were investigated before and after immobilization using Brunauer–Emmett–Teller and Barrett–Joyner–Halenda methods, respectively. RML@ZIF-8 exhibited high recovery activity of up to 2632%, representing a 26-fold increase in its free lipase. Encapsulated RML was used for biodiesel production from soybean oil in an isooctane system with a conversion yield of 95.6% under optimum conditions. The resulting reusability of the immobilized enzyme indicated no substantial decline in the conversion yield, which remained at 84.7% of the initial activity after 10 cycles. The stability and high performance of the immobilized enzyme are attributed to the harmony between RML and ZIF-8 based on the easy synthesis of ZIF-8 and the short time required to immobilize RML.

A hierarchical mesoporous zeolitic imidazolate framework (ZIF-8) was processed based on cetyltrimethylammonium bromide (CTAB) as a morphological regulating agent and amino acid (l-histidine) as assisting template agent. Burkholderia cepacia lipase (BCL) was successfully immobilized by ZIF-8 as the carrier via an adsorption method (BCL-ZIF-8). The immobilized lipase (BCL) showed utmost activity recovery up to 1279%, a 12-fold boost in its free counterpart. BCL-ZIF-8 was used as a biocatalyst in the transesterification reaction for the production of biodiesel with 93.4% yield. There was no significant lowering of conversion yield relative to original activity for BCL-ZIF-8 when continuously reused for eight cycles. This work provides a new outlook for biotechnological importance by immobilizing lipase on the hybrid catalyst (ZIF-8) and opens the door for its uses in the industrial field.

This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9 K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350 U/mL under the optimal culture conditions of a 500 mL shaking flask containing 20 mL culture medium with the addition of 1.2 % (w/v) methanol, cultivation for 144 h at pH 7.0 and 27 °C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600 U/mL, 6,000 U/mL and 4,800 U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000 U/mL after 130 h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500 U/mL after 145 h incubation when methanol was used as the sole carbon source.